HK1257032A1 - Compositions and methods of aloe polysaccharides - Google Patents

Description

Method and composition for preparing fine powder of polymannan extract

The application is a divisional application of an invention patent application with the application date of 2011, 01, and 06, the application number of 201180064415.X, and the name of the invention patent application is aloe polysaccharide composition and method.

Technical Field

The present invention relates generally to the field of aloe polysaccharides, and more particularly to aloe polysaccharide compositions and the use of the compositions as immunomodulators and in the treatment of different types of cancer.

Background

Without limiting the scope of the invention, its background is described in connection with compositions, methods of preparation, and therapeutic uses of the polysaccharide of the claw of sheep (Aloe Vera).

U.S. Pat. No.7,196,072(2007) issued to Pasco et al describes a complex water-soluble polysaccharide fraction isolated from the carpel of sheep having potent immunostimulatory activity. The polysaccharide fraction has an apparent molecular weight of more than 2,000,000 daltons and has glucose, galactose, mannose and arabinose as its main components. The invention further describes pharmaceutical compositions containing the polysaccharide component, optionally in combination with acceptable pharmaceutical carriers and/or excipients. The pharmaceutical composition can be used to provide immune stimulation to an individual in need of such treatment by administering to the individual an effective amount of the composition.

U.S. Pat. No.6,083,508(2000), issued to Avalos and Danhof, describes a process for producing an aloe product from only the leaf residue obtained after slicing the aloe leaves with the inner mesophyll removed. According to the' 508 patent, the residue is slurried by grinding and the aloe product is produced from the slurry. In addition, the step of preparing the aloe product includes washing the aloe leaves prior to slicing the aloe leaves, separating the resulting slurry into a liquid and a solid, and further processing the separated liquid to remove laxative prior to forming the aloe product. In addition, a process comprising all of the above steps may also be carried out to produce the liquid.

U.S. patent application No.2006/0084629 (Needleman and Needleman,2006) discloses a composition of two specific high molecular weight-long chain components isolated from the caralluma foetida and grifola frondosa (Maitake TD) that stimulate immune system activity, comprising a long chain-high molecular weight polysaccharide that activates the body's natural immune response, triggering an increase in the production of macrophages, T cells, B cells, natural killer cells, cytokines and antibodies. The long chain polysaccharides together with other active ingredients can provide appropriate support to the immune system to prevent debilitating diseases such as cancer, heart disease and aging.

Disclosure of Invention

The present invention describes an aloe polysaccharide composition and the use of the composition as an immunomodulator and in the treatment of different types of cancer selected from leukaemia and lymphoma, prostate cancer, breast cancer and colon cancer, and in the treatment of immunological diseases, especially immunologically related tumour diseases.

The present invention provides a method for preparing fine powder of polymannan extract, comprising the steps of: (i) weighing a specified amount of freeze-dried aloe powder, wherein the amount is corrected for moisture content, (ii) dissolving the freeze-dried aloe powder in deionized water to form a solution, (iii) adding an organic solvent to the solution to form a first mixture; wherein the ratio of organic solvent to deionized water is at least 2.5:1, (iv) settling the first mixture for at least 8 hours, (v) removing a specified volume of supernatant from the first mixture and adding an excess volume of the supernatant to form a second mixture, (vi) centrifuging the second mixture, (vii) observing the formation of a precipitate in the second mixture, (vii) adding another amount of organic solvent to the first mixture if some precipitate is observed in the second mixture, (viii) decanting the supernatant of the first mixture in a siphonic manner, wherein decanting is performed only when no precipitate is observed in the second mixture, (ix) filtering the precipitate from the first mixture with a filter paper and a suction filter funnel under vacuum, (x) recovering the polymannan extract powder from the filter funnel by scraping, (xi) Placing the polymannan extract powder in a capped freeze-dried bottle in a freezer for at least 8 hours, (xii) freeze-drying the frozen polymannan extract powder in the freeze-dryer, and (xiii) grinding the polymannan extract freeze-dried powder to a very fine-textured powder in a grinder.

