Injection for treating AIDS (acquired immune deficiency syndrome) and preparation method thereof
By optimizing the extraction process, a high-purity compound injection of sulfated algae polysaccharide and lentinan was prepared, which solved the problems of limited anti-HIV efficacy and side effects of existing AIDS treatment drugs, and achieved safe and efficient AIDS treatment.
Patent Information
- Application Number
- CN202511844791.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-09
- Publication Date
- 2026-01-16
AI Technical Summary
Existing HIV treatments have limited anti-HIV efficacy, insufficient polysaccharide extraction purity, lack of compound preparations with synergistic polysaccharide effects and supporting clinical protocols, and highly effective antiretroviral therapies suffer from drug resistance and side effects.
High-purity sulfated alginate polysaccharide was extracted from seaweed using an optimized extraction process and combined with lentinan to prepare an injection. Through the direct antiviral effect of sulfated alginate polysaccharide and the immunomodulatory effect of lentinan, it synergistically blocks HIV invasion and replication, and rebuilds immune function.
The preparation of high-purity sulfated algal polysaccharide has been achieved, which significantly enhances the anti-HIV effect, has high safety and no side effects, can effectively block HIV invasion and replication, and at the same time rebuild the body's immune function.
Smart Images
Figure SMS_2
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and in particular to an injectable solution for treating AIDS and its preparation method. Background Technology
[0002] AIDS is a serious infectious disease caused by human immunodeficiency virus (HIV). Its core pathogenic mechanism is that HIV attacks human CD4+ T lymphocytes, leading to the gradual collapse of the immune system. Currently, the mainstream clinical treatment is highly active antiretroviral therapy (HAART), which can inhibit viral replication and prolong patient survival. However, it has drawbacks such as drug resistance, significant side effects (such as gastrointestinal reactions and liver and kidney damage), inability to completely eliminate the virus, and difficulty in rebuilding immune function.
[0003] Natural polysaccharides have attracted much attention in the field of HIV treatment due to their good biocompatibility, low toxicity, and multi-target effects. Studies have shown that lentinan, a type of dextran polysaccharide, can stimulate the release of interferon from reticular cells and leukocytes, promote the secretion of cytokines such as IL-2, IL-12, and IFN-γ, and inhibit the production of IL-4, IL-10, and TNF-α. It induces the production of pro-inflammatory or immunosuppressive cytokines, thereby regulating immune function and restoring HIV-infected T lymphocytes, with no side effects. Sulfated algal polysaccharides, as polyanionic compounds, can bind to HIV envelope glycoprotein gp120, blocking the adsorption of the virus to CD4+ T cells, interfering with the viral infection process, and exerting a direct antiviral effect.
[0004] However, the existing technologies have the following shortcomings: (1) Single polysaccharide preparations have limited anti-HIV effects; lentinan lacks the ability to directly block viral invasion, and sulfated algae polysaccharides have weak immunomodulatory capabilities; (2) Polysaccharide extraction and purification processes are complex and the purity is insufficient, affecting the safety and effectiveness of the injection; (3) There is a lack of compound preparations targeting the synergistic effects of the two polysaccharides and corresponding clinical treatment protocols. Therefore, developing a compound polysaccharide injection with optimized process, high safety, and both antiviral and immune reconstitution functions is of great significance for the treatment of AIDS. Summary of the Invention
[0005] The purpose of this invention is to overcome the shortcomings of existing AIDS treatment drugs and provide an injection solution for treating AIDS and its preparation method.
[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for extracting sulfated algal polysaccharides, comprising the following steps: (1) Mix seaweed powder, cellulase and water, and hydrolyze for 2-3 hours to obtain hydrolysate; (2) The enzymatic hydrolysate was extracted at 85-95°C for 10-12 hours to obtain the extract; (3) Filter the extract, add magnesium chloride hexahydrate to the filtrate, let stand for 20-30 min, centrifuge, take the supernatant 1, add anhydrous ethanol to supernatant 1 to the final concentration of 15-25%, let stand for 2-3 h, centrifuge, take the supernatant 2; add anhydrous ethanol to supernatant 2 to the final concentration of 25-35%, let stand for 2-3 h, centrifuge, take the supernatant 3; add anhydrous ethanol to supernatant 3 to the final concentration of 55-65%, let stand for 10-12 h, centrifuge, collect precipitate 1; (4) Mix precipitate 1 with water to obtain a polysaccharide solution; mix the polysaccharide solution with hexadecylpyridine chloride solution, let stand for 12-15 h, centrifuge, and collect precipitate 2; mix precipitate 2 with calcium chloride solution, stir for 6-8 h, centrifuge, and take the supernatant 4; (5) Dialyze the supernatant 4, take the polysaccharide solution in the dialysis bag, freeze-dry it, and obtain sulfated algae polysaccharide.
