阅读说明：本技术 多糖的乙酰化 (Acetylation of polysaccharides ) 是由 朴炷祥 鲍志超 郑群怡 何侃 谢朝阳 特洛伊·斯迈利 于 2019-03-26 设计创作，主要内容包括：提供了乙酰化的多糖及其制备方法和使用方法。制备乙酰化的多糖的一种方法包括提供多糖,将多糖纯化至1-90重量％的纯度,提供乙酰化试剂,提供催化剂,将乙酰化试剂和催化剂与多糖混合,从而制备乙酰化的多糖,并纯化乙酰化的多糖。(Acetylated polysaccharides and methods of making and using the same are provided. One method of preparing acetylated polysaccharides includes providing a polysaccharide, purifying the polysaccharide to a purity of 1-90% by weight, providing an acetylating agent, providing a catalyst, mixing the acetylating agent and the catalyst with the polysaccharide to thereby prepare an acetylated polysaccharide, and purifying the acetylated polysaccharide.)

1. A method of preparing an acetylated polysaccharide, the method comprising:

a) providing a polysaccharide;

b) purifying the polysaccharide to a purity of 1-90 wt.%;

c) providing an acetylation agent;

d) providing a catalyst;

e) mixing the acetylating agent and catalyst with the polysaccharide to thereby produce an acetylated polysaccharide, wherein the acetylation exceeds the acetylation of the polysaccharide in step a); and

f) purifying the acetylated polysaccharide.

2. The method of claim 1, wherein the polysaccharide is a powder formulation.

3. The method of claim 1, wherein the polysaccharide comprises glucomannan, galactomannan, mannan, and acetylated forms thereof.

4. The method of claim 1, wherein the polysaccharide comprises hydroxyl groups, and wherein the hydroxyl groups can be substituted with acetyl groups.

5. The method of claim 1, wherein the polysaccharide comprises a mannose moiety.

6. The method of claim 5, wherein the mannose moiety comprises a hydroxyl group, wherein the hydroxyl group is capable of being acetylated.

7. The method of claim 6, wherein the acetylated polysaccharide comprises acetyl groups on the mannose moiety at mannose sites 2,3 and/or 6.

8. The process of claim 1, wherein the acetylating reagent is acetic anhydride (CH)₃CO)₂O, acetyl chloride and acetic acid.

9. The process of claim 1, wherein the catalyst is pyridine, sodium hydroxide, potassium hydroxide, sodium carbonate, or potassium carbonate and a solvent.

10. The method of claim 1, wherein the purification step of step b) is performed by ethanol precipitation.

11. The method of claim 1, wherein the purification step of step f) is performed by dialysis.

12. An acetylated polysaccharide prepared by the method of any one of claims 1-11.

13. The acetylated polysaccharide of claim 12, wherein the acetylated polysaccharide comprises at least one, at least two, or at least three acetyl groups per mannosyl group.

14. The acetylated polysaccharide of claim 12 wherein the acetylated polysaccharide comprises mannose groups.

15. The acetylated polysaccharide of claim 12, wherein the polysaccharide comprises glucomannan, galactomannan, mannan, and/or acetylated forms thereof.

16. The acetylated polysaccharide of claim 12 having a molecular weight of about 1 kilodaltons to about 70 kilodaltons.

17. The acetylated polysaccharide of claim 12 having a molecular weight of about 18 kilodaltons or about 29 kilodaltons.

18. A pharmaceutical formulation comprising:

an acetylated polysaccharide prepared by the method of any one of claims 1 to 12; and

a pharmaceutically acceptable excipient.

19. The pharmaceutical formulation of claim 18, further comprising non-toxic auxiliary substances such as wetting agents, pH buffering agents and absorption enhancing agents.

20. The pharmaceutical formulation of claim 19, wherein the formulation is a pill, tablet, gum, capsule, lozenge or liquid.

21. A method of treating, ameliorating, or preventing an inflammatory response in a subject, the method comprising administering to the subject the pharmaceutical formulation of claim 18.

22. The method of claim 20 wherein the acetylated polysaccharide is administered at least three times daily, twice daily, once weekly, or once monthly.

23. The method of claim 21, wherein the individual has an immune disorder.

24. The method of claim 23, wherein the immune disorder is systemic lupus, scleroderma, hemolytic anemia, vasculitis, type I diabetes, graves 'disease, rheumatoid arthritis, multiple sclerosis, Goodpasture's syndrome, myopathy, severe combined immunodeficiency, DiGeorge syndrome, hyperimmunoglobulin E syndrome, common variant immunodeficiency, chronic granulomatous disease, Wiskott-Aldrich syndrome, autoimmune lymphoproliferative syndrome, hyper IgM syndrome, leukocyte adhesion deficiency, NF- κ B essential regulatory protein (NEMO) mutations, selective immunoglobulin a deficiency, X-linked agammaglobulinemia, X-linked lymphoproliferative disorder, or ataxia-telangiectasia.

25. The method of claim 21, further comprising providing additional treatment to the individual before, during, or after administration of the acetylated polysaccharide.

26. A method of treating an immune disorder in a subject, the method comprising administering to the subject the pharmaceutical formulation of claim 18.

27. The method of claim 26 wherein the acetylated polysaccharide is administered at least three times daily, twice daily, once weekly, or once monthly.

28. The method of claim 26, wherein the immune disorder is systemic lupus, scleroderma, hemolytic anemia, vasculitis, type I diabetes, graves 'disease, rheumatoid arthritis, multiple sclerosis, Goodpasture's syndrome, myopathy, severe combined immunodeficiency, DiGeorge syndrome, hyperimmunoglobulin E syndrome, common variant immunodeficiency, chronic granulomatous disease, Wiskott-Aldrich syndrome, autoimmune lymphoproliferative syndrome, hyper IgM syndrome, leukocyte adhesion deficiency, NF- κ B essential regulatory protein (NEMO) mutations, selective immunoglobulin a deficiency, X-linked agammaglobulinemia, X-linked lymphoproliferative disorder, or ataxia-telangiectasia.

29. A method of increasing the expression of anti-inflammatory IL-10 (interleukin 10) in a subject, the method comprising administering the pharmaceutical formulation of claim 18 to the subject.

30. The method of claim 29 wherein the acetylated polysaccharide is administered at least three times daily, twice daily, once weekly, or once monthly.

31. The method of claim 29, further comprising monitoring the expression of anti-inflammatory IL-10 (interleukin 10).

32. A method of increasing the expression of an anti-inflammatory cytokine in a subject, the method comprising administering the pharmaceutical formulation of claim 18 to the subject.

33. The method of claim 32 wherein the acetylated polysaccharide is administered at least three times daily, twice daily, once weekly, or once monthly.

34. A method of treating a tumor in a subject, the method comprising administering to the subject the pharmaceutical formulation of claim 18.

35. The method of claim 34 wherein the acetylated polysaccharide is administered at least three times daily, twice daily, once weekly, or once monthly.

36. The method of claim 34, wherein the individual has cancer, and wherein the tumor is from lung cancer, skin cancer, liver cancer, brain cancer, kidney cancer, or uterine cancer.

37. The method of claim 34, further comprising providing additional treatment to the individual before, during, or after administration of the acetylated polysaccharide.

38. A method of treating a viral or bacterial infection in a subject, the method comprising administering to the subject the pharmaceutical formulation of claim 18.

39. The method of claim 38, further comprising monitoring the level of a cytokine released during viral or bacterial infection, wherein the cytokine is selected from the group consisting of IL-1 β, IL-6, IL-7, IL-8, IL-10, Tumor Necrosis Factor (TNF) - α, Interferon (IFN) - γ, and IFN- α/β.

Description of the Related Art

Polysaccharides are a natural resource for supplements and pharmaceuticals that have received increasing attention over the years. Natural polysaccharides have been shown to have fewer side effects, but their biological activity is difficult to compare with that of synthetic drugs due to their inherent physicochemical properties. Thus, researchers have altered the structure and properties of natural polysaccharides based on structure-activity relationships and have obtained better functionally improved polysaccharides. However, major modifications of polysaccharides may be necessary as they may affect their physicochemical properties and biological activity. Molecular modification methods mainly include chemical, physical and biological changes. Chemical modification is the most widely used method; by grafting to other groups, the water solubility and bioactivity of the polysaccharide can be obviously improved. Physical and biological modifications only alter the molecular weight of the polysaccharide, thereby altering its physicochemical properties and biological activity. Most molecular modifications result in increased antioxidant activity of polysaccharides, and among these modifications, sulfation and acetylation modifications are very common. Furthermore, modifications are the most common applications for increasing anti-inflammatory activity as discussed herein.

