Preparation process of astragalus polysaccharide camouflage monosaccharide
By using supercritical fluid extraction and ultrasonic degradation technology, Astragalus polysaccharides are disguised as monosaccharides or oligosaccharides, solving the problems of purity and targeting of Astragalus polysaccharides and achieving highly effective anti-tumor treatment.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- 容城县正秀略文化工作室
- Filing Date
- 2026-02-10
- Publication Date
- 2026-05-19
AI Technical Summary
Existing technologies struggle to obtain high-purity Astragalus polysaccharides, and their large molecular structure leads to low targeting and uptake efficiency by tumor cells. Existing modification methods suffer from issues such as altering activity, complex processes, and high costs.
A method combining supercritical fluid extraction and ultrasonic degradation was adopted. Astragalus polysaccharides were extracted by supercritical carbon dioxide and degraded into monosaccharide or oligosaccharide structures. Ultrasonic waves were used to break specific glycosidic bonds to achieve high purity and targeted camouflage.
It significantly improved the biocompatibility and anti-tumor targeting of Astragalus polysaccharides, enhanced their ability to recognize and absorb cancer cells, activated immune cell function, reversed cancer cell drug resistance, blocked nutrient absorption, and improved therapeutic effects.
Smart Images
Figure CN122060004A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of bioproduct manufacturing technology, specifically a process for preparing Astragalus polysaccharide disguised as monosaccharide. Background Technology
[0002] Astragalus, an important qi-tonifying herb in traditional Chinese medicine, has had its active ingredient, astragalus polysaccharide, confirmed by modern pharmacological studies to possess a wide range of biological activities, including immunomodulatory, antioxidant, anti-inflammatory, and anti-tumor effects. In the field of anti-tumor research, astragalus polysaccharide has shown the potential to exert its effects through multiple pathways, such as enhancing the body's immune function, inhibiting tumor cell proliferation, and inducing tumor cell apoptosis. However, despite its promising prospects, astragalus polysaccharide still faces a series of fundamental limitations due to its own physicochemical properties in clinical translation and practical therapeutic applications.
[0003] In existing technologies, the extraction of Astragalus polysaccharides mainly employs water extraction and alcohol precipitation, hot water extraction, or enzymatic hydrolysis. These methods generally suffer from limited extraction efficiency, cumbersome processes, and long processing times. Furthermore, they struggle to completely remove proteins, pigments, and small molecule impurities, resulting in low purity and bioavailability of the final product. More critically, as a biomolecule, the complex spatial structure and large molecular weight of polysaccharides severely limit their distribution, absorption, and targeting of specific cells in vivo. Tumor cells exhibit high selectivity in nutrient uptake, with significantly higher transport and absorption efficiency for monosaccharides or small oligosaccharides compared to large polysaccharides. Therefore, unmodified natural Astragalus polysaccharides are difficult for tumor cells to effectively internalize, resulting in a weak direct effect on cancer cells and limiting their application as a direct antitumor agent.
[0004] To improve the targeting and bioavailability of polysaccharides, existing technologies have explored various physical and chemical modification methods, such as carboxymethylation, sulfation, and selenization, or their preparation into delivery systems like nanoparticles and liposomes. However, chemical modifications may introduce unpredictable toxicity or alter their inherent bioactivity profile, while complex nanocarrier fabrication processes face challenges in stability, reproducibility, and large-scale production costs. Furthermore, these strategies primarily focus on improving delivery, failing to fundamentally address the core contradiction of the mismatch between polysaccharide structure and cancer cell uptake mechanisms.
[0005] On the other hand, using monosaccharides or oligosaccharides as carriers or "disguise" shells to enhance the targeting of drugs to specific cells is one of the research directions in the field of drug delivery. However, how to precisely and controllably "disassemble" and "disguise" natural polysaccharides with clear activity into monosaccharide / oligosaccharide structures that can retain or even enhance their biological functions, while achieving green, efficient and standardized processes, is a problem that existing technologies have not been able to solve well.