[0007] Preferably, the seaweed powder mentioned in step (1) is derived from one or more of kelp, Sargassum fusiforme, Undaria pinnatifida, and Fucus vesiculosus; The mass-to-volume ratio of seaweed powder to water in step (1) is 8-12 g: 1 L; The mass ratio of seaweed powder to cellulase is 8~12:0.04~0.06; The enzymatic hydrolysis temperature in step (1) is 45~55℃; In step (3), the final concentration of magnesium chloride hexahydrate added to the filtrate is 0.04~0.06 mol / L; In step (4), the mass-to-volume ratio of precipitate 1 to water is 1g: 20~30mL; The initial concentration of the hexadecylpyridine chloride solution in step (4) is 2-4 wt%; In step (4), the volume ratio of the polysaccharide solution to the hexadecylpyridine chloride solution is 3-5:1. The initial concentration of the calcium chloride solution in step (4) is 3~5 mol / L; In step (4), the volume ratio of precipitate 2 to calcium chloride solution is 1:2~3; The dialysis time in step (5) is 45-52 hours; In step (5), the molecular weight cutoff of the dialysis bag during dialysis is 3000~4000 Da.
[0008] The present invention also provides a sulfated algae polysaccharide extracted by the extraction method described above, wherein the sulfated algae polysaccharide has a purity of ≥95% and a sulfate group content of ≥22%.
[0009] The present invention also provides the use of the sulfated algal polysaccharide extracted by the extraction method described above, or the application of the sulfated algal polysaccharide in the preparation of drugs for treating AIDS.
[0010] The present invention also provides an injectable solution for treating AIDS, using water for injection as a solvent, comprising components at the following concentrations: 4~6g / L Lentinan, 7~9g / L Sulfated Algal Polysaccharide, 8~10g / L Sodium Chloride, 40~60g / L Mannitol; The sulfated algal polysaccharide is the sulfated algal polysaccharide extracted by the extraction method described above, or the sulfated algal polysaccharide described above.
[0011] Preferably, the purity of the lentinan is ≥98%, and the molecular weight is 50,000~80,000 Da.
[0012] Preferably, the pH of the injection solution is 6.5 to 7.5.
[0013] The present invention also provides a method for preparing an injectable solution for treating AIDS, comprising the following steps: Weigh each component according to the concentrations specified, mix lentinan, sulfated algae polysaccharide, sodium chloride, mannitol and water for injection, stir for 30-40 minutes to obtain a mixture; adjust the pH of the mixture to 6.5-7.5, filter through a microporous membrane, collect the filtrate, sterilize, and obtain the injection solution.
[0014] Preferably, the stirring speed is 200~300 rpm.
[0015] Preferably, the pore size of the microporous filter membrane is 0.2~0.3μm; the sterilization temperature is 118~125℃, and the sterilization time is 15~20min.
[0016] Advantages of the method of the present invention over the prior art: (1) Sulfated algal polysaccharides can be extracted according to the method of the present invention, and sulfated algal polysaccharides with a purity of ≥95% and a sulfate content of ≥22% can be obtained, which provides favorable support for the preparation of injection solutions.
[0017] (2) Synergistic effect: Combining the immunomodulatory effect of lentinan with the direct antiviral effect of sulfated algae polysaccharide can both block HIV invasion and replication and rebuild the body's immune function, resulting in more significant efficacy than single polysaccharide preparations or traditional drugs.
[0018] (3) High safety: The lentinan and sulfated algae polysaccharide of the present invention are both natural polysaccharides. When they are prepared into injections, they are highly safe and have no side effects. Detailed Implementation
[0019] The following detailed description of the solutions provided by the present invention, in conjunction with the embodiments, should not be construed as limiting the scope of protection of the present invention.
[0020] Example 1
[0021] Preparation of Lentinan
[0022] Select high-quality shiitake mushroom fruiting bodies, wash them, dry them at 60℃, crush them, and pass them through an 80-mesh sieve to obtain shiitake mushroom powder.