Background

The field relates to the processing of plants and plant parts for the development of acetylated sugars or carbohydrates for therapeutic applications. Also described herein are efficient methods of obtaining highly acetylated sugars or carbohydrates that have been extracted from plants and/or plant parts.

Aloe is a fleshy plant species of the genus aloe. It grows and cultivated wild in tropical climates around the world for agricultural and medical use. Aloe is widely recognized in the cosmetic and alternative pharmaceutical industries for its moisturizing and healing properties.

Aloe has been used for decades to relieve pain caused by irritation such as dry skin or sunburn. Extraction and characterization of aloe vera gel has revealed that the gel contains many types of mucopolysaccharides.

In medical practice, polysaccharides are known as signaling molecules that may explain their therapeutic properties. Gel-forming complex carbohydrates are known to be useful as emollients or emollients. In some cases, several types have been recognized as anti-inflammatory and immunomodulatory agents.

The polysaccharide, acetylated mannan, is a polysaccharide consisting of a main backbone of β - (1 → 4) -linked D-mannose residues interspersed with a small number of glucose residues, acetylated at O-2, O-3, O-2/O-3 or O-6, containing side chains consisting of O-6-linked single α -D-galactose and α -L-arabinose residues. The aloe acetylated mannan may have immunostimulatory activity. Studies of structural details of polysaccharides have shown that acetylated mannans exhibit complex acetylation patterns. However, during extraction and processing of polysaccharides, the polysaccharides may be deacetylated. Acetylated polysaccharides have been attributed to promoting immunomodulatory activity. Therefore, a method of efficiently acetylating the hydroxyl groups of the polysaccharide at a high content is required to maintain its immunomodulatory activity.

SUMMARY

In a first aspect, a process for preparing acetylated polysaccharides is provided. The method comprises a) providing a polysaccharide; b) purifying the polysaccharide to a purity of 90 wt.%; c) providing an acetylation agent; d) providing a catalyst; e) mixing the acetylating agent and catalyst with the polysaccharide to produce an acetylated polysaccharide, wherein acetylation exceeds acetylation of the polysaccharide in step a); and f) purifying the acetylated polysaccharide. In some embodiments, the resulting polysaccharide is characterized by acetylation over acetylation of a naturally occurring polysaccharide. In some embodiments, the polysaccharide is from a plant or plant part thereof. In some embodiments, the polysaccharide is a powder formulation. In some embodiments, the polysaccharide is from an extraction process of a plant or plant part thereof. In some embodiments, the plant or portion thereof comprises a whole leaf powdered extract or a plant interior clear gel powdered extract. In some embodiments, the plant is a succulent plant of the genus aloe. In some embodiments, the plant part thereof comprises an outer green bark and/or a plant inner transparent gel. In some embodiments, the polysaccharide comprises glucomannan, galactomannan, mannan, and acetylated forms thereof. In some embodiments, the polysaccharide comprises hydroxyl groups, wherein the hydroxyl groups can be substituted with acetyl groups. In some embodiments, the polysaccharide comprises a mannose moiety. In some embodiments, the mannose moiety comprises a hydroxyl group, wherein the hydroxyl group is capable of being acetylated. In some embodiments, the acetylated polysaccharide comprises acetyl groups on the mannose moiety at mannose sites 2,3 and/or 6. In some embodiments, the polysaccharide is purified to 1-90% purity by weight. In some embodiments, the polysaccharide is purified to 90% purity by weight. In some embodiments, the acetylated polysaccharide is purifiedTo 90% purity by weight. In some embodiments, the method further comprises determining the amount of acetylated polysaccharide and the position of acetyl groups on the acetylated polysaccharide. In some embodiments, the determination is by infrared spectroscopy (IR) and/or Nuclear Magnetic Resonance (NMR) spectroscopy. In some embodiments, the acetylation reagent is acetic anhydride (CH)₃CO)₂O, acetyl chloride or acetic acid. In some embodiments, the catalyst is pyridine, sodium hydroxide, potassium hydroxide, sodium or potassium carbonate and/or a solvent. In some embodiments, the purification step of step b) is performed by ethanol precipitation. In some embodiments, the purification step of step d) is performed by dialysis.

In a second aspect, there is provided an acetylated polysaccharide made by the method of any embodiment herein. In some embodiments, the method comprises a) providing a polysaccharide; b) purifying the polysaccharide to 90 wt% purity; c) providing an acetylation agent; d) providing a catalyst; e) mixing an acetylation agent and a catalyst with the polysaccharide, thereby producing an acetylated polysaccharide, wherein acetylation exceeds acetylation of the polysaccharide in step a); and f) purifying the acetylated polysaccharide. In some embodiments, the resulting polysaccharide is characterized by acetylation over acetylation of a naturally occurring polysaccharide. In some embodiments, the polysaccharide is from a plant or plant part thereof. In some embodiments, the polysaccharide is a powder formulation. In some embodiments, the polysaccharide is from an extraction process of a plant or plant part thereof. In some embodiments, the plant or portion thereof comprises a whole leaf powdered extract or a plant interior clear gel powdered extract. In some embodiments, the plant is a succulent plant of the genus aloe. In some embodiments, the plant part thereof comprises an outer green bark and/or a plant inner transparent gel. In some embodiments, the polysaccharide comprises glucomannan, galactomannan, mannan, and/or acetylated forms thereof. In some embodiments, the polysaccharide comprises hydroxyl groups, wherein the hydroxyl groups can be substituted with acetyl groups. In some embodiments, the polysaccharide comprises a mannose moiety. In some embodiments, the mannose moiety comprises a hydroxyl group, wherein the hydroxyl group is capable of being acetylatedAnd (4) transforming. In some embodiments, the acetylated polysaccharide comprises acetyl groups on the mannose moiety at mannose sites 2,3 and/or 6. In some embodiments, the polysaccharide is purified to 1-90% purity by weight. In some embodiments, the polysaccharide is purified to 90% purity by weight. In some embodiments, the acetylated polysaccharide is purified to 90% purity by weight. In some embodiments, the method further comprises determining the amount of acetylated polysaccharide and the position of acetyl groups on the acetylated polysaccharide. In some embodiments, the determination is by infrared spectroscopy (IR) and/or Nuclear Magnetic Resonance (NMR) spectroscopy. In some embodiments, the acetylation reagent is acetic anhydride (CH)₃CO)₂O, acetyl chloride or acetic acid. In some embodiments, the catalyst is pyridine, sodium hydroxide, potassium hydroxide, sodium carbonate, or potassium carbonate and a solvent. In some embodiments, the purification step of step b) is performed by ethanol precipitation. In some embodiments, the purification step of step d) is performed by dialysis. In some embodiments, the acetylated polysaccharide comprises at least one, at least two, or at least three acetyl groups. In some embodiments, the acetylated polysaccharide comprises mannose groups. In some embodiments, the polysaccharide comprises glucomannan, galactomannan, mannan, and/or acetylated forms thereof.

In a third aspect, there is provided a pharmaceutical formulation comprising an acetylated polysaccharide of any of the embodiments provided herein or prepared by a method of any of the embodiments provided herein and a pharmaceutically acceptable excipient. In some embodiments, the formulation is a pill. In some embodiments, the formulation is a liquid. In some embodiments, the formulation is a tablet, gum (gummy), capsule, or lozenge. In some embodiments, the formulation further comprises non-toxic auxiliary substances, such as wetting agents, pH buffering agents, and absorption enhancing agents.

In a fourth aspect, a method of treating, ameliorating or preventing an inflammatory response in a subject in need thereof is provided. The method comprises administering to a patient in need thereof an acetylated polysaccharide of any embodiment provided herein or an acetylated polysaccharide prepared by a method of any embodiment provided herein or a pharmaceutical formulation of any embodiment provided herein. In some embodiments, the acetylated polysaccharide is administered at least three times daily, twice daily, once weekly, or once monthly. In some embodiments, the patient has an immune disorder. In some embodiments, the immune disorder is systemic lupus, scleroderma, hemolytic anemia, vasculitis, type I diabetes, graves 'disease, rheumatoid arthritis, multiple sclerosis, Goodpasture's syndrome, myopathy, severe combined immunodeficiency, DiGeorge syndrome, hyperimmunoglobulin E syndrome, common variant immunodeficiency, chronic granulomatosis, Wiskott-Aldrich syndrome, autoimmune lymphoproliferative syndrome, hyper IgM syndrome, leukocyte adhesion deficiency, NF- κ B essential regulatory protein (NEMO) mutation, selective immunoglobulin a deficiency, X-linked agammaglobulinemia, X-linked lymphoproliferative disorder, or ataxia-telangiectasia. In some embodiments, the patient is identified as receiving treatment for inflammation. In some embodiments, the patient is identified as receiving treatment for an immune disorder. In some embodiments, the method further comprises measuring or evaluating inhibition of the inflammatory response. In some embodiments, the method further comprises providing additional treatment to the individual before, during, or after administering the acetylated polysaccharide of any embodiment herein or prepared by the method of embodiments herein or the pharmaceutical formulation of embodiments herein. In some embodiments, the subject is a patient. In some embodiments, the patient is an individual. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human, horse, dog, cat.