In one aspect, the method further comprises the steps of weighing, labeling, and storing the fine polymannan extract powder in a container. The freeze-dried Aloe powder described in a particular embodiment of the present invention is derived from an Aloe species selected from the group consisting of Aloe vera (aloeevera), Aloe barbadensis (Aloe arborescens), damask brocade (Aloe arista), Aloe bifidus (Aloedichotoma), alue nyeriensis (Aloe nyeriensis), Aloe assortment (Aloe variegate), Aloe barbadensis (aloebarbidensis), and Aloe lildii. The freeze-dried aloe powder used in the present invention comprises aloe polysaccharides, wherein the aloe polysaccharides comprise one or more short-chain, medium-chain, long-chain, very-long-chain polysaccharides, or any combination thereof. In a particular aspect, the organic solvent is selected from the group consisting of methanol, ethanol, isopropanol, and propanol. In another aspect, the aloe polysaccharides further comprise a monosaccharide selected from glucose, mannose, arabinose, and galactose, and have a molecular weight of 11,500 daltons to over 10,000,000 daltons. In another aspect, the freeze-dried aloe powder has at least 25% aloe polysaccharides. In another aspect the freeze-dried aloe powder has 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 95% aloe polysaccharides. The freeze-dried aloe powder described in the preparation method of the present invention comprises at least 14% of aloe polysaccharides having a molecular weight of 66,000 daltons, at least 9% of aloe polysaccharides having a molecular weight of 480,000 daltons, at least 3.5% of aloe polysaccharides having a molecular weight of 1,000,000 daltons, and at least 2.4% of aloe polysaccharides having a molecular weight of 2,000,000 daltons.

In a particular aspect of the methods relating to aloe polysaccharide compositions, about 1.32% -6.36% of the aloe polysaccharides in the aloe freeze-dried powder have a molecular weight of 2,000,000 daltons, 2.55% -3.89% have a molecular weight of 1,000,000 daltons, and 63.85% -73.36% of the aloe polysaccharides have a molecular weight of 480,000 daltons. In one aspect, the freeze-dried aloe powder may contain one or more residual small molecular weight substances selected from the group consisting of glucose, organic acids, lactic acid, malic acid, citric acid, and aspartic acid. In another aspect, the one or more residual small molecular weight species are present in an amount of 14% to 24%. In another aspect, the fine powder of polymannan extract can be used to prepare a pharmaceutical composition for the treatment of one or more malignancies selected from the group consisting of leukemia and lymphoma, prostate cancer, breast cancer, and colon cancer, and for the treatment of one or more immune disorders.

In another embodiment, the present invention discloses a sterile injectable formulation of a polymannan extract comprising a specified amount of a polymannan extract dissolved in deionized water and one or more pharmaceutically acceptable preservatives. One or more pharmaceutically acceptable preservatives that may be used in the formulations described above are selected from the group consisting of parabens, benzoic acid and its salts, mercurial, quaternary ammonium salts, benzyl alcohol and other related alcohols, and phenols. In a particular aspect, the preservative is benzyl alcohol. In one aspect, the polymannan extract comprises aloe polysaccharides, wherein the aloe polysaccharides comprise one or more short-chain, medium-chain, long-chain, very-long-chain polysaccharides, or any combination thereof. In another aspect, the aloe polysaccharides further comprise monosaccharides selected from glucose, mannose, arabinose, and galactose.

In another particular aspect, the aloe polysaccharides have a molecular weight of 11,500 daltons to greater than 10,000,000 daltons and the polymannan extract has at least 25% aloe polysaccharides, wherein the polymannan extract has 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 95% aloe polysaccharides. In a related aspect, the polymannan extract comprises at least 14% aloe polysaccharides having a molecular weight of 66,000 daltons, at least 9% aloe polysaccharides having a molecular weight of 480,000 daltons, at least 3.5% aloe polysaccharides having a molecular weight of 1,000,000 daltons, and at least 2.4% aloe polysaccharides having a molecular weight of 2,000,000 daltons. The composition of the invention is used for treating one or more cancers selected from leukemia and lymphoma, prostate cancer, breast cancer, and colon cancer, for immunomodulation, immunostimulation, or for treating individuals with a compromised immune system or immune disease. The compositions of the present invention can result in a 75-80% increase in one or more natural killer cells (NK).