[0023] Add 10g of shiitake mushroom powder to 1L of distilled water and extract in a 90℃ water bath for 8 hours to obtain the extract. Sonicate the extract at 40kHz for 30 minutes, filter, and concentrate the filtrate to 200mL. Add 600mL of 95% pre-cooled ethanol to the concentrate, let stand at 4℃ for 12 hours, centrifuge at 10000rpm for 20 minutes, and collect the precipitate. Mix the precipitate with distilled water at a volume ratio of 1:3 to obtain a crude polysaccharide extract. Mix the crude polysaccharide extract with a chloroform-n-butanol mixture (chloroform:n-butanol volume ratio of 4:1) at a volume ratio of 5:1, vortex for 15 minutes, centrifuge at 8000rpm for 15 minutes, collect the supernatant, and repeat the above steps for the chloroform-n-butanol mixture three times to thoroughly remove protein from the polysaccharide solution, obtaining a deproteinized polysaccharide solution.
[0024] The deproteinized polysaccharide solution was filtered through a 0.45 μm filter membrane to remove insoluble impurities, and then subjected to Sephadex G-100 gel chromatography with deionized water as the eluent, maintaining a flow rate of 0.8 mL / min. The eluent was concentrated and passed through a DEAE-cellulose column, followed by linear gradient elution with 0–1 mol / L NaCl solution at a flow rate of 0.8 mL / min. After elution, the collected eluent was the polysaccharide solution to be dialyzed.
[0025] A dialysis bag with a molecular weight cutoff of 3500 Da was selected. The polysaccharide solution to be dialyzed was transferred into the dialysis bag, the bag opening was sealed with a dialysis clamp, and the bag was placed in deionized water. Dialysis was performed at 4°C for 48 hours, with the deionized water changed every 8 hours. After dialysis, the solution in the dialysis bag was removed, concentrated under reduced pressure to 10 mL, and then freeze-dried in a -50°C freeze dryer for 36 hours to obtain lentinan.
[0026] High performance liquid chromatography was used to determine the purity and molecular weight of lentinan.
[0027] After testing, the purity of the extracted lentinan was 98.5%, and the molecular weight of the lentinan ranged from 50,000 to 80,000 Da, with the highest molecular weight being 65,000 Da.
[0028] Example 2
[0029] Extraction of sulfated algal polysaccharides
[0030] Select kelp, rinse to remove salt and sand, dry in a 45℃ oven, pulverize, and pass through a 60-mesh sieve to obtain seaweed powder.
[0031] Add 10g of seaweed powder to 1L of distilled water, add 0.05g of cellulase, and hydrolyze at 50℃ for 2h to obtain the hydrolysate. Extract the hydrolysate in a 90℃ water bath for 10h to obtain the extract. Filter the extract, collect the filtrate, add magnesium chloride hexahydrate to a final concentration of 0.05mol / L, let stand at 4℃ for 30min, centrifuge at 10000rpm for 15min, and collect the supernatant. Add anhydrous ethanol to supernatant 1 to achieve an ethanol concentration of 20%, and let stand at 4°C for 2 hours; centrifuge at 10,000 rpm for 15 minutes, collect supernatant 2, add anhydrous ethanol to supernatant 2 to achieve an ethanol concentration of 30%, and let stand at 4°C for 2 hours (the purpose of 20% and 30% ethanol precipitation is to remove small molecular weight monosaccharides, oligosaccharides and small molecule proteins); centrifuge at 10,000 rpm for 15 minutes, collect supernatant 3, add anhydrous ethanol to supernatant 3 to achieve an ethanol concentration of 60%, let stand at 4°C for 12 hours, centrifuge at 10,000 rpm for 15 minutes, discard the supernatant, collect the precipitate, and obtain precipitate 1.
[0032] Place precipitate 1 in a clean beaker, add deionized water at a mass-to-volume ratio of precipitate 1 to deionized water of 1g:25mL, place in a magnetic stirrer, and stir at room temperature for 3 hours until the precipitate is completely dissolved to obtain a polysaccharide solution.
[0033] A 3 wt% hexadecylpyridine solution was slowly added dropwise to a polysaccharide solution (the volume ratio of hexadecylpyridine solution to polysaccharide solution was 1:4). The mixture was stirred at room temperature for 30 min, then allowed to stand at 4 °C for 12 h. After centrifugation at 10,000 rpm for 15 min, the supernatant was discarded and the precipitate was collected to obtain precipitate 2.