In a fifth aspect, a method of treating an immune disorder in an individual in need thereof is provided. The method comprises administering to a patient in need thereof an acetylated polysaccharide of any embodiment provided herein or an acetylated polysaccharide prepared by a method of any embodiment provided herein or a pharmaceutical formulation of any embodiment provided herein. In some embodiments, the acetylated polysaccharide is administered at least three times daily, twice daily, once weekly, or once monthly. In some embodiments, the immune disorder is systemic lupus, scleroderma, hemolytic anemia, vasculitis, type I diabetes, graves 'disease, rheumatoid arthritis, multiple sclerosis, Goodpasture's syndrome, myopathy, severe combined immunodeficiency, DiGeorge syndrome, hyperimmunoglobulin E syndrome, common variant immunodeficiency, chronic granulomatosis, Wiskott-Aldrich syndrome, autoimmune lymphoproliferative syndrome, hyper IgM syndrome, leukocyte adhesion deficiency, NF- κ B essential regulatory protein (NEMO) mutation, selective immunoglobulin a deficiency, X-linked agammaglobulinemia, X-linked lymphoproliferative disorder, or ataxia-telangiectasia.

In a sixth aspect, a method of increasing the expression of anti-inflammatory IL-10 (Interleukin 10) in a subject in need thereof is provided. The method comprises administering to a patient in need thereof an acetylated polysaccharide of any embodiment provided herein or an acetylated polysaccharide prepared by a method of any embodiment provided herein or a pharmaceutical formulation of any embodiment provided herein. In some embodiments, the acetylated polysaccharide is administered at least three times daily, twice daily, once weekly, or once monthly. In some embodiments, the patient has an immune disorder. In some embodiments, the method further comprises monitoring the expression of anti-inflammatory IL-10 (interleukin 10). In some embodiments, the subject in need thereof is a mammal. In some embodiments, the subject is a human, horse, dog, cat.

In a seventh aspect, a method of increasing the expression of an anti-inflammatory cytokine in a subject in need thereof is provided. The method comprises administering to a patient in need thereof an acetylated polysaccharide of any embodiment provided herein or an acetylated polysaccharide prepared by a method of any embodiment provided herein or a pharmaceutical formulation of any embodiment provided herein. In some embodiments, the acetylated polysaccharide is administered at least three times daily, twice daily, once weekly, or once monthly. In some embodiments, the patient has an immune disorder. In some embodiments, the method further comprises monitoring anti-inflammatory expression. In some embodiments, the subject in need thereof is a mammal. In some embodiments, the subject is a human, horse, dog, cat. In some embodiments, the anti-inflammatory cytokine is selected from the group consisting of Interleukin (IL) -1 receptor antagonists, IL-4, IL-6, IL-10, IL-11, and IL-13.

In an eighth aspect, a method of treating a tumor in an individual in need thereof is provided. The method comprises administering to a patient in need thereof an acetylated polysaccharide of any embodiment provided herein or an acetylated polysaccharide prepared by a method of any embodiment provided herein or a pharmaceutical formulation of any embodiment provided herein. In some embodiments, the acetylated polysaccharide is administered at least three times daily, twice daily, once weekly, or once monthly. In some embodiments, the patient has cancer. In some embodiments, the tumor is selected from lung cancer, skin cancer, liver cancer, brain cancer, kidney cancer, uterine cancer. In some embodiments, the patient is identified as receiving treatment for cancer. In some embodiments, the method further comprises measuring or assessing tumor growth. In some embodiments, the method further comprises providing additional treatment to the individual before, during, or after administering the acetylated polysaccharide of any embodiment herein or an acetylated polysaccharide prepared by the method of any embodiment herein or the pharmaceutical formulation of any embodiment herein. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human, horse, dog, cat.

In a ninth aspect, a method of treating a viral or bacterial infection in an individual in need thereof is provided. The method comprises administering to a patient in need thereof an acetylated polysaccharide of any embodiment provided herein or an acetylated polysaccharide prepared by a method of any embodiment provided herein or a pharmaceutical formulation of any embodiment provided herein. In some embodiments, the method further comprises monitoring the level of cytokine released during viral or bacterial infection. The method comprises administering to a patient in need thereof an acetylated polysaccharide of any embodiment provided herein or an acetylated polysaccharide prepared by a method of any embodiment provided herein or a pharmaceutical formulation of any embodiment provided herein. In some embodiments, the cytokine is selected from the group consisting of IL-1 β, IL-6, IL-7, IL-8, IL-10, Tumor Necrosis Factor (TNF) - α, Interferon (IFN) - γ, and IFN- α/β.

In some embodiments, the acetylated polysaccharide has a molecular weight of about 1 kilodaltons to about 70 kilodaltons. In some embodiments, the acetylated polysaccharide has a molecular weight of about 5 kilodaltons to about 50 kilodaltons. In some embodiments, the acetylated polysaccharide has a molecular weight of about 10 kilodaltons to about 40 kilodaltons. In some embodiments, the acetylated polysaccharide has a molecular weight of about 15 kilodaltons to about 30 kilodaltons. In some embodiments, the acetylated polysaccharide has a molecular weight of about 18 kilodaltons. In some embodiments, the acetylated polysaccharide has a molecular weight of about 29 kilodaltons. In some embodiments, the acetylated polysaccharide has a molecular weight of less than, greater than, or about 1, 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 kilodaltons or any number or range therebetween.

Brief description of the drawings

The features and advantages of the compositions, systems, devices, and methods described herein will become apparent from the following description when taken in conjunction with the accompanying drawings. These drawings depict only several embodiments in accordance with the disclosure and are not to be considered limiting of its scope. In the drawings, like reference numbers or symbols generally identify like components, unless context dictates otherwise. The drawings may not be to scale.

FIGS. 1A-1C show a method of acetylating natural aloe glucomannan. Figure 1A) the general structure of natural aloe glucomannans with inserted glucosyl groups, with 1,4- β -linked backbones and acetylated at the 2,3 or 6 position; FIG. 1B) polysaccharides with less acetyl groups after processing; FIG. 1C) acetylated highly acetylated polysaccharide. And (2) Manp: a mannopyranosyl group; glcp: glucopyranosyl radical.

FIG. 2A shows the IR spectrum of crude polysaccharide 83008-.

FIG. 2B shows IR spectra of acetylated products 83018-3 (2) from crude polysaccharides 83008-175-15 (1).

FIG. 3 shows A) the crude polysaccharides 83008-¹H NMR spectrum; B) from the acetylated product 83018-3 (2) of the crude polysaccharide¹H NMR spectrum.

FIG. 4A shows IR spectra of enriched polysaccharide 83018-7-11 (3).

Figure 4B shows IR spectra of acetylated products 83018-9-21(4) from enriched polysaccharide 83018-7-11 (3).

FIG. 5 shows A) preparation of enriched polysaccharides 83018-7-11(3)¹H NMR spectrum; B) from the acetylated product 83018-9-21(4) of the enriched polysaccharide¹H NMR spectrum.

FIG. 6 shows A) the carbon-13 spectrum of enriched polysaccharide 83018-7-11 (3); B) carbon-13 spectra of acetylated products 83018-9-21(4) from the enriched polysaccharide. 6M: c-6 of mannopyranosyl; 6M 6: 6-acetylmannopyranosyl C-6; 3M 23: 2, 3-diacetylmannopyranosyl C-3.

FIG. 7 shows HSQC spectra of enriched polysaccharide 83018-7-11 (3); HSQC is used to directly associate C-H coupled cores. In the contour diagram, the horizontal axis and the vertical axis are proton chemical shift and carbon chemical shift, respectively. When protons are bonded to carbon, a cross peak can be found at the intersection of a horizontal line from the carbon signal and a vertical line from the proton signal.

FIG. 8 shows HSQC spectra of enriched polysaccharide 83018-9-21 (4).

