Compositions and methods of aloe polysaccharides

Abstract

The present invention describes a method for preparing a polymannan extract from freeze-dried aloe powder. The polymannan extract of the present invention is further used to formulate a sterile injectable formulation for the treatment of one or more cancers, leukemias and lymphomas, prostate cancer, breast cancer, and colon cancer, immune diseases, particularly immune related neoplasms, acquired immune deficiency syndrome, and hepatitis C.

Claims

1. A method for the treatment of one or more cancers, wherein the one or more cancers are selected from the group consisting of leukemias and lymphomas, prostate cancer, breast cancer, and colon cancer, comprising the steps of: identifying an individual in need of treatment against the one or more cancers; and injecting a sterile injectable polymannan extract formulation from Aloe vera two to three times in a week in a dosage sufficient to treat the one or more cancers, wherein the sterile injectable polymannan extract formulation has a molecular weight of 66,000 to 2,000,000 Daltons, and comprises an amount of the polymannan extract formulation is sufficient to treat the leukemias and lymphomas, prostate cancer, breast cancer, and colon cancer specified quantity of very fine polymannan extract dissolved in deionized water; and one or more pharmaceutical preservatives.

2. The method of claim 1, wherein the polymannan extract further comprises aloe polysaccharides that have a molecular weight of 10,000,000 Daltons.

3. The method of claim 1, wherein the polymannan extract has at least 25% of aloe polysaccharides.

4. The method of claim 1, wherein the polymannan extract causes a 75-80% increase in one or more natural killer (NK) cells.

5. A method of immunomodulation or immunostimulation in an individual comprising the steps of: identifying the individual in need of immunomodulation or immunostimulation; administering intravenously a specified dosage of a sterile injectable polymannan extract formulation, wherein the sterile injectable polymannan extract formulation comprises a specified quantity of very fine polymannan extract dissolved in deionized water; and one or more pharmaceutical preservatives, wherein the dosage of the sterile injectable polymannan extract formulation is dependent on a weight, an age, an ethnicity, and a gender of the individual.

6. The method of claim 5, wherein the polymannan extract comprises aloe polysaccharides, wherein the aloe polysaccharides comprise one or more small chain, medium chain, large chain, very-large chain polysaccharides, or any combinations thereof.

7. The method of claim 5, wherein the polymannan extract causes a 75-80% increase in one or more natural killer (NK) cells.

8. The method of claim 5, wherein the polymannan extract comprises aloe polysaccharides, wherein the aloe polysaccharides comprise one or more small chain, medium chain, large chain, very-large chain polysaccharides, or any combinations thereof.

9. The method of claim 5, wherein the aloe polysaccharides have a molecular weight ranging from 480,000 Daltons to 2,000,000 Daltons.

10. The method of claim 5, wherein the polymannan extract has at least 25% of aloe polysaccharides.

11. The method of claim 5, wherein the individual has one or more cancers, wherein the one or more cancers are selected from the group consisting of leukemias and lymphomas, prostate cancer, breast cancer, and colon cancer.

12. The method of claim 5, wherein the individual has one or more cancers, wherein the one or more cancers are selected from the group consisting of leukemias and lymphomas, prostate cancer, breast cancer, and colon cancer, comprising the steps of: identifying an individual in need of treatment against the one or more cancers; and injecting a sterile injectable polymannan extract formulation two to three times in a week in a dosage sufficient to treat the one or more cancers, wherein the sterile injectable polymannan extract formulation comprises a specified quantity of very fine polymannan extract dissolved in deionized water; and one or more pharmaceutical preservatives.

13. The method of claim 5, wherein the polymannan extract has at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% aloe polysaccharides.

14. The method of claim 5, wherein the polymannan extract comprises at least 14% of aloe polysaccharides having a molecular weight of at least one of 66,000 Daltons; 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; or at least 2.4% of aloe polysaccharides having a molecular weight of 2,000,000 Daltons.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

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

(2) FIG. 1 shows a size-exclusion chromatogram of an aloe polysaccharide showing the retention times of the different glucose and mannose sub-units;

(3) FIG. 2 shows the size-exclusion chromatogram of an aloe polysaccharide showing the peaks corresponding to the different glucose and mannose sub-units;

(4) FIG. 3 is the proton-nuclear magnetic resonance profile of the polymannan extract of the present invention;