In another embodiment, the present invention describes a method of treatment for treating one or more cancers selected from the group consisting of leukemia and lymphoma, prostate cancer, breast cancer, and colon cancer, comprising the steps of: identifying an individual in need of treatment for one or more cancers and injecting a polymannan extract sterile injection at a dose sufficient to treat the one or more cancers 2 to 3 times a week, wherein the polymannan extract sterile injection comprises a specified amount of a very fine polymannan extract dissolved in deionized water; and one or more pharmaceutically acceptable preservatives. The method further comprises the steps of: blood samples are taken from the individual at one or more designated time intervals and the concentration of caspase 3(caspase 3) protein in the blood is determined and the resulting concentration is compared to the concentration prior to injection, wherein an increase in caspase 3 concentration is directly correlated with an increase in the level of apoptosis in one or more cancer cells.

In one aspect, the dose of the polymannan extract sterile injection depends on the weight, age, race, and sex of the individual. In another aspect, the polymannan extract comprises aloe polysaccharides, wherein the aloe polysaccharides comprise one or more short-chain, medium-chain, long-chain, very-long-chain polysaccharides, or any combination thereof. In another aspect, the aloe polysaccharides have a molecular weight of 11,500 daltons to greater than 10,000,000 daltons. The polymannan extract described in the methods of the invention has at least 25% aloe polysaccharides. The polymannan extract in the methods of the invention results in a 75-80% increase in one or more natural killer cells (NK).

in one embodiment, the present invention discloses a method of immunomodulation or immunostimulation in an individual having a compromised immune system or an immune disorder, comprising the steps of (i) identifying an individual having a compromised immune system or an immune disorder and in need of immunomodulation or immunostimulation, (ii) intravenously administering a specified dose of a sterile injectable formulation of a polymannan extract, wherein the sterile injectable formulation of the polymannan extract comprises a specified amount of a very fine polymannan extract dissolved in deionized water, and one or more pharmaceutically acceptable preservatives, wherein the dose of the sterile injectable formulation of the polymannan extract is dependent on the weight, age, race, and sex of the individual, (iii) withdrawing a blood sample from the individual at one or more specified time intervals, and determining the concentration of tumor necrosis factor-alpha (TNF α) in the blood and comparing the obtained concentration with the concentration prior to injection, wherein an increased concentration of TNF α is indicative of immunomodulation or immunostimulation.

Drawings

For a more complete understanding of the features and advantages of the present invention, reference is made to the detailed description of the invention along with the following figures, in which:

FIG. 1 shows a size exclusion chromatogram of aloe polysaccharides showing different residence times of glucose and mannose subunits (sub-units);

FIG. 2 is a graph showing size exclusion chromatography of aloe polysaccharides corresponding to different glucose and mannose subunit peaks;

FIG. 3 is a proton-nuclear magnetic resonance spectrum of a polymannan extract of the present invention;

FIG. 4A is an HPLC chromatogram showing the amount of polysaccharide in each group of polysaccharide molecules in a methanol precipitated aloe polysaccharide concentrate;

FIG. 4B is a proton-nuclear magnetic resonance spectrum of methanol precipitated aloe polysaccharide concentrate;

FIG. 5A is an HPLC chromatogram showing the amount of polysaccharide in each group of polysaccharide molecules in an ethanol precipitated aloe polysaccharide concentrate;

FIG. 5B is a proton-nuclear magnetic resonance spectrum of ethanol precipitated aloe polysaccharide concentrate;

FIG. 6A is an HPLC chromatogram showing the amount of polysaccharide in each group of polysaccharide molecules in an isopropanol-precipitated aloe polysaccharide concentrate;

FIG. 6B is a proton-nuclear magnetic resonance spectrum of isopropanol-precipitated aloe polysaccharide concentrate;

FIG. 7A is an HPLC chromatogram showing the amount of polysaccharide in each group of polysaccharide molecules in propanol-precipitated aloe polysaccharide concentrate; and

figure 7B is a proton-nmr spectrum of propanol precipitated aloe polysaccharide concentrate.