Figure 9A shows that acetylated polysaccharides reduce anti-inflammatory activity. The figure shows the effect of 1-4 on the cytokines IL-1. beta., IL-6, IL-8, IL-10, MIP-1. alpha. and TNF. alpha.

FIG. 9B shows the effect of 1-4 on the cytokine IL-10.

Definition of

As described herein, a "polysaccharide" is a polymeric carbohydrate molecule consisting of long chains of monosaccharide units joined together by glycoside bonds. During hydrolysis, these polysaccharides are cleaved into the component mono-or oligosaccharides. The polysaccharides can range in structure from linear polysaccharides to highly branched polysaccharides. There are several types of polysaccharides. Polysaccharides may include, without limitation, structural polysaccharides, neutral polysaccharides, acidic polysaccharides, bacterial capsular polysaccharides, and storage polysaccharides. The function of polysaccharides in living organisms such as plants can be structure-related or storage-related. Starch (a polymer of glucose) is used in plants as a storage polysaccharide, which is present in the form of amylose and branched amylopectin.

"purification" is a technique known to those skilled in the art in which a compound, chemical, protein or DNA acts as a foreign or contaminating substance. Without limitation, purification may be by affinity purification, filtration, evaporation, liquid-liquid extraction, crystallization, recrystallization, and fractional distillation. In some embodiments, the crude polysaccharide is purified to 1-90% purity by weight. In some embodiments herein, the crude polysaccharide is purified to 90% purity by weight. In some embodiments herein, the acetylated product is purified to 90% purity by weight. In some embodiments, the product produced by the methods described herein is purified by ethanol precipitation. In some embodiments, the purification is performed by dialysis.

As used herein, "glucomannan" is the major polysaccharide in aloe, consisting of mannose with a 1,4- β -linked backbone and glucose, of which mannose is the major.

"acetylation" as described herein refers to a reaction that introduces acetyl functionality into a compound. Acetylation refers to the process of introducing an acetyl group (resulting in an acetoxy group) into a compound, i.e., the acetyl group replaces an active hydrogen atom. In some embodiments herein, the polysaccharide is acetylated at the hydroxyl group. The term "acetyl" refers to "-C (═ O) CH₃A "group.

An "acetylation agent" as described herein is a chemical substance used for the acetylation of a compound. Without limitation, acetylation with an acid anhydride may be carried out in the presence of an acid catalyst or a base catalyst. Various kinds ofMetal salts, e.g. CoCl₂、TiCl₄-AgClO₄TaCl and TaCl₅-SiO₂Ce (III) triflate, Sn (IV) porphyrins and some metal triflates such as Sc (OTf)₃、MeSiOTf、In(OTf)₃、Cu(OTf)₂And Bi (OTf)₃Bis (cyclopentadienyl) zirconium dichloride, I₂1, 3-dibromo-5, 5-dimethylhydantoin (hydantoin) or trichloroisocyanuric acid may also be used in the reaction to increase efficiency.

"catalysis" as described herein refers to an increase in the rate of a chemical reaction in the presence of an additional substance referred to as a "catalyst". The process for acetylation may also be provided with a catalyst, as described herein. In some embodiments herein, the catalyst is pyridine, sodium hydroxide, potassium hydroxide, sodium or potassium carbonate and a solvent.

As described herein, "infrared spectroscopy" (IR spectroscopy or vibrational spectroscopy) is spectroscopy involving the infrared region of the electromagnetic spectrum, i.e., light having a longer wavelength and lower frequency than visible light. It covers a range of techniques based primarily on absorption spectroscopy. IR spectroscopy is known to those skilled in the art and can be used for identification and characterization of compounds and chemicals. In some embodiments herein, IR spectroscopy is used to determine the content of the polysaccharide or to characterize the polysaccharide.

As used herein, "nuclear magnetic resonance spectroscopy" or "NMR spectroscopy" is a research technique that utilizes the magnetism of certain nuclei. It can be used to determine the physical and chemical properties of an atom or a molecule containing the atom. Analysis of 1D, 2D or 3D NMR spectra can use studies of chemical shifts of molecules for determining the properties, characteristics and structure of molecules of interest. This technique is known to those skilled in the art and can be applied using, for example, 200, 300, 400, 500, 600, 800, 900MHz NMR instruments. In some embodiments herein, the acetylation content of the polysaccharide is analyzed using NMR spectroscopic techniques.

As used herein, "pharmaceutical excipient" refers to a substance formulated with an active ingredient of a drug. It may be included for long-term stability purposes, to fill (bulking up) solid formulations containing the active ingredient (hence commonly referred to as "bulking agents", "fillers" or "diluents"), or to impart a therapeutic enhancement to the active ingredient in the final dosage form, e.g. to promote drug absorption, to reduce viscosity or to enhance solubility. Excipients may also be used in the manufacturing process to help handle the active involved, for example by promoting powder flowability or non-stick properties, and to help in vitro stability, for example to prevent denaturation or aggregation over the expected shelf life. The choice of suitable excipients also depends on the route of administration and the dosage form, as well as the active ingredient and other factors.

As used herein, "immune disorder" refers to a dysfunction of the immune system. The immune disorder may be a "primary immunodeficiency disease," which is a disease caused by a genetic gene mutation. An immune disorder can also be "secondary or acquired immunodeficiency" which is caused by something outside the body such as a virus or immunosuppressive drug. Without limitation, examples are systemic lupus, scleroderma, hemolytic anemia, vasculitis, type I diabetes, Graves 'disease, rheumatoid arthritis, multiple sclerosis, Goodpasture's syndrome, myopathy, severe combined immunodeficiency, DiGeorge syndrome, hyperimmune E syndrome, common variant immunodeficiency, chronic granulomatosis, Wiskott-Aldrich syndrome, autoimmune lymphoproliferative syndrome, hyper IgM syndrome, defective leukocyte adhesion, NF- κ B essential regulatory protein (NEMO) mutations, selective immunoglobulin A deficiency, X-linked agammaglobulinemia, X-linked lymphoproliferative disorders or ataxia-telangiectasia. For example, when detected in the serum or cerebrospinal fluid of a patient, immune disorders can be analyzed by examining the profile of nerve-specific autoantibodies or other biomarkers.

As used herein, "cytokine" refers to a small protein important in cell signaling. Cytokine release has an effect on the behavior of the cells around them. Cytokines are involved as immunomodulators in, for example, autocrine signaling, paracrine signaling, and endocrine signaling. Examples of anti-inflammatory cytokines may include, but are not limited to, Interleukin (IL) -1 receptor antagonists, IL-4, IL-6, IL-10, IL-11, and IL-13.

Detailed Description

Polysaccharides are one of the main components in aloe. The main polysaccharide in aloe is glucomannan, which consists of mannose with a 1, 4-beta-linked backbone and glucose, of which mannose is the major. The hydroxyl groups of the mannose moiety may be substituted with acetyl groups and the aloe polysaccharides are referred to as acetylated glucomannans or interchangeably as acetylated mannans (Acemannan). Acetylated mannans are commonly used trade names in the literature. Acetylated glucomannans are reported to be responsible for a variety of biological activities of aloe vera, including immunostimulating, anti-inflammatory, hypoglycemic, hypolipidemic, antibacterial, antiviral and antitumor effects.

The position of the acetylated group found on the mannose moiety is 2-, 3-or 6-acetyl, 2, 3-; 2, 6-; or 3, 6-diacetyl and 2,3, 6-triacetyl. The reported acetyl content found in natural acetylated glucomannan is 15-26% acetylation in aloe.

The aloe leaves comprise an outer green skin and an inner clear gel. Both the outer green skin and the inner gel contain polysaccharides. In the aloe preparation method, the aloe leaf product may be prepared from the whole leaf extract using an outer green skin and gel (referred to as 100X), or from an extract using only an inner gel (referred to as 200X), based on the plant parts to be used. During the aloe product preparation process, enzymes and/or heat are applied to the plant parts. Both enzymes and heat can deacetylate polysaccharides, resulting in a reduction of acetyl groups on the polysaccharide. Thus, when polysaccharides are subjected to the manufacturing process, the content of acetyl groups is generally less than their natural precursors. To restore or increase the content of acetyl groups lost during the preparation process, acetylation reagents are used in the reaction with glucomannan to synthesize highly acetylated polysaccharides.

In embodiments herein are described aloe vera powder, which is used as a reaction starting material, which is first purified by ethanol precipitation. Filtering the ethanol-insoluble polysaccharide and separating from the ethanol-soluble molecules to obtain crude aloe polysaccharide. The crude polysaccharide was then dissolved in water and dialyzed in a container of purified water. The water is often replaced with fresh water until the dialysis process is complete. Centrifuging the dialysate, followed by freeze-drying, to obtain enriched acetylated glucomannan having a purity of 70-90% by weight.