[0006] In summary, existing technologies for processing Astragalus polysaccharides for highly effective anti-tumor therapy suffer from the following drawbacks: First, traditional extraction and purification techniques struggle to obtain ultra-high purity raw materials suitable for intravenous injection; second, the inherent structure of the large-molecule polysaccharide leads to low targeting and uptake efficiency by tumor cells; and third, existing structural modification or delivery system modification methods suffer from issues such as altering activity, complex processes, or high costs. Therefore, there is an urgent need to develop an innovative preparation technology that can fundamentally alter the molecular morphology of Astragalus polysaccharides, enabling them to possess high purity, high targeting, strong bioactivity, and good process feasibility. Summary of the Invention
[0007] The purpose of this invention is to provide a process for preparing Astragalus polysaccharides disguised as monosaccharides, thereby solving the problems mentioned in the background art.
[0008] To achieve the above objectives, the present invention provides a process for preparing Astragalus polysaccharides disguised as monosaccharides, comprising the following steps: S1. Extraction: Astragalus polysaccharides were extracted from Astragalus raw materials using supercritical fluid extraction technology; S2. Purification: The Astragalus polysaccharide obtained in step one is purified to meet the purity standard for intravenous injection. S3. Degradation and Disguise: The purified Astragalus polysaccharide from step two is dissolved in a solvent and subjected to ultrasonic degradation treatment to degrade specific glucan structural units in the polysaccharide chain and disguise them as monosaccharide or oligosaccharide structures. S4. Separation and product acquisition: The solution after step three is separated and dried to obtain the Astragalus polysaccharide disguised as a monosaccharide product.
[0009] Furthermore, in step one, the supercritical fluid extraction technology uses supercritical carbon dioxide as the extraction fluid and a mixed solution of ethanol and water as the entrainer.
[0010] Furthermore, in step two, the purity of the purified Astragalus polysaccharide is not less than 99.99%, and the total impurity content is not higher than 100 ppm.
[0011] Furthermore, in step three, the solvent is water for injection or phosphate buffer.
[0012] Furthermore, in step three, the ultrasonic degradation treatment has a power of 100 W to 1000 W, a frequency of 20 kHz to 100 kHz, a treatment temperature of 20°C to 50°C, and a treatment time of 10 minutes to 120 minutes.
[0013] Furthermore, in step three, the specific dextran structural unit includes glucose units linked by α-(1→4) glycosidic bonds, glucose units linked by α-(1→6) glycosidic bonds, and segments composed of glucose units linked by both types of glycosidic bonds. The ultrasonic degradation treatment can target and break the α-(1→4) and α-(1→6) glycosidic bonds in the segments, exposing the terminal α-D-glucopyran ring structure, thereby achieving structural camouflage from polysaccharide to monosaccharide or oligosaccharide.
[0014] Furthermore, in step four, the separation method includes at least one of membrane filtration, chromatographic separation, or alcohol precipitation.
[0015] Furthermore, the product is mainly an oligosaccharide or monosaccharide fragment composed of α-D-glucose pyran rings, with an average degree of polymerization between 1 and 10.
[0016] A pharmaceutical composition comprising the above-described Astragalus polysaccharide masquerading as a monosaccharide product and a pharmaceutically acceptable carrier or excipient.
[0017] The use of the above-mentioned Astragalus polysaccharide masquerading as a monosaccharide product or the above-mentioned pharmaceutical composition in the preparation of a medicine for the following purposes includes, but is not limited to: inducing apoptosis of cancer cells, activating immune cells, reversing drug resistance of cancer cells, and serving as a dendritic cell trainer to enable dendritic cells to target and recognize cancer cells with stress response and block the nutrient supply channels of cancer cells.
[0018] This invention provides a process for preparing Astragalus polysaccharides disguised as monosaccharides, which has the following beneficial effects: (1) This invention uses a combination of supercritical fluid extraction and ultrasonic degradation to successfully degrade macromolecular Astragalus polysaccharide into monosaccharide or oligosaccharide structure, which significantly improves the bioacceptability of the product. This structure is more easily recognized and taken up by the monosaccharide transport mechanism on the surface of cancer cells, thus providing a structural basis for directly acting on cancer cells and inducing their apoptosis, thereby enhancing the targeting and therapeutic effect of antitumor treatment.
[0019] (2) The monosaccharide product of the present invention, after being disguised, can effectively activate the human immune system, especially key immune cells such as dendritic cells, and enhance their ability to recognize and deal with cancer cells. At the same time, the product can be further reduced in the body to release polysaccharide fragments with the function of training immune cells, thereby achieving targeted training of dendritic cells, enabling them to continuously recognize and attack cancer cells with stress response, and forming long-term immune surveillance.