(5) FIG. 4A is a HPLC chromatogram showing the amounts of polysaccharide in each polysaccharide molecular group in a methanol precipitated aloe polysaccharide concentrate;

(6) FIG. 4B is the proton-nuclear magnetic resonance profile of a methanol precipitated aloe polysaccharide concentrate;

(7) FIG. 5A is a HPLC chromatogram showing the amounts of polysaccharide in each polysaccharide molecular group in a ethanol precipitated aloe polysaccharide concentrate;

(8) FIG. 5B is the proton-nuclear magnetic resonance profile of an ethanol precipitated aloe polysaccharide concentrate;

(9) FIG. 6A is a HPLC chromatogram showing the amounts of polysaccharide in each polysaccharide molecular group in an isopropyl alcohol precipitated aloe polysaccharide concentrate;

(10) FIG. 6B is the proton-nuclear magnetic resonance profile of an isopropyl alcohol precipitated aloe polysaccharide concentrate;

(11) FIG. 7A is a HPLC chromatogram showing the amounts of polysaccharide in each polysaccharide molecular group in a propanol precipitated aloe polysaccharide concentrate; and

(12) FIG. 7B is the proton-nuclear magnetic resonance profile of a propanol precipitated aloe polysaccharide concentrate.

DETAILED DESCRIPTION OF THE INVENTION

(13) While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that 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.

(14) To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as a, an and the are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.

(15) The present invention describes a process for preparing a polymannan extract and the use of the said extract in the form of an injection as an immune stimulatory compound. Immune stimulation is assessed using macrophages/monocytes of human origin and the cell type is assessed for the secretion of tumor necrosis factor alpha (TNF).

(16) Aloe polysaccharides are generally considered to be those molecules composed predominantly of glucose and mannose simple sugars having chain lengths of 10,000 Daltons to those with molecular weights of 10,000,000 Daltons. The higher the mannose content and longer the chain length the greater is the immunomodulatory activity expressed by the polysaccharides. The different long, unbranched chains comprising these aloe polysaccharides are listed in Table 1.

(17) TABLE-US-00001 TABLE 1 Aloe polysaccharide compositions and pharmacological actions. # of Sugar Residues Mol. Wt (Daltons) Pharmacological Actions Small Chain 70-650 11,500 > 100,000 Diabetes Polysaccharides Tyrosinase inhibition (skin lightening) Anti-inflammatory activity (Cox-2 inhibition) Medium Chain 1500 250,000 Anti-oxidant Polysaccharides Protects heart, lungs (emphysema), and nervous system (Parkinsonism) Large Chain 4000-5000 650,000 Antibacterial Polysaccharides Induction of healing Very Large 8000-9000 2,000,000 Immunomodulatory Polysaccharides 10,000,000 activity Stimulation of - lymphocytes with the elaboration of antibodies Increasing level of natural killer cells Release of large quantities of TNF to cause angiogenesis in wounds and promotion of healing

(18) Aloe polysaccharides with molecular weights of 100,000 Daltons or more are listed in Table 2.

(19) TABLE-US-00002 TABLE 2 Compositions and molecular weights of aloe polysaccharides having Mol. Wts of 100,000 or greater. Mol. Wt (Daltons) Major Components Aloeride 2,000,000-10,000,000 Arabinose, galactose, glucose, mannose Acemannan 900,000-1,500,000 Glucose, mannose Manapol 500,000-900,000 Glucose, mannose Aloemannan 100,000-500,000 Glucose, Mannose

(20) The precursor material for the polymannan extract is described by the inventors in a previous patent (U.S. Pat. No. 6,083,508Avalos and Danhof, 2000) titled Method of Processing Aloe Leaves. The certificate of analysis of the polymannan extract precursor material is presented in Table 3.

(21) FIG. 1 is a size-exclusion chromatogram of an aloe polysaccharide preparation showing retention times of various-sized glucose and mannose subunits. FIG. 2 is a size-exclusion chromatogram of an aloe polysaccharide preparation identifying molecular weights ranging from 100 to 10,000,000 Daltons. FIG. 3 is the proton-nuclear magnetic resonance profile of the polymannan extract of the present invention. FIG. 3 shows: (i) the absence of the standard preservativessodium benzoate and potassium sorbate, (ii) the presence of smaller monohexoses, (iii) the peaks of isocitric acid indicating a whole leaf methodology was employed in processing the raw aloe material, (iv) the presence of malic acid peaksa primary marker for Aloe vera, (v) the presence of the aloeride/acemannan peak in the polysaccharide portion of the profile confirming the presence of the large polysaccharide species, and (vi) acetyl groups are present confirming the presence of the partially acetylated polysaccharide glucomannans.