Acetylation reagent acetic anhydride (CH)₃CO)₂O is used to prepare highly acetylated glucomannans. Acetyl chloride and acetic acid may also be used as acetylating agents. The crude or enriched polysaccharide is treated with acetic anhydride and the reaction product is purified by dialysis to remove unreacted acetylating agent, catalyst (e.g., pyridine, sodium hydroxide, potassium hydroxide, sodium or potassium carbonate and solvent). The purified reaction product was subjected to spectral analysis and chemical analysis to determine the polysaccharide content and acetyl content.

Infrared spectroscopy (IR) and Nuclear Magnetic Resonance (NMR) spectroscopy are used for identification and quantification of the starting materials and products of the reaction.

In some embodiments, the patient is identified as receiving treatment for inflammation after being diagnosed by a physician as having inflammation. In some embodiments, the inflammation is manifested by one or more of heat, pain, redness, swelling, and loss of function of the skin or internal organs. In some embodiments, the patient is identified as receiving treatment for the immune disorder after being diagnosed by a physician with the immune disorder. In some embodiments, inhibition of the inflammatory response is measured by monitoring an anti-inflammatory cytokine in the patient, the anti-inflammatory cytokine selected from the group consisting of an Interleukin (IL) -1 receptor antagonist, IL-4, IL-6, IL-10, IL-11, and IL-13. In some embodiments, the patient is identified as receiving treatment for cancer after being diagnosed with cancer by a physician. In some embodiments, tumor growth is measured and assessed by monitoring the size of the tumor with one or more of the following techniques: computed tomography, Magnetic Resonance Imaging (MRI), X-ray, mammography, ultrasound, and Positron Emission Tomography (PET).

Materials and methods

Aloe leaf juice powder (200X or 100X) was purchased from Aloe barbadensis bioengineering (USA) Inc. (Hainan Aloecorp Co., Ltd.) (Hainan, China) or Pharmachem Laborates, Inc., Improvive USA (Anaheim, Calif., USA). Anhydrous ethanol and dimethyl sulfoxide (DMSO) were purchased from Sinopharm Chemical Reagent co. Dialysis membranes were purchased from Shanghai Yuanye Biological technology co., Ltd) (Shanghai, china). Acetic anhydride was purchased from Kyoto Kelong Chemical company, Inc. (Chengdu Kelong Chemical Reagent Inc.) (Chinese Goodu). Pyridine was purchased from Shanghai, China reagent works (Shanghai, China). All reagents and chemicals were used without further purification. Fourier transform infrared spectroscopy was performed on a PerkinElmer Frontier FT-IR spectrometer (PerkinElmer Instruments, Norwalk, CT, USA).¹H NMR (400MHz) and¹³c NMR (100MHz) spectra were recorded on a Bruker Ultrashield 400 Plus or Bruker 400 Avance III instrument (Fremont, CA, USA) with D₂O as solvent (Sigma-Aldrich, st.louis, MO, USA). Chemical shifts are reported in parts per million (ppm, δ). By weighing approximately 2-10mg of sample and 5mg of internal standard nicotinamide (Sigma-Aldrich, St. Louis, Mo., USA)¹Quantification of acetyl by H NMR. Dissolve both sample and Nicotinamide in 1mL of D₂O and incubated at 4 ℃ for 24h, then subjected to NMR analysis. Acquisition by heteronuclear single-quantum coherence (HSQC) spectroscopy using standard gradient pulse sequences of Bruker Topspin software¹H-¹³C correlation spectra were performed on a PABBO BB-1H/D Z-GRD probe. The molecular weight of the polysaccharides was determined on Size Exclusion Chromatography (SEC) using a PolySep-GFC-P-Linear column (300x7.5mm) (Phenomenex, Inc., Torrance, Calif., USA) equipped with a TSKgel PWXL guard column (40x6mm) (Sigma-Aldrich). The mobile phase was 0.1M NaCl, which was pre-filtered through a 0.22 μ M GSWP membrane (Merck Millipore, Darmstadt Germany) before use. During analysis, the mobile phase was further passed through an in-line filter (0.10 μm x 25mm durapore membrane filter, Millipore co., Bedford, MA) before reaching the SEC column. The flow rate was 0.7mL/min, the injection volume was 100. mu.L, and column chromatography was performed at 35 ℃. The sample was initially dissolved in 0.1M NaCl at a concentration of 0.25-2mg/mL, allowed to swell at 4 ℃ for 20-24 hours, and then filtered through a 0.45 μ M MCE filter prior to injection. Light Scattering measurements were performed using the Dawn Enhanced Optical System (DAWN HELEOS II) MALS testThe detector was equipped with a 100mW 662nm Ga/As laser and an 18 ℃ research grade light scattering photometer (Wyatt Technology Co, Santa Barbara, Calif., USA). The chromatography system consisted of a 2690 separation module with a Waters 2410 differential refractometer (Waters Corp, Milford, MA, USA). The instrument was validated by the manufacturer using 200kDa polystyrene in toluene for the MALS detector and 66,000Da Bovine Serum Albumin (BSA) for the MALS/RI detector. The SEC/MALS/RI detectors are connected in series. The inter-detector delay was measured to be 255.6. mu.L using BSA. Data from the light scattering and differential refractometer were analyzed using Wyatt ASTRA 6.1.5.22 software. A refractive index delta (dn/dc) of 0.14mL/g was used throughout the experiment. Data from MALS measurements were processed using Zimm extrapolation. For the MW and MWD analyses of samples by SEC/RI systems calibrated with pullulan or dextran standards, calibration curves were obtained by plotting the logarithm of molecular weight versus retention time, and using Empower 3GPC/SEC software (Waters Corp, Milford, MA, USA), linear regression was performed on narrow polydispersity pullulan and a third order polynomial fit was performed on broad polydispersity dextran. For pullulan calibration in combination with broad polydispersity aloe polysaccharides, a third order polynomial fit was used.

Embodiment 1: preparation of acetylated polysaccharides from crude Aloe polysaccharide

A total of 100 grams of 200X aloe leaf juice powder was dissolved in 900ml of water to prepare a 10% aloe solution. To the 10% aloe solution, 3,600 ml of ethanol was slowly added with stirring, and the ethanol solution was left at 4 ℃ for 18 hours. A white precipitate formed in the ethanol solution and was obtained by filtration system. The precipitate was rinsed twice with 250mL of ethanol and freeze-dried to yield 39g of crude polysaccharide, 83008-175-15 (1).

A total of 500mg of 83008-. After stirring the DMSO solution at room temperature for 24 hours, 3ml of pyridine and 2.5ml of acetic anhydride were introduced into the DMSO solution in this order. The reaction shown below was carried out under stirring in an ice-water bath for 30 minutes. The reaction solution was then warmed to room temperature for 1.5 hours. After 2 hours, water was added to the reaction product and the solution was transferred to a dialysis membrane tube having a molecular weight cut-off (MWCO) of 8,000-14,000 Da. The reaction product was dialyzed until pyridine odor was removed. The dialysate was freeze dried to give white acetylated polysaccharide 83018-3-3, 47mg total weight of product with a purity of 86% by weight (2).

The acetylated product (2) was analyzed by infrared spectroscopy (IR) at-1741 cm^-1And a carbonyl group (═ O) at-1247 cm^-1The stretched bond (stretchbond) at the ester group (-O-) was shown to be stronger in the acetylated product (2) than in the crude polysaccharide (1) (fig. 2A and 2B), indicating that more acetyl groups were present in the acetylated product (2) and indicating that acetylation has occurred. This reaction leads to the surprising result that higher acetylation is obtained compared to the plant process of acetylation by this reaction.

In that¹In the H NMR spectrum, acetyl groups appeared at 2.0 to 2.2ppm and were quantified. By passing¹H NMR showed that the polysaccharide produced 7.5% acetylation in the crude polysaccharide (1) and 17.8% acetylation in the acetylated product (2), respectively, which confirmed acetylation of 2 (fig. 3). The entire sample showed multiple acetylated hydroxyl groups of the sugar.

Embodiment 2: preparation of acetylated polysaccharides from enriched polysaccharides

The ethanol-undissolved crude polysaccharide 83008-175-15(1) was dialyzed in a membrane tube with MWCO 8,000-14,000Da to yield enriched polysaccharide 83018-7-11 (3). Polysaccharide 3 was then treated with acetic anhydride-pyridine under the same conditions as used in example 1 to give acetylated product 83018-9-21, (4). The product 4 was purified by dialysis using a membrane with a molecular weight cut-off (MWCO) of 8,000-14,000Da and freeze-dried to give a white powder.