[0020] (3) The monosaccharide structure prepared by the present invention can interfere with the metabolic pathways of cancer cells and reverse their resistance to traditional chemotherapy drugs, providing a new auxiliary means for combined drug use and improving the effect of existing tumor treatment programs; in addition, by blocking the absorption channels of nutrients by cancer cells, the product can indirectly inhibit tumor growth and proliferation, and exert anti-cancer effects from the perspective of nutrient supply.
[0021] (4) The supercritical fluid extraction process used in this invention can make the purity of Astragalus polysaccharide reach the high standard required for intravenous injection, with extremely low impurity content, thereby ensuring the safety of the product and the reliability of clinical application; the high purity of polysaccharide raw materials also lays a quality foundation for subsequent controllable degradation and structural camouflage, making the consistency and bioactivity of the final product more stable.
[0022] (5) The steps of this invention are clear and the conditions are controllable, making it easy to scale up production. Ultrasonic degradation, as a physical means, avoids the use of strong chemical reagents, reduces the generation of by-products and the impact on the environment, which is in line with the development trend of green pharmaceuticals. At the same time, the product form obtained by this process is suitable for the development of various drug dosage forms and has broad application prospects and industrialization potential. Attached Figure Description
[0023] Figure 1 This is a schematic flowchart of a process for preparing Astragalus polysaccharide disguised as monosaccharide according to the present invention. Detailed Implementation
[0024] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.
[0025] Examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the invention, and should not be construed as limiting the invention.
[0026] The present invention will be further described below with reference to the accompanying drawings and embodiments: Example 1, please refer to Figure 1 A process for preparing Astragalus polysaccharides disguised as monosaccharides includes the following steps: Step 1: Raw material preparation: Take 100g of dried Astragalus root slices, grind them and pass them through a 40-mesh sieve.
[0027] Step 2, Supercritical fluid extraction: Astragalus powder is placed in a supercritical carbon dioxide extraction device, and an aqueous solution containing 10% ethanol is used as an entrainer. Extraction is carried out for 2 hours under the conditions of 35 MPa pressure and 55℃ temperature, and the extract is collected.
[0028] Step 3: Purification: The extract was filtered through a 0.22 μm microfiltration membrane and then separated using an ultrafiltration membrane (molecular weight cutoff 10 kDa). The permeate was collected. After vacuum concentration and freeze-drying, 25.2 g of high-purity Astragalus polysaccharide powder was obtained. High-performance liquid chromatography-evaporative light scattering (HPLC-ELSD) analysis showed a polysaccharide purity of 99.992% and an impurity content of 85 ppm.
[0029] Step 4: Ultrasonic Degradation of Camouflage Weigh 5.0 g of the purified Astragalus polysaccharide and dissolve it in 500 mL of phosphate buffer at pH 7.4 to prepare a 10 mg / mL solution.
[0030] The solution was placed in an ultrasonic cell disruptor with a power of 500W and a frequency of 40kHz, and degraded in a 25℃ water bath for 30, 60 and 90 minutes, respectively.
[0031] After the treatment was completed, the solution was immediately placed in an ice bath to cool and terminate the reaction.
[0032] Step 5: Product Separation: The degraded solution was passed through an ultrafiltration membrane with a molecular weight cutoff of 1 kDa, and the filtrate (mainly oligosaccharide and monosaccharide fragments) was collected. The filtrate was freeze-dried to obtain white powder products, which were labeled as products APS-M30, APS-M60, and APS-M90, respectively.
[0033] Structural characterization of the products: The raw polysaccharide and degradation products were analyzed by proton nuclear magnetic resonance (NMR) spectroscopy and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (Matrix-Assisted Laser Ionization Time-of-Flight Mass Spectrometry). The raw Astragalus polysaccharide mainly showed characteristic signals of long-chain glucans linked by α-(1→4) and α-(1→6) glycosidic bonds. In the spectrum of the degradation product APS-M60, the long-chain signal of the raw material was significantly weakened, while a distinct terminal α-D-glucopyran ring (α-D-Glcp) anodic hydrogen characteristic signal appeared (δ 5.40 ppm, J = 3.8 Hz). Mass spectrometry showed that its molecular weight distribution was concentrated between 162-1458 Da, corresponding to monosaccharide and oligosaccharide fragments with a degree of polymerization (DP) mainly of 1-9. This indicates that ultrasonic treatment effectively broke the glycosidic bonds (→4 and →6) in the polysaccharide chains, exposing a large number of terminal glucose units, achieving structural camouflage from polysaccharide to monosaccharide / oligosaccharide.