Detailed description while various specific embodiments of the invention are discussed in detail below, it should also be appreciated that the present invention provides many applicable inventive concepts which can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention, and do not delimit the scope of the invention.

To facilitate an understanding of the present invention, a number of terms are defined below. The terms defined herein have meanings as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms such as "a", "an" and "the" are not meant to refer to only a single entity (singular entity), but also include the general class of which a particular embodiment may serve as an example. The terminology herein is used to describe specific embodiments of the invention, but its use is not intended to be limiting of the invention, except as outlined in the claims.

the use of human macrophages/monocytes to assess immunostimulation, and to assess the cell type with respect to the secretion of tumor necrosis factor α (TNF α).

Aloe polysaccharides are generally considered to be molecules consisting primarily of glucose and mannose monosaccharides, with long chains having molecular weights of 10,000 to 10,000,000 daltons. The higher the mannose content and the longer the chain length, the greater the immunomodulatory activity expressed by the polysaccharide. Listed in table 1 are the different length linear chains comprising the aloe polysaccharides.

Table 1: aloe polysaccharide composition and its pharmacological action.

Aloe polysaccharides having a molecular weight of 100,000 daltons or greater are listed in Table 2.

Table 2: the molecular weight and composition of aloe polysaccharides having a molecular weight of 100,000 or more.

The inventors have described the precursor material of polymannan extracts in a previous patent entitled "Method of Processing aloe leaves" (U.S. Pat. No.6,083,508-Avalos and Danhof, 2000). The analytical validation of the polymannan extract precursor raw material is listed in table 3.

FIG. 1 is a size exclusion chromatography of aloe polysaccharide preparations showing residence times of different sizes of glucose and mannose subunits. Figure 2 is a size exclusion chromatography to identify aloe polysaccharide preparations in the molecular weight range of 100 to 10,000,000 daltons. FIG. 3 is a proton-nuclear magnetic resonance spectrum of the polymannan extract of the present invention. FIG. 3 shows: (i) the absence of standard preservatives-sodium benzoate and potassium sorbate, (ii) the presence of smaller monohexoses, (iii) the peaks of isocitric acid indicating that whole leaf methodology was employed in processing aloe raw materials, (iv) the presence of malic acid peaks-a major hallmark of the caralluma capricolum, (v) the presence of barbaloin/acetylated mannan peaks in the polysaccharide portion of the spectrum confirming the presence of large polysaccharide species, and (vi) the presence of acetyl groups confirming the presence of partially acetylated polysaccharide glucomannan.

Size Exclusion Chromatography (SEC) of aloe vera product prior to extraction of polymannan:

the instrument comprises the following steps: the HPLC system was a Hitachi L-7100 pump and 7250 autosampler coupled to a Waters 410 differential refractometer. The SEC is a Tosoh BiosepG6000PWXL TSK gel column of 30cm x7.8mm volume operated in a column heater heated to 70 ℃. Molecular weight standards were purchased from Sigma-2,000,000 daltons, 1,000,000 daltons, 480,000 daltons, 66,000 daltons, and 180 daltons (glucose). The mobile phase was deionized water at a flow rate of 0.70 mL/min. The injection volume was 10 uL. The SEC method is performed by Pugh et al (2001)¹A description is given.

Table 3: and (3) analysis and verification of a polymannan extract precursor raw material.

Table 4: SEC results of aloe vera product before polymannan extraction.