Polysaccharide 4 showed a greater intensity of about 1742cm in the IR spectrum than polysaccharide 3^-1Carbonyl signal of about 1244cm^-1Ester signal (FIGS. 4A and 4B). In that¹Analysis of H NMR spectra (FIG. 5) for polysaccharide 3 and polysaccharideThe acetyl group content of 4 was 17.2% and 24.5%, respectively, indicating successful acetylation as shown in polysaccharide product 4. Thus, the acetylation reaction surprisingly produces a high percentage of acetylated products compared to the crude polysaccharide.

Comparing polysaccharide 3 with polysaccharide 4¹³C NMR, signal increase corresponding to acetyl (see fig. 6). For example, the signal at 60.3ppm comes from C-6 and moves to the low field to 63.5ppm when 6-O is substituted with acetyl. The signal intensity at 60.3ppm was higher in 3 than at 63.5ppm, indicating less acetyl groups at 6-OH of 3. However, in polysaccharide 4, the strength from the acetylate C-6 is significantly higher than its non-acetylated C-6. Similarly, the signals generated by C-2 and C-3 also increased due to acetylation of O-2 and O-3 at about 72-74ppm (FIG. 6).

Although it is not limited to¹³The C NMR spectra clearly show the difference between the polysaccharide before and after acetylation, but due to its low sensitivity and low resolution here, 2D HSQC (heteronuclear single quantum coherence) spectra were obtained on polysaccharide 3 and polysaccharide 4. In the HSQC spectra of polysaccharide 3, characteristic cross peaks from proton-carbon coherence at 5.35,5.42/69.7 are shown, which correspond to the signal of C-2 due to the substitution of acetyl groups at both O-2 and O-3 positions and are designated 2M23(M represents the mannopyranosyl moiety). The cross peaks at 5.10/71.7, 5.34,5.40/71.6 and 4.78/73.1 are due to the signal of C-3 due to 2, 3-O-diacetyl (3M23) substitution, the signal of C-2 due to 2-O-acetyl (2M2) substitution and the signal of C-3 due to 3-O-acetyl (3M3) substitution, respectively. The cross-peaks at 3.55,3.78/60.3 were assigned as signals for C-6(6M), i.e., the 6-O position without acetyl substitution, while the 6-acetylated proton-carbon cross-peak was found at 4.19,4.28/63.5(6M 6). The distribution of other cross peaks is shown in fig. 7.

Comparing the HSQC spectrum of polysaccharide 4 (FIG. 8) with the HSQC spectrum of polysaccharide 3, the cross-peaks caused by the C-3 substituted signals from the C-2 and 3-O-acetyl (3M3) of 2-O-acetyl (2M2) become weak or disappear, while the cross-peaks due to the C2(2M23) signal at 5.34,5.41/69.5 and the cross-peaks due to the C-3(3M23) signal at 5.10/71.8, respectively, of 2, 3-O-diacetyl substitution become dominant. Similarly, the signal intensity of the 6-acetylated cross peak at 4.16,4.37/62.6(6M6) was stronger in 4 than the non-acetylated signal at 3.69,3.79/60.1(6M), whereas the opposite signal intensity was observed at 6M6 and 6M in polysaccharide 3. All these results indicate that more acetyl groups are present in polysaccharide 4 than in polysaccharide 3, confirming that polysaccharide 4 is an acetylated product of polysaccharide 3.

Acetylation of the processed Aloe polysaccharide 100X (033694-. NMR analysis confirmed that polysaccharides 7,10 and 13 are acetylated polysaccharides of their precursor polysaccharides 6, 9 and 12, respectively. The content of acetylated groups determined by proton NMR before and after acetylation is shown in table 1.

TABLE 1 content of acetylated groups of polysaccharides before and after acetylation

Analysis of polysaccharide molecular weight

The molecular weight was found to decrease after acetylation (table 2). Theoretically, the molecular weight of the acetylated polysaccharide should be increased because more acetyl groups are added. The reason for the reduction may be that acetylation disrupts the network between the polysaccharides due to hydrogen bonding. The formation of acetyl bonds makes the number of hydroxyl groups less available for hydrogen bonding.

TABLE 2 molecular weight of polysaccharide before and after acetylation

Embodiment 5: immunomodulatory Activity of acetylated polysaccharides

As shown in Table 3, the non-acetylated aloe preparation of inner gel 030604-3500PS,9 showed immunomodulatory activity, as its treatment of PBMC in the presence of LPS resulted in a significant reduction of pro-inflammatory cytokines (IL-1. beta. and IL-6) and a significant increase of anti-inflammatory IL-10, compared to their expression levels stimulated by LPS. Interestingly, the acetylation 83018-51-20,10 of this formulation, the acetylated polysaccharide increased from 13.5% to 19.7% (an increment of 46%), resulting in a significant decrease in proinflammatory IL-1 β and TNF- α, suggesting that the increase in acetylation of this particular aloe formulation causes a change in its immunomodulatory activity. On the other hand, non-acetylated aloe vera gel powder (200X)3 showed immunomodulatory activity, as its treatment of PBMC in the presence of LPS resulted in a significant reduction of IL-6, TNF- α and IFN- γ, and in a significant increase of IL-10, compared to their expression levels stimulated by LPS. Acetylation 4 of this preparation, whose acetylated polysaccharide increased from 14.2% to 24.5% (increment of 42%), resulted in a significant increase in IL-10, but not significant changes in proinflammatory cytokines, indicating that an increase in acetylation of this particular aloe preparation causes a change in its immunomodulatory activity. The results indicate that increased acetylation of the tested aloe preparations causes a significant change in their immunomodulatory activity.

TABLE 3 content of acetylated groups of polysaccharides and Effect on cytokine expression from human PBMC

Embodiment 6: acetylation of crude polysaccharide 83018-3 (2)

A500 mg sample of crude polysaccharide 83008-. After stirring the solution at ambient temperature for 24 hours, 3mL of pyridine and 2.5mL of acetic anhydride were added successively in an ice-water bath while stirring for 30 minutes, and then warmed to room temperature for 1.5 hours. After 2 hours, the reaction was quenched by the addition of 20mL of water. The reaction product was transferred to a dialysis membrane tube with MWCO 8,000-14,000Da until no pyridine odor was smelled. The dialysate was lyophilized yielding 47mg of white acetylated polysaccharide 83018-3 (2). IR (KBr) v max 3437,1741,1638,1376,1247,1037cm^–1.¹H NMR(400MHz,D₂O)δ(ppm):2.01–2.20(br.,m,-COCH₃)；¹³C NMR(100MHz,D₂O) delta (ppm)100.2(C-1M),99.6(C-1M3),98.7(C-1M2),98.4(C-1M23),76.9(C-4M),75.0(C-5M),73.3(C-3M3),71.8(C-3M23),71.4(C-3M, C-2M2),69.8(C-2M),69.5(C-2M23),68.4(C-2M3),62.8(C-6M6) and 60.3 (C-6M). The content of acetyl groups was 17.8%.

Embodiment 7: acetylation of enriched polysaccharide 83018-9-21(4)

500mg of enriched polysaccharide 83018-7-11(3) was weighed accurately into a 50ml round bottom flask and 24ml DMSO was added. The solution was allowed to stand at ambient temperature for 24 hours, and 1.8ml of pyridine and 1.5ml of acetic anhydride were added successively in an ice-water bath while stirring for 30 minutes, and then warmed to room temperature for 1.5 hours. After 2 hours, water was added to quench the reaction and the solution was transferred to dialysis membrane tubes with an MWCO of 8,000-14,000 Da. The reaction product was dialyzed until no pyridine odor was smelled, and the dialysate was lyophilized to yield a white enriched acetylated polysaccharide product 83018-9-21(4), 362 mg. IR (KBr) v max 3474,1742,1638,1375,1244,1043cm^–1.¹H NMR(400MHz,D₂O)δ(ppm):1.98-2.22(br.,m,-COCH₃)；¹³C NMR(100MHz,D₂O) delta (ppm)100.2(C-1M), 99.5(C-1M3),99.8(C-1M2),98.3(C-1M23),77.0(C-4M),75.0(C-5M),73.4(C-4M2),73.3(C-3M, C-3M3),71.8(C-3M23),69.7(C-3M),69.5(C-2M23),68.6(),62.6(C-6M6) and 60.0 (C-6M). The content of acetyl groups was 24.5%.