[0034] Add twice the volume of precipitate 2 to 4 mol / L calcium chloride solution, stir magnetically at room temperature for 7 h, centrifuge at 10000 rpm for 15 min, collect the supernatant to obtain supernatant 4.
[0035] A dialysis bag with a molecular weight cutoff of 3500 Da was selected. The supernatant was transferred into the dialysis bag, the bag opening was sealed with a dialysis clamp, and the bag was placed in deionized water. Dialysis was performed at 4°C for 48 hours, with the deionized water changed every 8 hours. After dialysis, the solution in the dialysis bag was removed, concentrated under reduced pressure to 10 mL, and then freeze-dried at -50°C for 36 hours to obtain sulfated algal polysaccharides.
[0036] High performance liquid chromatography (HPLC) was used to determine the purity, molecular weight, and sulfate group content of sulfated algal polysaccharides.
[0037] The results showed that the purity of the extracted sulfated algal polysaccharide was 96.5%, the sulfate group content was 23.5%, and the molecular weight ranged from 60,000 to 100,000 Da, with the highest content at 80,000 Da.
[0038] Example 3
[0039] Preparation of Lentinan-Sulfated Algal Polysaccharide Complex Injection
[0040] Add 0.8g sodium chloride and 5g mannitol to 80mL of water for injection and heat at 60℃ to dissolve, to obtain intermediate solution 1.
[0041] Add 0.5g of lentinan and 0.8g of sulfated algae polysaccharide to intermediate solution 1, stir at 200rpm for 30min to dissolve all components, and obtain a mixed solution. Make up to 100mL with water for injection. Adjust the pH to 7.0. Filter through a 0.22μm microporous membrane, collect the filtrate, and sterilize at 121℃ for 15min to obtain the injection solution.
[0042] Experimental Example 1
[0043] The injection solution of Example 3 was placed in a 40°C constant temperature incubator and continuously placed. Samples were taken and measured at 0, 1, 3 and 6 months. The results are shown in Table 1.
[0044] Table 1. Stability test results of the compound injection solution
[0045] Table 1 shows that the polysaccharide content and pH value of the composite injection solution of the present invention remained stable after being placed at 40 degrees Celsius for 6 months. This indicates that the composite injection solution of the present invention is stable.
[0046] Experiment Example 2
[0047] Healthy New Zealand white rabbits were selected, with 3 rabbits as the experimental group and 3 as the control group. The drug was administered to the right ear of each rabbit. The experimental group rabbits were injected with 1 mL of the injection solution described in Example 3, while the control group rabbits were injected with 1 mL of physiological saline. This was done once daily for 5 consecutive days. Forty-eight hours after the last administration, the rabbits were observed for any irritation reactions such as congestion and edema at the injection site and throughout their bodies. The results were compared with the control group.
[0048] As a result, no irritation reactions such as redness, swelling, congestion, or necrosis were observed in the local area or throughout the body of the New Zealand white rabbits injected with the injection solution of Example 3, and there was no difference compared with the control. This indicates that the injection solution of the present invention is non-irritating and meets the safety inspection requirements for injection solutions.
[0049] Application Example 1
[0050] Mr. Qian, 54 years old, presented with cough and sputum for two months and was diagnosed with HIV complicated by Mycobacterium tumefaciens infection. He was treated with antiretroviral therapy (ART) (3TC (lamivudine) + TDF (tenofovir disoproxil fumarate) + EFV (efavirenz)) and anti-tuberculosis therapy (RFP (rifampin) + INH (isoniazid) + PZA (pyrazinamide) + EMB (ethambutol)). During treatment, the concentration of EFV in the patient's blood did not reach the recommended therapeutic range, at 0.25 μg / mL. Therefore, with the consent of the patient and his family, ART was replaced with the injection solution prepared in Example 3 of this invention, administered intravenously at 100 mL daily. After 7 consecutive days of injections, the patient's vital signs stabilized, and intravenous infusion continued for another 7 days. All indicators returned to normal, and the patient was discharged after recovery.
[0051] As shown in the above embodiments, this invention provides a compound injection of lentinan and alginate polysaccharides. The lentinan and alginate polysaccharides in this injection work synergistically, directly combining antiviral effects to both block HIV invasion and replication and rebuild the body's immune function. Furthermore, this injection is safe and stable, providing a basis for the treatment of AIDS.