(22) Size exclusion chromatography (SEC) of an aloe preparation prior to polymannan extraction:

(23) Equipment: HPLC system is a Hitachi L-7100 pump and 7250 autosampler paired to a Waters 410 differential refractometer. The SEC is a Tosoh Biosep G6000 PWXL TSK Gel 30 cm7.8 mm operated in a column heater at 70 C. Molecular weight standards are from Sigma2,000,000 Daltons, 1,000,000 Daltons, 480,000 Daltons, 66,000 Daltons, and 180 Daltons (glucose). The mobile phase is de-ionized water with flow rate of 0.70 mL/min. The injection volume is 10 uL. The SEC method is described by Pugh et al. (2001).

(24) TABLE-US-00003 TABLE 3 Certificate of analysis of Polymannan Extract Precursor Material. Parameter Constituent Range Determined Value Assessment Powder: Color: White to light tan Offwhite Complies Characteristics: Finely granular Finely granular Complies Rheology: Free-flowing Free-flowing Complies Taste: Salty Salty Complies Iodine test: Negative Negative Complies Moisture: 2.2-7.0% 3.70% Complies Solubility: Complete Complete Complies Minerals: Ca.sup.++ >25 mg/gram 56.3 mg/gram Complies Mg.sup.++ >10 mg/gram 14.3 mg/gram Complies Organic Acids: Citric: Present Present Complies Isocitric: Present Present Complies Lactic: <25 mg/gram 12 ppm Complies Malic: >250 mg/gram 410 mg/gram Complies Anthraquinones; Aloin A: <0.05 ppm 0.02 ppm Complies Aloin B: <0.03 ppm 0.01 ppm Complies Aloe-emodin: <0.02 ppm 0.005 ppm Complies Emodin: <0.01 ppm 0.001 ppm Complies Brix Value: <1.0 Negative Complies

(25) TABLE-US-00004 TABLE 4 SEC results for aloe preparation prior to polymannan extraction. Aloe #09116 Refraction Refractive Molecular Time Index Area Area % Area Size 0:30 0.0000 0.0000 1:00 0.0000 0.0000 1:30 0.0000 0.0000 2:00 0.0000 0.0000 2:30 0.0000 0.0000 3:00 0.0000 0.0000 3:30 0.0000 0.0000 4:00 0.0000 0.0000 4:30 0.0000 0.0000 5:00 0.0000 0.0000 5:30 0.0000 0.0000 6:00 0.0000 0.0000 6:30 0.0000 0.0000 7:00 0.0000 0.0000 7:30 0.0000 0.0000 8:00 0.0000 0.0000 8:30 0.0000 0.0000 9:00 0.0000 0.0000 9:30 0.0010 0.0014 10:00 0.0020 0.0046 10:30 0.0015 0.0058 0.012 2.44% 2 10.sup.6 11:00 0.0010 0.0042 11:30 0.0015 0.0039 12:00 0.0015 0.0049 12:30 0.0015 0.0049 0.018 3.69% 1 10.sup.6 13:00 0.0015 0.0049 13:30 0.0020 0.0056 14:00 0.0020 0.0065 14:30 0.0025 0.0072 15:00 0.0030 0.0088 15:30 0.0040 0.0111 0.044 9.09% 4.80 10.sup.5 16:00 0.0050 0.0144 16:30 0.0055 0.0169 17:00 0.0060 0.0186 17:30 0.0070 0.0209 0.071 14.59% 6.6 10.sup.4 18:00 0.0080 0.0241 18:30 0.0090 0.0274 19:00 0.0110 0.0320 19:30 0.0130 0.0385 20:00 0.0155 0.0457 20:30 0.0170 0.0524 21:00 0.0240 0.0649 21:30 0.0005 0.0457 22:00 0.0004 0.0015 22:30 0.0005 0.0014 23:00 0.0004 0.0015 23:30 0.0003 0.0012 24:00 0.0000 0.0000 0.340 70.20% 0.0000 0.4849 100.00%