Embodiment 8: general Process for the preparation of crude polysaccharides 033694AIRs (5)

A total of 50 grams of aloe leaf juice powder (100X,033694) was accurately weighed into a 1L glass shredder and 450 ml of water was added. The solution was sonicated until the aloe powder was completely dissolved and then slowly poured into 1800 ml of absolute ethanol with stirring. The alcohol solution was placed in a refrigerator at 4 ℃ overnight. A white precipitate was obtained by filtration through filter paper under vacuum and washed with 25 ml 80% ethanol. After removing 25 ml of 80% ethanol washing solution, the washing was repeated by adding 25 ml of 80% ethanol. Under vacuumThe precipitate was dried to obtain a total of 27 g of crude polysaccharides 033694AIRs (5). IR (KBr) v max 3376,1591,1420,1259, and1093cm^–1.¹H NMR(400MHz,D₂O)δ(ppm):2.03-2.17(br.,m,-COCH₃). The content of acetyl groups was 2.6%.

Embodiment 9: preparation of enriched polysaccharide 100X-AIRs-3500DaPs (6)

A total of 2 grams of crude polysaccharides 033694AIRs (5) were weighed and dissolved in about 100ml of water and dialyzed in a membrane tube with MWCO 3500Da for 2 days. During dialysis, the solution is often replaced with fresh water until the process is completed. The dialysate was lyophilized to yield 202 mg of white enriched polysaccharide 100X-AIRs-3500DaPs (6). IR (KBr) v max 3412,1737,1629,1376,1248, and 1034cm^–1.¹H NMR(400MHz,D₂O):δ(ppm):2.00-2.20(br.,m,-COCH₃). The content of acetyl groups was 12.1%.

Embodiment 10: acetylation 83018-51-22(7) of enriched polysaccharide

500mg of enriched polysaccharide 100X-AIRs-3500DaPs (6) were accurately weighed into a 50mL round bottom flask and 40mL of DMSO was added. The solution was allowed to stand at ambient temperature for 24 hours, 3mL pyridine and 2.5mL acetic anhydride were added successively in an ice-water bath while stirring for 30 minutes, and then allowed to warm to room temperature for 1.5 hours. After 2 hours, water was added to quench the reaction and the solution was transferred to dialysis membrane tubes with 3500 Da. The reaction product was dialyzed until no pyridine odor was smelled, and the dialysate was lyophilized to yield a white enriched acetylated polysaccharide product, 83018-51-22(7)430 mg. IR (KBr) v max 3329,1740,1641,1376,1247, and 1042cm^–1.¹H NMR(400MHz,D₂O)δ(ppm):1.99-2.24(br.,m,-COCH₃). The content of acetyl groups was 20.3%.

Embodiment 11: general Process for the preparation of crude polysaccharides 030604AIRs (8)

A total of 100 grams of aloe gel powder (200:1,030604) was accurately weighed into a 1-L glass beaker and 900mL of water was added. Subjecting the solution to ultrasonic treatment until the aloe powder is completely dissolvedAfter hydrolysis, it was slowly poured into 3600mL of absolute ethanol with stirring. The alcohol solution was placed in a refrigerator at 4 ℃ overnight. A white precipitate was obtained by filtration through filter paper and washed twice with 50mL of 80% ethanol. The precipitate was dried in vacuo to yield a total of 41g of crude polysaccharides 030604AIRs (8). IR (KBr) v max 3385,1735,1591,1413,1250, and1091cm^–1.¹H NMR(400MHz,D₂O):δ(ppm):2.01-2.17(br.,m,-COCH₃). The content of acetyl groups was 3.6%.

Embodiment 12: preparation of enriched polysaccharide 100X-030604-3500DaPs (9)

A total of 2g of crude polysaccharides 030604AIRs (8) were weighed and dissolved in about 100mL of water, then dialyzed in a membrane tube with MWCO 3500Da for 2 days. During dialysis, the solution is often replaced with fresh water until the process is completed. The dialysate was lyophilized yielding 405mg of white enriched polysaccharide 100X-030604-3500DaPs (9). IR (KBr) v max 3420,1740,1644,1376,1246, and 1068cm^–1.¹H NMR(400MHz,D₂O)δ(ppm):2.02-2.20(br.,m,-COCH₃). The content of acetyl groups was 13.5%.

Embodiment 13: acetylation of rich polysaccharides 83018-51-20(10)

A300 mg sample of enriched polysaccharide 100X-030604 and 3500DaPs (9) was accurately weighed into a 50mL round-bottom flask and 24mL of DMSO was added. The solution was allowed to stand at ambient temperature for 24 hours, and 1.8mL of pyridine and 1.5mL of acetic anhydride were added successively in an ice-water bath while stirring for 30 minutes, and then allowed to warm to room temperature for 1.5 hours. After 2 hours, water was added to quench the reaction and the solution was transferred to dialysis membrane tubes with 3500 Da. The reaction product was dialyzed until no pyridine odor was smelled, and the dialysate was lyophilized to yield a white enriched acetylated polysaccharide product 83018-51-20(10)191 mg. IR (KBr) v max 3440,1741,1638,1376,1246, and 1042cm^-1.¹H NMR(400MHz,D₂O)δ(ppm):1.97-2.24(br.,m,-COCH₃). The content of acetyl groups was 19.7%.

Embodiment 14: preparation of crude polysaccharides 032889AIRs (11)) General method of

A total of 100g of aloe leaf juice powder (100X,032889) was accurately weighed into a 1L glass shredder and 900mL of water was added. The solution was sonicated until the aloe powder was completely dissolved, then slowly poured into 3600mL of absolute ethanol with stirring. The alcohol solution was placed in a refrigerator at 4 ℃ overnight. A white precipitate was obtained by filtration and washed twice with 50mL of 80% ethanol. The precipitate was dried in vacuo to give a total of 50g of crude polysaccharides 032889AIRs (11). IR (KBr) v max 3374,1591,1418,1254, and 1088cm^–1；¹H NMR(400MHz,D₂O)δ(ppm):2.02-2.17(br.,m,-COCH₃). The content of acetyl groups was 3.3%.

Embodiment 15: preparation of enriched polysaccharide 100X-032889-3500DaPs (12)

A total of 1g of crude polysaccharides 032889AIRs (11) were weighed and dissolved in 100mL of water, then dialyzed in a membrane tube with MWCO 3500Da for 2 days. During dialysis, the solution is often replaced with fresh water until the process is completed. The dialysate was lyophilized, yielding 126mg of white enriched polysaccharide 100X-030604-3500DaPs (12). IR (KBr) v max 3401,1739,1634,1376,1248, and 1067cm^–1.¹H NMR(400MHz,D₂O)δ(ppm):2.01-2.20(br.,m,-COCH₃). The acetyl content was 11.6%. V is

Embodiment 16: acetylation of enriched polysaccharides, 83018-16 (13)

A total of 300mg of enriched polysaccharide 100X-032889-3500DaPs (12) was accurately weighed into a 50mL round-bottomed flask and 24mL of DMSO was added. The solution was allowed to stand at ambient temperature for 24 hours, and 1.8mL of pyridine and 1.5mL of acetic anhydride were added successively in an ice-water bath while stirring for 30 minutes, and then warmed to room temperature for 1.5 hours. After 2 hours, water was added to quench the reaction and the solution was transferred to dialysis membrane tubes with 3500 Da. The reaction product was dialyzed until no pyridine odor was smelled, and the dialysate was lyophilized to yield a white enriched acetylated polysaccharide product, 83018-16 (13). IR (KBr) v max 3428,1738,1639,1375,1246, and 1042cm^–1.¹H NMR(400MHz,D₂O)δ(ppm):2.00-2.19(br.m,-COCH₃). The content of acetyl groups was 20.2%.

Further embodiments

Pharmaceutical preparation

In some embodiments, the active ingredient and mixtures of active ingredients may be used, for example, in pharmaceutical formulations comprising pharmaceutically acceptable carriers, to prepare them for storage and subsequent administration. In addition, some embodiments include the use of the active ingredients described above with a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the Pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences,18th ed., Mack Publishing co., Easton, Pa, (1990), which is incorporated herein by reference in its entirety. Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical formulations. For example, sodium benzoate, ascorbic acid and esters of p-hydroxybenzoic acid may be added as preservatives. In addition, antioxidants and suspending agents may be used.

Pharmaceutical formulations of the active ingredient may be formulated and used as tablets, capsules or elixirs for oral administration. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for dissolution or suspension in liquid prior to injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride and the like. In addition, if desired, the injectable pharmaceutical formulations may contain minor amounts of non-toxic auxiliary substances, such as wetting agents, pH buffering agents and the like. If desired, absorption enhancing agents (e.g., liposomes) can be used.

