Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-04-21
2002-08-20
Prats, Francisco (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S101000, C536S123000, C536S123100, C536S127000, C536S128000
Reexamination Certificate
active
06436679
ABSTRACT:
FIELD OF THE INVENTION
The present application relates to methods for activating and purifying polysaccharides from Aloe. In particular, the invention relates to methods for isolating polysaccharides with immunomodulatory activity from Aloe. The present invention includes the activated mixture of polysaccharides (referred to herein as “Immuno-10” or “Immuno-10 polysaccharide”), produced by the methods of the invention. The invention also includes the use of the polysaccharides as immunostimulating, immunomodulating and wound healing agents.
BACKGROUND OF THE INVENTION
Aloe is an intricate plant which contains many biologically active substances. (Cohen et al. in
Wound Healing/Biochemical and Clinical Aspects
, 1st ed. W B Saunders, Philadelphia (1992)). Over 300 species of Aloe are known, most of which are indigenous to Africa. Studies have shown that the biologically active substances are located in three separate sections of the aloe leaf—a clear gel fillet located in the center of the leaf, in the leaf rind or cortex of the leaf and in a yellow fluid contained in the pericyclic cells of the vascular bundles, located between the leaf rind and the internal gel fillet, referred to as the latex. Historically, Aloe products have been used in dermatological applications for the treatment of burns, sores and other wounds. These uses have stimulated a great deal of research on identifying compounds from Aloe that have clinical efficacy, particularly anti-inflammatory activity. (See, e.g., Grindlay and Reynolds (1986) J. of Ethnopharmacology 16:117-151; Hart et al. (1988) J. of Ethnopharmacology 23:61-71). As a result of these studies there have been numerous reports of Aloe compounds having diverse biological activities, including anti-tumor activity, anti-acid activity (Hirata and Suga (1977) Z. Naturforsch 32c:731-734), anti-diabetic activity, tyrosinase inhibiting activity (Yagi et al. (1987) Planta medica 515-517) and antioxidant activity (International Application Serial No. PCT/US95/07404, published Dec. 19, 1996, publication number WO 96/40182).
It has also been reported that Aloe products can stimulate the immune system. The ability of Aloe to stimulate the immune system has been attributed to polysaccharides present in the gel. (See, e.g., Day et al. (1922) J. Am. Pharm. Assoc. 11:462-463; Flagg (1959) American Perfumes and Aromatics 74:27-28, 61; Waller et al. (1978) Proc. Okla. Acad. Sci. 58:69-76; Shcherbukhin et al. (1979) Applied Biochemistry & Microbiology 15:892-896; Mandal et al. (1980) Carbohydrate Research 86:247-257; Mandal et al. (1980) Carbohydrate Research 87:249-256; Winters et al. (1981) Eco. Botany 35:89-95; Robson et al. (1982) J. Burn Care Rehab. 3:157-163; Ivan etal. (1983) Drug & Cosmetic Ind. 52-54, 105-106; Smothers (1983) Drug & Cosmetic Ind. 40:77-80; Mandal et al. (1983) Indian J. of Chem. 22B:890-893; Vilkas etal. (1986) Biochimie 68:1123-1127; Waller etal. (1994) Cosmetic Toiletries Manufacturing Worldwide 64-80; U.S. Pat. No. 5,308,838 of McAnalley et al. ).
Aloe products are also used extensively in the cosmetic industry to protect skin against the harmful effects of ultraviolet radiation. (Grollier et al. U.S. Pat. No. 4,656,029, issued Apr. 7, 1987). Chronic exposure of the skin to ultraviolet radiation causes skin cancer in humans and laboratory animals. Exposure of the skin of laboratory animals to ultraviolet B (UVB) radiation (280-320 nm) causes suppression of the skins immune system, which impairs its ability to develop an immune response to UV-induced skin cancers, contact-sensitizing haptens and a variety of infectious microorganism. (See, Strickland (1994) J. Invest. Dernatol. 102:197-204, and references cited therein). Studies by Strickland et al. show that topical application of Aloe vera gel reduces the suppression of the immune system caused by UVB exposure. (Strickland (1994) J. Invest. Dermatol. 102:197-204).