Aloe vera precipitant evaluation study: 25ml COATS concentrated aloe samples were pipetted into each 200ml beaker and 125ml of each polysaccharide precipitant liquid was added separately to the beaker and stirred well. The precipitated polysaccharide was collected by filtration through a weighed amount of dehydrated filter paper, which was placed in a dry box overnight after filtration. The next morning, the dry weight of the precipitated polysaccharide was determined. The present inventors have conducted studies on four kinds of alcoholic precipitants including methanol, ethanol, isopropanol, and propanol. The powder was passed through an HPLC procedure to determine the different content of all kinds of molecules, which recorded the measured amount of polysaccharide in each group of polysaccharide molecules, including each group of polysaccharide molecules greater than 2,000,000 daltons, greater than 1,000,000 daltons, greater than 480,000 daltons, greater than 66,000 daltons, and the residue fraction, which included very small molecular species such as glucose with a molecular weight of 180 (HPLC data as shown in tables 6-9). The HPLC profiles corresponding to the four precipitants methanol, ethanol, isopropanol, and propanol are shown in fig. 4A, 5A, 6A, and 7A, respectively. Proton nuclear magnetic resonance spectra of the precipitates were also obtained and are shown in FIGS. 4B, 5B, 6B, and 7B. The data collected are shown in table 5.

TABLE 5 Aloe Vera precipitant evaluation test data.

TABLE 6 HPLC data showing the amount of polysaccharide in each group of polysaccharide molecules in methanol precipitated aloe polysaccharide concentrates.

Table 7: HPLC data showing the amount of polysaccharide in each group of polysaccharide molecules in ethanol precipitated aloe polysaccharide concentrate.

TABLE 8 HPLC data showing the amount of polysaccharide in each group of polysaccharide molecules in isopropanol precipitated aloe polysaccharide concentrate.

Table 9: HPLC data showing the amount of polysaccharide in each group of polysaccharide molecules in propanol precipitated aloe polysaccharide concentrate.

The polymannan extract is prepared by precipitation. After correct calibration for moisture content, the freeze-dried aloe powder described above was weighed. For example, if the water content is 3.7% and we need 80g, then the inventors weigh 82.96g (80g + (3.7% x 80) g). The weighed aloe powder was completely dissolved in 1 gallon of deionized water (d.i) in a stainless steel precipitation vessel. 2.5 gallons of 95% ethanol was added and stirred to ensure complete mixing. The vessel was covered with a stainless steel lid and the mixture was allowed to stand overnight.

The next day, 2mL of the clear supernatant was taken and 5mL of 95% ethanol was added and the sample was centrifuged at 3000rpm for 20 minutes. The sample was checked for precipitation and if no precipitation was observed the precipitation was considered complete. If any significant precipitation is observed, another quantity of 95% ethanol is added to the precipitation vessel before proceeding to the next step. Decanting the clarified liquid in the settling vessel by siphoning without agitating the sediment at the bottom of the vesselSupernatant liquid. By using a suction filter funnel (Whatman)Quantitative ashless filter paper) the bottom white precipitate was separated. The precipitate was removed by scraping it into a 600ml virtis lyophilization flask and the material was spread over the entire face of the flask to form a thin layer with a large exposed surface area. The lyophilized bottle was placed in a shell-freezer (shell-freezer) overnight. The following day the frozen lyophilization flask with frozen contents was placed on a lyophilizer operating at-90 ℃ and 1/3 atm for 24 hours. The lyophilizer was shut down and the lyophilized powder was placed in a small powder mill until it was ground to a uniformly finely divided powder. The pulverized fine powder was weighed and placed in a small plastic container, and then the container was stored in a freezer.