For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers, such as Hanks 'solution, Ringer' solution or physiological saline buffer. For such transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. It is within the scope of the present invention to formulate the ingredients disclosed herein for practicing the invention into a dosage form suitable for systemic administration using a pharmaceutically acceptable carrier. By appropriate selection of the carrier and appropriate manufacturing quality management specifications, the pharmaceutical formulations disclosed herein, particularly those formulated as solutions, can be administered parenterally, for example by intravenous injection. The active ingredients can be readily formulated into dosage forms suitable for oral administration using pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.

Pharmaceutical formulations for parenteral administration comprise aqueous solutions of the active ingredient in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or other organic oils such as soybean oil, grape seed oil or almond oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, for example sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the ingredients to allow for the preparation of highly concentrated solutions. In some embodiments of the pharmaceutical formulation, the vehicle is a lipophilic solvent, a fatty oil, an organic oil, or a liposome. In some embodiments, the vehicle is sesame oil, soybean oil, grape seed oil, or almond oil, or a synthetic fatty acid ester, such as ethyl oleate or triglycerides, or liposomes.

Pharmaceutical preparations for oral use can be obtained by mixing the active ingredient with solid excipients, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are in particular fillers, for example sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations, for example maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbomer gel, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the tablets or dragee coatings for identifying or characterizing different combinations of active ingredient doses. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbomer gel, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the tablets or dragee coatings for identifying or characterizing different combinations of active ingredient doses. Such formulations may be prepared using methods known in the art. See, for example, U.S. patent No. 5,733,888 (injectable pharmaceutical formulations); 5,726,181 (poorly water soluble compounds); 5,707,641 (therapeutically active proteins or peptides); 5,667,809 (lipophilic agents); 5,576,012 (solubilizing polymerization agents); 5,707,615 (antiviral preparations); 5,683,676 (granular medication); 5,654,286 (topical formulations); 5,688,529 (oral suspension); 5,445,829 (extended release formulation); 5,653,987 (liquid formulations); 5,641,515 (controlled release formulation) and 5,601,845 (sphere formulation); all of these documents are incorporated herein by reference in their entirety. The pharmaceutical preparations can be prepared in a manner known per se, for example by means of conventional mixing, dissolving, granulating, dragee-making, levitating, emulsifying, encapsulating, entrapping or lyophilizing processes. In some embodiments, the pharmaceutical formulation further comprises an excipient. In some embodiments, pharmaceutical formulations for oral use are prepared. In some embodiments, the excipient is a sugar, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, for example maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone (PVP).

For formulating a dose comprising one or more of the active ingredients disclosed herein, known surfactants, excipients, smoothing agents, suspending agents, and pharmaceutically acceptable film-forming and coating aids and the like, may be used. Preferably, alcohols, esters, sulfated fatty alcohols, and the like may be used as the surfactant; sucrose, glucose, lactose, starch, crystalline cellulose, mannitol, light anhydrous silicate, magnesium aluminate, magnesium metasilicate aluminate, synthetic aluminum silicate, calcium carbonate, acid sodium carbonate, calcium hydrogen phosphate, carboxymethylcellulose calcium, etc. may be used as the excipient; magnesium stearate, talc, hardened oil, etc. as a smoothing agent; coconut oil, olive oil, sesame oil, peanut oil, soybean oil may be used as suspending or lubricating agents; cellulose acetate phthalate as a carbohydrate, such as cellulose or sugar derivatives, or methyl acetate-methacrylate copolymers as polyethylene derivatives may be used as suspending agents; plasticizers such as phthalates and the like may be used as suspending agents. In addition to the above ingredients, sweeteners, flavors, colorants, preservatives and the like may be added to the administration preparations of the compound of the present invention, particularly when the compound is administered orally.

Also disclosed herein are various pharmaceutical formulations well known in the pharmaceutical arts for delivery including intraocular, intranasal, and otic. Pharmaceutical formulations include aqueous ophthalmic solutions of the active ingredient in water-soluble form, such as eye drops, or gellan gum (Shedden et al, clin.ther.,23(3):440-50(2001)) or hydrogels (Mayer et al, Ophthalmologica,210(2):101-3 (1996)); ophthalmic ointments; ophthalmic suspensions, e.g., microparticles, small polymer particles containing a drug, suspended in a liquid carrier medium (Joshi, a., j. ocul. pharmacol.,10(l):29-45(1994)), lipid-soluble formulations (Alm et al, prog.clin.biol.res.,312:447-58(1989)), and microspheres (Mordenti, toxicol.sci.,52(1):101-6 (1999)); and an ocular insert. For example, these formulations are useful as anti-inflammatory agents for the eye. All of the above references are incorporated herein by reference in their entirety. Such suitable pharmaceutical formulations are most often and preferably formulated as sterile, isotonic and buffered formulations for stability and comfort. As disclosed in Remington's Pharmaceutical Sciences,18th ed., Mack Publishing co., Easton, Pa. (1990), which is incorporated herein by reference in its entirety, and well known to those skilled in the art, suitable formulations are most often and preferably isotonic, lightly buffered to maintain a pH of 5.5 to 6.5, and most often and preferably include an antimicrobial agent and a suitable Pharmaceutical stabilizer.

The pharmaceutical formulations described herein may be administered by the oral or non-oral route. When administered orally, the pharmaceutical formulation may be administered in the form of capsules, tablets, granules, sprays, syrups, or other such forms. The pharmaceutical preparation may also be brewed with tea, or formed by dissolving a powdered pharmaceutical preparation in a fluid, typically water, fruit or vegetable juice or milk. When administered non-orally, it may be administered as an aqueous suspension, an oily preparation, etc., or as drops, suppositories, ointments, etc., when administered by injection, subcutaneous, intraperitoneal, intravenous, intramuscular, etc. Similarly, the compositions of the present invention may be administered topically as deemed appropriate by those skilled in the art for optimal contact with living tissue.

Agents intended for intracellular administration may be administered using techniques well known to those of ordinary skill in the art. For example, such agents may be encapsulated into liposomes and then administered by any of the methods described herein. All molecules present in the aqueous solution at the time of liposome formation are incorporated into the aqueous interior. The liposome contents are protected from the external microenvironment and are efficiently delivered into the cytoplasm as the liposomes fuse with the cell membrane. In addition, small organic molecules can be administered directly intracellularly due to their hydrophobicity.

In some embodiments, the pharmaceutical formulations described herein are formulated as a single pill or tablet or gel or capsule or lozenge. In some embodiments, the pill or tablet has a mass of 10mg to 2000 mg. In some embodiments, the pill or tablet has a mass of 100mg to 1500 mg. In some embodiments, the pill or tablet has a mass of 500mg to 1200 mg. In some embodiments, the pill or tablet has a mass of 800mg to 1100 mg.

With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. For clarity, various singular/plural permutations may be expressly set forth herein.

It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to," etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles "a" or "an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an" (e.g., "a" and/or "an" should be interpreted to mean "at least one" or "one or more" and the use of definite articles to introduce claim recitation. The direct recitation of "two recitations," without other modifiers, means at least two recitations, or two or more recitations). Further, in those instances where a convention analogous to "at least one of A, B and C, etc." is used, in general such a construction is intended to mean that one of ordinary skill in the art would understand the meaning of the convention (e.g., "a system having at least one of A, B and C" would include, but not be limited to, systems that have a alone, B alone, C alone, a and B together, a and C together, B and C together, and/or A, B and C together, etc.). In those instances where a convention analogous to "A, B or at least one of C, etc." is used, in general such a construction is intended that one skilled in the art will understand the meaning of the convention (e.g., "a system having at least one of A, B and C" will include, but not be limited to, systems that have a alone, B alone, C alone, a and B together, a and C together, B and C together, and/or A, B and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "a or B" will be understood to include the possibility of "a" or "B" or "a and B".

Further, where features or aspects of the disclosure are described in terms of markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the markush group.

As will be understood by those skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges thereof, and combinations of subranges thereof. Any listed range can be easily considered to be sufficiently descriptive and enable the same range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. By way of non-limiting example, each range discussed herein can be readily broken down into a lower third, a middle third, and an upper third, etc. As will also be understood by those of skill in the art, all languages such as "at most," "at least," "greater than," "less than," and the like include the recited number and refer to ranges that may be subsequently broken down into sub-ranges as set forth above. Finally, as will be understood by those skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1-5 items refers to groups having 1, 2,3, 4, or 5 items, and so forth.

While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.