[0052] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for extracting alginic acid sulfate, characterized by, Comprising the following steps: (1) mixing seaweed powder, cellulase and water, enzymolysis for 2-3 h to obtain an enzymolysis product; (2) placing the enzymolysis product in 85-95℃ for 10-12 h to obtain an extract; (3) filtering the extract, adding magnesium chloride hexahydrate to the filtrate, standing for 20-30 min, centrifuging, taking supernatant 1, adding anhydrous ethanol to supernatant 1 to a final concentration of 15-25%, standing for 2-3 h, centrifuging, taking supernatant 2, adding anhydrous ethanol to supernatant 2 to a final concentration of 25-35%, standing for 2-3 h, centrifuging, taking supernatant 3, adding anhydrous ethanol to supernatant 3 to a final concentration of 55-65%, standing for 10-12 h, centrifuging, and collecting precipitate 1; (4) mixing precipitate 1 with water to obtain a polysaccharide solution, mixing the polysaccharide solution with a cetylpyridinium chloride solution, standing for 12-15 h, centrifuging, and collecting precipitate 2, mixing precipitate 2 with a calcium chloride solution, stirring for 6-8 h, centrifuging, and taking supernatant 4; (5) dialyzing supernatant 4, taking the polysaccharide solution in the dialysis bag, and freeze-drying to obtain algal sulfated polysaccharides.
2. The extraction method according to claim 1, characterized in that, The seaweed powder in step (1) is derived from one or more of Laminaria japonica, Sargassum fusiforme, Undaria pinnatifida and Ascophyllum nodosum; The mass-volume ratio of the seaweed powder to water in step (1) is 8-12 g: 1 L; The mass ratio of the seaweed powder to cellulase in step (1) is 8-12: 0.04-0.06; The temperature of the enzymolysis in step (1) is 45-55℃; The final concentration of magnesium chloride hexahydrate added to the filtrate in step (3) is 0.04-0.06 mol / L; The mass-volume ratio of precipitate 1 to water in step (4) is 1 g: 20-30 mL; The initial concentration of the cetylpyridinium chloride solution in step (4) is 2-4 wt%; The volume ratio of the polysaccharide solution to the cetylpyridinium chloride solution in step (4) is 3-5: 1; The initial concentration of the calcium chloride solution in step (4) is 3-5 mol / L; The volume ratio of precipitate 2 to the calcium chloride solution in step (4) is 1: 2-3; The dialysis time in step (5) is 45-52 h; The molecular weight cut-off of the dialysis bag during dialysis in step (5) is 3000-4000 Da.
3. The sulfated phycocolloid obtainable by the extraction process according to claim 1 or 2, characterized in that, The purity of the algal sulfated polysaccharides is ≥95%, and the content of sulfate groups in the algal sulfated polysaccharides is ≥22%.
4. Use of algal sulfated polysaccharides obtained by the extraction method of any one of claims 1-2 or the algal sulfated polysaccharides of claim 3 in the preparation of a medicament for treating AIDS.
5. An injection for treating AIDS, characterized in that, Using water for injection as the solvent, the following components are included at the following concentrations: 4-6 g / L lentinan, 7-9 g / L algal sulfated polysaccharides, 8-10 g / L sodium chloride, and 40-60 g / L mannitol; The algal sulfated polysaccharides are algal sulfated polysaccharides obtained by the extraction method of any one of claims 1-2 or the algal sulfated polysaccharides of claim 3.
6. The injectable solution according to claim 5, characterized in that, The purity of the lentinan is ≥98%, and the molecular weight of the lentinan is 50000-80000 Da.
7. The injection fluid according to claim 5 or 6, characterized in that The pH of the injection is 6.5-7.
5.
8. A method of preparing an injection for treating AIDS, characterized by, Comprising the following steps: The components are weighed according to any one of claims 5-7, and lentinan, fucoidin, sodium chloride, mannitol and water for injection are mixed to obtain a mixed solution; the pH of the mixed solution is adjusted to 6.5-7.5, the mixed solution is filtered through a microporous filter membrane, the filtrate is obtained, sterilized to obtain an injection.
9. The production method according to claim 8, characterized by, The stirring speed is 200-300 rpm.
10. The preparation method according to claim 8, characterized in that, The pore size of the microporous filter membrane is 0.2-0.3 μm; the sterilization temperature is 118-125 ℃, and the sterilization time is 15-20 min.