(26) Aloe precipitant evaluation study: 25 ml samples of COATS concentrated aloe were pipetted into 200 ml beakers, and 125 ml of various polysaccharide precipitant liquids were added to the beaker and thoroughly stirred. The precipitated polysaccharides were collected by filtration through tared dehydrated filter papers which, following filtration, were placed in the drying oven overnight. The next morning the dry weight of the precipitated polysaccharide was determined. The inventors studied four alcoholic precipitants including, methanol, ethanol, isopropyl alcohol, and propanol. The powders were passed through a HPLC procedure which determined the various quantities of all of the molecular species which was recorded with determination of the amounts of polysaccharides in each of the polysaccharide molecular groups, including greater than 2,000,000 Daltons, greater than 1,000,000 Daltons, greater than 480,000 Daltons, greater than 66,000 Daltons, and the residual fraction containing the very small molecular species, e.g. glucose having a M. W. of 180. (HPLC data shown in Tables 6-9). The HPLC chromatograms corresponding to the four precipitants methanol, ethanol, isopropyl alcohol, and propanol is shown in FIGS. 4A, 5A, 6A, and 7A, respectively. Proton Nuclear Magnetic Resonance Profiles of the precipitates were also obtained and are showing in FIGS. 4B, 5B, 6B, and 7B. The data collected is shown in Table 5.

(27) TABLE-US-00005 TABLE 5 Data from the aloe precipitant evaluation study. M.W. M.W. M.W. M.W. M.W. Precipitant 2.0 10.sup.6 1.0 10.sup.6 4.8 10.sup.5 6.6 10.sup.4 Residual METHANOL 6.36% 3.89% 68.56% 6.92% 14.27% ETHANOL 2.52% 3.21% 70.70% 6.94% 16.57% ISOPROPYL 1.32% 3.43% 73.36% 5.91% 15.99% ALCOHOL PROPANOL 2.74% 2.55% 63.85% 6.92% 23.94%

(28) TABLE-US-00006 TABLE 6 HPLC data showing the amounts of polysaccharide in each polysaccharide molecular group in an ethanol precipitated aloe polysaccharide concentrate. Aloe #09423-A Retention Refractive Molecular Time Index Area Area % Area Size 0:30 0.0000 0.0000 1:00 0.0000 0.0000 1:30 0.0000 0.0000 2:00 0.0000 0.0000 2:30 0.0000 0.0000 3:00 0.0000 0.0000 3:30 0.0010 0.0014 4:00 0.0010 0.0033 4:30 0.0010 0.0033 5:00 0.0005 0.0026 5:30 0.0005 0.0016 6:00 0.0005 0.0016 6:30 0.0080 0.0119 7:00 0.0030 0.0191 0.045 6.36% 2 10.sup.6 7:30 0.0020 0.0084 8:00 0.0030 0.0079 8:30 0.0040 0.0111 0.027 3.89% 1 10.sup.6 9:00 0.0060 0.0158 9:30 0.0070 0.0209 10:00 0.0090 0.0255 10:30 0.0130 0.0348 11:00 0.0180 0.0491 11:30 0.0220 0.0640 12:00 0.0250 0.0756 12:30 0.0230 0.0785 13:00 0.0170 0.0665 13:30 0.0040 0.0374 14:00 0.0050 0.0144 0.482 68.56% 4.80 10.sup.5 14:30 0.0050 0.0163 15:00 0.0050 0.0163 15:30 0.0050 0.0163 0.049 6.93% 6.6 10.sup.4 16:00 0.0060 0.0176 16:30 0.0070 0.0209 17:00 0.0050 0.0200 17:30 0.0040 0.0149 18:00 0.0030 0.0116 18:30 0.0020 0.0084 19:00 0.0010 0.0051 19:30 0.0000 0.0019 20:00 0.0000 0.0000 20:30 0.0000 0.0000 21:00 0.0000 0.0000 21:30 0.0000 0.0000 22:00 0.0000 0.0000 22:30 0.0000 0.0000 23:00 0.0000 0.0000 23:30 0.0000 0.0000 24:00 0.0000 0.0000 0.100 14.27% 0.0000 0.7036 100.00%