Preparation of polymannan extract injection solution: after correction for water content and determination of at least 2% barbaloin content by size exclusion chromatography, polymannan extract Powder (PME) prepared as described above (1.5g) was weighed. 1ml of concentrated HCl was added to 125ml of warm D.I. (deionized) water and stirred, followed by slow addition of the weighed PME powder with continuous stirring. Stirring was continued until all the PME powder was dissolved and the solution was clear and colorless, and an additional amount of concentrated HCl was added to obtain a pH of 1.6 to 1.7 (continuously measured with a pH meter). Added d.i water was added to adjust the volume to 150ml, followed by monitoring of pH to ensure pH 1.6-1.7. The PME solution was then poured into a 0.45 μm pore size150ml filtration system flask. The flask system was placed in a refrigerator and the filtrate was transferred to a 0.22 μm pore sizeA150 ml filtration system flask was placed in a refrigerator overnight. The filter top of the filtration system was removed under sterile conditions and the vial was sealed with a sterile cap. The bottle was then transferred to a compounderThe laboratory, and under a sterile hood 0.9% benzyl alcohol as a preservative (since the final product is for multi-dose use) was added, and the solution was placed in a 10mL sterile glass vial and sealed with a multidose closure (multidose closure). The vials were labeled with lot, control number, date of manufacture, 6 month expiration date, and physician and patient names.

evaluation of PME immunomodulatory Activity human macrophage/monocyte cells obtained from the American Type Culture Collection (ATCC) located in Maryland were used to evaluate the immunostimulatory activity.the cell type was evaluated for TNF α secretion.under standard cell conditions, small amounts of the final PME product were introduced into the culture.samples were taken at 6, 12, and 24 hours and TNF α concentrations were evaluated.

binding of PME to macrophage/monocyte mannose-binding proteins results in the release of a large number of cell transmitters (cytocommunications) including TNF- α, IL-1 β, INF- γ, IL-2, and IL-6, which restore impaired immune system surveillance, allowing the patient's immune system to recognize and remove malignant cells, wherein the impaired immune system surveillance disables the tumor detection function of the cancer patient's immune system.

The aloe polysaccharides having molecular weights of 1,000,000, 300,000, 100,000, 50,000 and 25,000 in polymannan extracts all showed caspase (caspase) activity. Since caspase 3 is a mediator of tumor cell apoptosis, the activity of caspase 3, caspase 9, and cytochrome-C is critical to the treatment of malignant tumors with the compositions of the present invention. Immunomodulatory activities of initiator (most upstream) caspase (caspase)3 and effector (effector) caspase (caspase)9, as well as cytochrome-C, have been demonstrated to be present and considered as mediating systems of tumor cell apoptosis.

The inventors tested the compositions described herein in 104 patients with different types of cancer. Leukemia and lymphoma were most reactive (> 98%) to the polymannan extract of the invention. Prostate, breast, and colon cancers also respond to polymannan extracts of the present invention. For this test, the polymannan extract was administered by injection. 10mg of polymannan extract was re-dissolved in sterile water for injection to give a final concentration of-10 mg/mL. Injections were administered 2 to 3 times a week. Patient serum samples were then taken at regular intervals and assayed for caspase 3 activity.

It is contemplated that any particular embodiment discussed in this specification can be practiced according to any of the methods, apparatus, reagents, compositions of the invention, and vice versa. Furthermore, the compositions of the invention can be used to carry out the methods of the invention.

It is to be understood that the specific embodiments described herein are disclosed by way of example and not as limitations of the invention. The principal features of this invention can be employed in various specific embodiments without departing from the scope of the invention. Those skilled in the art can ascertain, using no more than routine experimentation, many equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of the invention and are encompassed by the claims.

All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

In the claims and/or the specification, the use of the words "a" or "an" when used in conjunction with the term "comprising" may mean "one," but it is also consistent with the meaning of "one or more," at least one, "and" one or more. Although the invention supports the definition of "and/or" and "in the claims, the use of the term" or "in the claims means" and/or "unless explicitly indicated to the contrary that the alternatives are unique or mutually exclusive. Throughout this application, the term "about" means that a value includes inherent variations in the apparatus, the method used to determine the value, or variations that exist between subjects.