The ability of native gel to reduce suppression of the immune system, is very low and irregular and also decreases with time. One hypothesis is that the UV-B protective factor is hydrolyzed by naturally occurring enzymes in the Aloe plant and/or by bacterial degradation. Therefore, it would seem likely that isolating polysaccharides from Aloe would help preserve this immunomodulatory activity. Previous prior art methods for the bulk isolation of polysaccharides from Aloe, however, do not effectively preserve the immunomodulatory activity. These methods, described for example in U.S. Pat. Nos. 4,957,907, 4,966,892 and 5,356,811, use lengthy (4-24 hours) alcohol precipitation and centrifugation steps. Given the failure of the prior art methods to effectively preserve the immunomodulatory activity of Aloe gel, it would be useful to have a procedure for the isolation of polysaccharides from Aloe that would allow the immunomodulatory activity to be retained and stabilized. The present invention provides such methods.
SUMMARY OF THE INVENTION
The present application relates to methods for activating and isolating a mixture of polysaccharides from Aloe. Included in the present invention is the activated mixture of polysaccharides produced and the use of said mixture as an immunostimulating, immunomodulating and wound healing agent. The activity of polysaccharides isolated by the method of this invention is much higher and much more stable and reproducible than that of native Aloe gel extracts.
The method of the present invention is comprised of (a) extracting Aloe gel juice from Aloe; (b) performing a controlled limited enzymatic hydrolysis of the total polysaccharides in said Aloe gel juice at a temperature and for a period of time suitable for limited carbohydrate hydrolysis; (c) terminating said hydrolysis; and (d) optionally decolorizing and filtering said hydrolyzed product. In a preferred embodiment the limited hydrolysis is performed by the addition of cellulase at 25° C.±1° C. for a period of 2-2.5 hours using a ratio of 0.5 g-2.5 g of cellulase to 216 L of gel extract. A schematic diagram of the instant method is provided in FIG.
1
.
The present invention includes the mixture of polysaccharides (referred to herein as “Immuno-10” or “Immuno-10 polysaccharide”) prepared and isolated by the methods of this invention. Said composition of matter is characterized in detail below.
The present invention also includes the use of Immuno-10 as an immunostimulating, immunomodulating and wound healing agent. Immuno-10 prevents suppression of contact hypersensitivity (CH) in mice exposed to UVB radiation and also inhibits UVB irradiation-induced tumor necrosis factor (TNF-&agr;) release in human epidermoid carcinoma cell line. The Immuno-10 isolated by the method of this invention can be used in an oral or topical formulation for the restoration or stimulation of the human immune system, for individuals suffering immunodeficiency or immune-suppressing diseases or for therapeutic treatment for diseases, such as HIV. The Immuno-10 isolated by the method of this invention is also useful for wound healing. The polysaccharides isolated by the method of this invention are more active and more stable than native Aloe gel.
The methods described herein include a limited and controlled hydrolysis of Aloe polysaccharides, which operates to increase the stability and immunomodulatory activity of Aloe polysaccharides. The method is faster, simpler and more amenable to scale-up than prior art methods, and does not involve the use of organic solvents. Moreover, the processes described herein increase the solubility of Aloe polysaccharide and reduce the viscosity of solutions thereof without loss of the immunomodulatory activity. Immuno-10 isolated using the method of this invention shows qualitatively-similar UVB protective activity as the activated bulk polysaccharide purified from the same Aloe gel extracts, but has a higher specific activity than the bulk polysaccharide. Additionally, the purified Immuno-10 exhibits UVB CH restorative activity that is at least twice as high as that of native Aloe gel.
It is to be understood that both th
Mahiou Belaid
Qiu Zhihua
Prats Francisco
Swanson & Bratschun L.L.C.
Unigen Pharmaceuticals, Inc.
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