(29) TABLE-US-00007 TABLE 7 HPLC data showing the amounts of polysaccharide in each polysaccharide molecular group in an ethanol precipitated aloe polysaccharide concentrate. Aloe #09423-B Retention Refractive Molecular Time Index Area Area % Area Size 0:30 0.0000 0.0000 1:00 0.0000 0.0000 1:30 0.0000 0.0000 2:00 0.0000 0.0000 2:30 0.0000 0.0000 3:00 0.0000 0.0000 3:30 0.0000 0.0000 4:00 0.0005 0.0007 4:30 0.0010 0.0023 5:00 0.0005 0.0026 5:30 0.0005 0.0016 6:00 0.0010 0.0023 6:30 0.0010 0.0033 7:00 0.0015 0.0039 0.017 2.52% 2 10.sup.6 7:30 0.0020 0.0056 8:00 0.0020 0.0065 8:30 0.0040 0.0093 0.021 3.21% 1 10.sup.6 9:00 0.0050 0.0144 9:30 0.0070 0.0190 10:00 0.0100 0.0269 10:30 0.0130 0.0366 11:00 0.0160 0.0464 11:30 0.0200 0.0575 12:00 0.0240 0.0705 12:30 0.0210 0.0739 13:00 0.0185 0.0648 13:30 0.0050 0.0416 14:00 0.0060 0.0176 0.469 70.76% 4.80 10.sup.5 14:30 0.0050 0.0181 15:00 0.0040 0.0149 15:30 0.0040 0.0130 0.046 6.94% 6.6 10.sup.4 16:00 0.0050 0.0144 16:30 0.0075 0.0197 17:00 0.0080 0.0251 17:30 0.0035 0.0198 18:00 0.0025 0.0100 18:30 0.0050 0.0116 19:00 0.0000 0.0094 19:30 0.0000 0.0000 20:00 0.0000 0.0000 20:30 0.0000 0.0000 21:00 0.0000 0.0000 21:30 0.0000 0.0000 22:00 0.0000 0.0000 22:30 0.0000 0.0000 23:00 0.0000 0.0000 23:30 0.0000 0.0000 24:00 0.0000 0.0000 0.110 16.57% 0.0000 0.6630 100.00%

(30) TABLE-US-00008 TABLE 8 HPLC data showing the amounts of polysaccharide in each polysaccharide molecular group in an isopropyl alcohol precipitated aloe polysaccharide concentrate. Aloe #09423-C Retention Refractive Molecular Time Index Area Area % Area Size 0:30 0.0000 0.0000 1:00 0.0000 0.0000 1:30 0.0000 0.0000 2:00 0.0000 0.0000 2:30 0.0001 0.0001 3:00 0.0000 0.0002 3:30 0.0000 0.0000 4:00 0.0000 0.0000 4:30 0.0001 0.0001 5:00 0.0001 0.0003 5:30 0.0001 0.0003 6:00 0.0001 0.0003 6:30 0.0015 0.0023 7:00 0.0015 0.0049 0.009 1.32% 2 10.sup.6 7:30 0.0020 0.0056 8:00 0.0025 0.0072 8:30 0.0035 0.0095 0.022 3.43% 1 10.sup.6 9:00 0.0060 0.0148 9:30 0.0080 0.0223 10:00 0.0095 0.0281 10:30 0.0120 0.0343 11:00 0.0165 0.0452 11:30 0.0190 0.0571 12:00 0.0200 0.0631 12:30 0.0215 0.0671 13:00 0.0200 0.0678 13:30 0.0100 0.0513 14:00 0.0045 0.0249 0.476 73.36% 4.80 10.sup.5 14:30 0.0040 0.0139 15:00 0.0035 0.0123 15:30 0.0040 0.0121 0.038 5.91% 6.6 10.sup.4 16:00 0.0065 0.0164 16:30 0.0096 0.0254 17:00 0.0075 0.0283 17:30 0.0030 0.0182 18:00 0.0020 0.0084 18:30 0.0010 0.0051 19:00 0.0000 0.0019 19:30 0.0000 0.0000 20:00 0.0000 0.0000 20:30 0.0000 0.0000 21:00 0.0000 0.0000 21:30 0.0000 0.0000 22:00 0.0000 0.0000 22:30 0.0000 0.0000 23:00 0.0000 0.0000 23:30 0.0000 0.0000 24:00 0.0000 0.0000 0.104 15.99% 0.0000 0.6487 100.00%