As used in this specification and claims (and claims), the words "comprising" (and any form of comprising, such as "comprises" and "comprising" where the subject is the plural and "comprising" where the subject is the singular), "having" (and any form of having, such as "having" where the subject is the plural and "having" where the subject is the singular), or "including" (and any form of including, such as "including" where the subject is the plural and "including" where the subject is the singular) or "containing" (and any form of containing, such as "containing" where the subject is the plural and "containing" where the subject is the singular) are inclusive or open-ended and do not exclude additional unrecited elements or method steps.

As used herein, the term "or combinations thereof refers to all permutations and combinations listed before that term. For example, "A, B, C, or a combination thereof," is meant to include at least one of: A. b, C, AB, AC, BC, or ABC, and if order is of importance in a particular context, BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with the example, combinations comprising one or more repetitions of the term or item are also expressly included, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and the like. It will be understood by those of skill in the art that, in general, there is no limitation to the number of items or terms in any composition, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein can be made and executed in accordance with the present invention without undue experimentation. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. It will be apparent to those skilled in the art that all such similar substitutes and modifications are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Reference to the literature

United States Patent No.7,196,072:High Molecular WeightPolysaccharide Fraction From Aloe Vera with Immunostimulatory Activity.

United States Patent No.6,083,508:Method of Processing Aloe Leaves.

United States Patent Application No.2006/0084629:Immune SystemActivating Formula Composed of Selected Long Chain Polysaccharides FromNatural Sources.

¹Pugh N.,Ross S.A.,ElSohly M.A.,andPasco,D.S.(2001).Characterizationof Aloeride,a new high-molecular weight polysaccharide from Aloe vera withpotent immunomodulatory activity.J Agr.Food Chem.,49,1030-1034.

Claims (33)

1. A method for preparing fine powder of polymannan extract, comprising the steps of:

weighing a specified amount of freeze-dried aloe powder, wherein the amount is corrected for moisture content, the freeze-dried aloe powder having at least 25% aloe polysaccharides, the freeze-dried aloe powder being derived from aloe barbadensis (Aloe barbadensis);

dissolving the freeze-dried aloe powder in deionized water to form a solution;

adding a first volume of an organic solvent to the solution to form a first mixture; wherein the ratio of the organic solvent to deionized water is at least 2.5: 1;

settling the first mixture for at least 8 hours;

removing a specified volume of supernatant from the first mixture and adding a second volume of the organic solvent to the specified volume of supernatant to form a second mixture;

centrifuging the second mixture;

observing the second mixture for the occurrence of a precipitate;

if some precipitation is observed in the second mixture, adding a third volume of organic solvent to the first mixture;

decanting the supernatant of the first mixture in a siphonic manner, wherein decanting is performed only when no precipitate is observed in the second mixture;

filtering the precipitate from the first mixture under vacuum with filter paper and a suction funnel;

recovering the polymannan extract powder from the suction funnel by scraping;

placing the polymannan extract powder in a capped freeze-dried bottle and placing in a freezer for at least 8 hours;

freeze-drying the frozen polymannan extract powder in a freeze-dryer; and

the polymannan extract freeze-dried powder is ground in a grinder to a powder of desired texture.

2. The method of claim 1, further comprising the steps of weighing, labeling, and storing the fine polymannan extract powder in a container.

3. The method of claim 1, wherein the organic solvent is selected from the group consisting of methanol, ethanol, isopropanol, and propanol.

4. The method of claim 1, wherein the freeze-dried aloe powder comprises aloe polysaccharides, wherein the aloe polysaccharides comprise one or more short-chain, medium-chain, long-chain, very-long-chain polysaccharides, or any combination thereof.

5. The method of claim 4, wherein the aloe polysaccharides further comprise monosaccharides selected from the group consisting of glucose, mannose, arabinose, and galactose.

6. The method of claim 4, wherein the aloe polysaccharides have a molecular weight of 11,500 daltons to 10,000,000 daltons.

7. The method of claim 1, wherein the freeze-dried aloe powder has 25% to 95% aloe polysaccharides.

8. The method of claim 1, wherein the freeze-dried aloe powder comprises at least 14% aloe polysaccharides having a molecular weight of 66,000 daltons.