(31) TABLE-US-00009 TABLE 9 HPLC data showing the amounts of polysaccharide in each polysaccharide molecular group in a propanol precipitated aloe polysaccharide concentrate. Aloe #09423-C Retention Refractive Molecular Time Index Area Area % Area Size 0:30 0,0000 0.0000 1:00 0.0000 0.0000 1:30 0.0000 0.0000 2:00 0.0000 0.0000 2:30 0.0001 0.0001 3:00 0.0000 0.0002 3:30 0.0000 0.0000 4:00 0.0000 0.0000 4:30 0.0001 0.0001 5:00 0.0001 0.0003 5:30 0.0001 0.0003 6:00 0.0001 0.0003 6:30 0.0015 0.0023 7:00 0.0015 0.0049 0.009 1.32% 2 10.sup.6 7:30 0.0020 0.0056 8:00 0.0025 0.0072 8:30 0.0035 0.0095 0.022 3.43% 1 10.sup.6 9:00 0.0060 0.0148 9:30 0.0080 0.0223 10:00 0.0095 0.0281 10:30 0.0120 0.0343 11:00 0.0165 0.0452 11:30 0.0190 0.0571 12:00 0.0200 0.0631 12:30 0.0215 0.0671 13:00 0.0200 0.0678 13:30 0.0100 0.0513 14:00 0.0045 0.0249 0.476 73.36% 4.80 10.sup.5 14:30 0.0040 0.0139 15:00 0.0035 0.0123 15:30 0.0040 0.0121 0.038 5.91% 6.6 10.sup.4 16:00 0.0065 0.0164 16:30 0.0096 0.0254 17:00 0.0075 0.0283 17:30 0.0030 0.0182 18:00 0.0020 0.0084 18:30 0.0010 0.0051 19:00 0.0000 0.0019 19:30 0.0000 0.0000 20:00 0.0000 0.0000 20:30 0.0000 0.0000 21:00 0.0000 0.0000 21:30 0.0000 0.0000 22:00 0.0000 0.0000 22:30 0.0000 0.0000 23:00 0.0000 0.0000 23:30 0.0000 0.0000 24:00 0.0000 0.0000 0.104 15.99% 0.0000 0.6487 100.00%

(32) The polymannan extract is prepared by precipitation. The freeze dried aloe powder is described above was weighed after correcting appropriately for the moisture content. For example, if the moisture content is 3.7% and we need 80 gms, the inventors weighed out 82.96 gms (80 gms+(3.7%80) gms). The weighed aloe powder was dissolved completely in one gallon of deionized water (DI) 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 settle overnight.

(33) The following day a 2 ml of clear supernatant was taken and 5 mL of 95% ethanol was added and the sample was centrifuged at 3000 rpm for 20 minutes. The sample was examined for precipitation, if no precipitate was observed then the precipitation was considered complete. If any significant degree of precipitation was observed then additional 95% ethanol was added to the precipitation vessel before proceeding. The clear supernatant fluid in the precipitation vessel was decanted by siphoning without disturbing the precipitate at the bottom of the vessel. The white precipitate at the bottom was separated by using a suction funnel (Whatman No. 42 quantitative ashless filter paper. The precipitated material was removed by scraping it into a 600 ml Virtis lyophilization flask, and by distributing the material over one side of the flask to form a thin layer with a large exposed surface area. The lyophilization flask was placed in a shell-freezer overnight. The next day the chilled lyophilization flask with its frozen contents, was placed on a lyophilizer operating at 90 C. and at atmosphere for 24 hours. The lyophizer was turned off and the lyophilized powder was placed into a small powder grinder until it reduced to an evenly ground fine powder. The ground powder was weighed and placed in plastic small containers and the containers were stored in a freezer.

(34) Preparation of an injectable solution of Polymannan extract: The polymannan extract powder (PME) prepared as described above was weighed (1.5 gms) after correcting for moisture content and having an aloeride content of at least 2% as determined by size exclusion chromatography. To 125 ml of warm D.I water 1 ml of concentrated HCl was added and stirred followed by the slow addition of the PME powder with constant stirring. The stirring was continued till all the PME powder dissolved and the solution was clear and colorless an additional amount of concentrated HCl was added to obtain a pH of 1.6 to 1.7 (measured continuously using a pH meter). Additional D.I water was added to adjust the volume to 150 ml followed by a pH monitoring to ensure a pH of 1.6-1.7. The PME solution was then poured into a Corning 150 ml filter system flask with a pore size of 0.45 m. The flask system was placed in a refrigerator and the filtrate was transferred to a Corning 150 ml filter system flask with a pore size of 0.22 m and placed in a refrigerator overnight. Under sterile conditions the filter top of the filter system was removed and the bottle was sealed with a sterile cap. The bottles were them transferred to a compounding lab, and under a sterile hood 0.9% benzyl alcohol was added as a preservative (because the final product is for multi-dosage use), and the solution was placed in sterile 10 mL glass vials and sealed with a multidosage closure. The vials are labeled with a batch number, control number, manufacturing date, expiration date of 6 months along with the names of the physician and patient.

(35) PME immunomodulatory activity assessment: The immune stimulatory activity is assessed using macrophages/monocytes of human origin obtained from the American Type Culture Collection (ATCC) in Maryland. The cell type was assessed for the secretion of TNF. Under standard cell conditions a small amount of the final PME product was introduced in the culture. Samples were drawn at 6, 12, and 24 hours and assessed for TNF levels. A specific quantity of TNF was not used because of the variability in the different cell batches. In a clinical setting the immunomodulatory response is expected to vary due to changing hemotological factors like the total leukocyte count, differential macrophage/monocyte count, number of surface mannose receptors on the white cells, amount of mannose-binding carrier protein, etc.

(36) The white blood cell profile varies with cells constantly entering and leaving the blood stream. The affinity of the cellular mannose receptors for the PME far exceeds that of the mannose binding protein. As new macrophages/monocytes enter the blood stream, the PME is transferred to the new cells from the circulating mannose-binding protein. PME binding to the macrophage/monocyte mannose-binding protein results in the release of an array of cytocommunicators. The cytocommunicators, including TNF-, IL-1, INF-, IL-2, and IL-6, restore to normal the impaired surveillance function of the immune system which had failed in its neoplasm detection function in the cancer patient permitting the patient's immune system of identifying and removing the malignant cells.

(37) Aloe polysaccharides in the polymannan extract having molecular weights of 1,000,000, 300,000, 100,000, 50,000 and 25,000 all showed caspase activity. This caspase 3, caspase 9, and cytochrome-C activity is key in the treatment of malignancies by the composition of the present invention, as caspase 3 is a mediator of tumor cell apoptosis. The immune modulatory activity of initiator (apical) caspase 3 and effector (executioner) caspase 3 as well as cytochrome-C have been demonstrated as being extant and are considered to be the mediator system of tumor cell apoptosis.

(38) The inventors tested the composition described herein on 104 patients with different types of cancers. Leukemia and lymphomas were most responsive to the polymannan extract of the present invention (>98%). Prostate, breast, and colon cancers were also responsive to the polymannan extract of the present invention. For the testing the polymannan extract was administered as an injection. 10 mg of the polymannan extract was reconstituted in sterile water for injection to give a final concentration of 10 mg/mL. This was injected 2 to 3 times a week. The serum samples from the patients were then taken at regular intervals and monitored for caspase 3 activity.

(39) It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

(40) It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.

(41) 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.

(42) The use of the word a or an when used in conjunction with the term comprising in the claims and/or the specification may mean one, but it is also consistent with the meaning of one or more, at least one, and one or more than one. The use of the term or in the claims is used to mean and/or unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and and/or. Throughout this application, the term about is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

(43) As used in this specification and claim(s), the words comprising (and any form of comprising, such as comprise and comprises), having (and any form of having, such as have and has), including (and any form of including, such as includes and include) or containing (and any form of containing, such as contains and contain) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

(44) The term or combinations thereof as used herein refers to all permutations and combinations of the listed items preceding the term. For example, A, B, C, or combinations thereof is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

(45) All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. 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. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

REFERENCES

(46) U.S. Pat. No. 7,196,072: High Molecular Weight Polysaccharide Fraction From Aloe Vera with Immunostimulatory Activity. U.S. Pat. No. 6,083,508: Method of Processing Aloe Leaves. U.S. Patent Application No. 2006/0084629: Immune System Activating Formula Composed of Selected Long Chain Polysaccharides From Natural Sources. .sup.1 Pugh N., Ross S. A., ElSohly M. A., and Pasco, D. S. (2001). Characterization of Aloeride, a new high-molecular weight polysaccharide from Aloe vera with potent immunomodulatory activity. J Agr. Food Chem., 49, 1030-1034.