9. The method of claim 1, wherein the freeze-dried aloe powder comprises at least 9% aloe polysaccharides having a molecular weight of 480,000 daltons.

10. The method of claim 1, wherein the freeze-dried aloe powder comprises at least 3.5% aloe polysaccharides having a molecular weight of 1,000,000 daltons.

11. The method of claim 1, wherein the freeze-dried aloe powder comprises at least 2.4% aloe polysaccharides having a molecular weight of 2,000,000 daltons.

12. The method of claim 1, wherein the amount of aloe polysaccharides having a molecular weight of 2,000,000 daltons in the freeze dried aloe powder is between 1.32% and 6.36%.

13. The method of claim 1, wherein the amount of aloe polysaccharides having a molecular weight of 1,000,000 daltons in the freeze dried aloe powder is between 2.55% and 3.89%.

14. The method of claim 1, wherein the amount of aloe polysaccharides having a molecular weight of 480,000 daltons in the freeze dried aloe powder is 63.85% to 73.36%.

15. The method of claim 1, wherein the freeze-dried aloe powder may contain one or more residual small molecular weight substances selected from the group consisting of glucose, organic acids, lactic acid, malic acid, citric acid, and aspartic acid.

16. The method of claim 15, wherein the one or more residual small molecular weight species are present in an amount of 14% to 24% by weight.

17. The method of claim 1, wherein the fine powder of polymannan extract is used to prepare a pharmaceutical composition.

18. The method of claim 17, wherein the pharmaceutical composition is used to treat one or more malignancies selected from the group consisting of leukemia and lymphoma, prostate cancer, breast cancer and colon cancer, and to treat one or more immune disorders.

19. An injectable composition prepared by the method of claim 1, comprising:

a specified amount of polymannan extract dissolved in deionized water; and

one or more pharmaceutically acceptable preservatives.

20. The composition of claim 19, wherein the injectable composition is sterile.

21. The composition of claim 19, wherein the one or more pharmaceutically acceptable preservatives are selected from the group consisting of parabens, benzoic acid and salts thereof, mercurials, quaternary ammonium salts, benzyl alcohol and other related alcohols, and phenols.

22. The composition of claim 19, wherein the preservative is benzyl alcohol.

23. The composition of claim 19, wherein the polymannan extract comprises aloe polysaccharides, wherein the aloe polysaccharides comprise one or more short-chain, medium-chain, long-chain, very-long-chain polysaccharides, or any combination thereof.

24. The composition of claim 23, wherein the aloe polysaccharides further comprise monosaccharides selected from the group consisting of glucose, mannose, arabinose, and galactose.

25. The composition of claim 23, wherein the aloe polysaccharides have a molecular weight of 11,500 daltons to 10,000,000 daltons.

26. The composition of claim 19, wherein the polymannan extract has at least 25% aloe polysaccharides.

27. The composition of claim 19, wherein the polymannan extract has 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 30%, 95% aloe polysaccharides.

28. The composition of claim 19, wherein the polymannan extract comprises at least 14% aloe polysaccharides having a molecular weight of 66,000 daltons.

29. The composition of claim 19, wherein the polymannan extract comprises at least 9% aloe polysaccharides having a molecular weight of 480,000 daltons.

30. The composition of claim 19, wherein the polymannan extract comprises at least 3.5% aloe polysaccharides having a molecular weight of 1,000,000 daltons.

31. The composition of claim 19, wherein the polymannan extract comprises at least 2.4% aloe polysaccharides having a molecular weight of 2,000,000 daltons.

32. The composition of claim 19, wherein said composition is used to treat one or more cancers selected from the group consisting of leukemia and lymphoma, prostate cancer, breast cancer, and colon cancer.

33. The composition of claim 19, wherein the injectable composition is used for immunomodulation, immunostimulation, or for the treatment of individuals with a compromised immune system or an immune disease.