[0034] Target structure: Degrade the glucan 4 structural unit in the polysaccharide and disguise it as a monosaccharide (α-D-Glcp) structure.
[0035] Structural diagram: Original polysaccharide chain structure: →6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→ After being degraded by ultrasound, some of the structure disguises itself as a monosaccharide: α-D-Glcp.
[0036] Another structure: →4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→4) is also degraded and disguised as a monosaccharide structure.
[0037] Example 2: Comparative Experiment on the Activity of Mimicked Monosaccharides in Inducing Apoptosis in Cancer Cells To verify the camouflage effect, the inhibitory effects of high-purity Astragalus polysaccharide (APS) and the camouflaged monosaccharide product (APS-M60) prepared in Example 1 on the proliferation inhibition and apoptosis induction of artificial hepatocytes (LO2) and human hepatocellular carcinoma cells (HepG2) were compared. Cell viability was detected by CCK-8 assay, and apoptosis rate was detected by flow cytometry (Annexin V-FITC / PI double staining). The treatment concentration was 200 μg / mL, and the treatment time was 48 hours. The results are shown in the table below: Table 1: Effects of Astragalus polysaccharides and their disguised monosaccharides on cell viability and apoptosis (after 48 hours of treatment)
[0038] Conclusion: The data show that APS-M60 treated with the masquerading monosaccharide process exhibits significantly stronger inhibitory effects and apoptosis-inducing abilities against HepG2 liver cancer cells than the original polysaccharide APS (p<0.01), while showing no significant increase in toxicity to normal LO2 liver cells. This confirms that structural masquerading effectively enhances the product's targeted killing effect on cancer cells.
[0039] Example 3: Comparative Experiment on the Activating Effect of Mimicked Monosaccharides on Immune Cells The effects of the product on the maturation and activation of mouse bone marrow-derived dendritic cells (BMDCs) were evaluated in vitro. BMDCs were isolated and cultured, and stimulated with APS and APS-M60 (both at a concentration of 50 μg / mL) for 24 hours, with lipopolysaccharide (LPS, 100 ng / mL) as a positive control. The expression rate of the cell surface co-stimulatory molecule CD86 was detected by flow cytometry, and the secretion level of interleukin-12 (IL-12) in the culture supernatant was detected by enzyme-linked immunosorbent assay. The results are shown in the table below: Table 2: Effects of Astragalus polysaccharides and their masquerading monosaccharides on dendritic cell activation
[0040] Conclusion: Compared with the raw polysaccharide APS, the masquerading monosaccharide APS-M60 more effectively promotes the expression of CD86 and the secretion of IL-12 in dendritic cells, indicating a significant enhancement in its immune activation capacity. This supports its functional claim of "training dendritic cells".
[0041] Example 4: Preliminary trial of reversing drug resistance in cancer cells A549 / DDP human lung cancer cells resistant to cisplatin were used as a model. Cells were co-treated with cisplatin (5 μM) and either APS or APS-M60 (100 μg / mL) for 48 hours. Cell viability was assessed by CCK-8 assay, and changes in the half-maximal inhibitory concentration (IC50) of cisplatin were calculated to evaluate the resistance reversal effect.
[0042] Table 3: Effects of masquerading monosaccharides on cisplatin resistance in A549 / DDP cells
[0043] Note: Resistance reversal factor = IC50 of cisplatin alone / IC50 of combined treatment.
[0044] Conclusion: The combination of APS-M60 and cisplatin significantly reduced the cisplatin IC50 value of drug-resistant cancer cells, with a reversal fold approximately 1.8 times that of the raw material APS. This indicates that the masquerading monosaccharide structure has superior potential in reversing drug resistance in cancer cells.
[0045] Overall conclusion: The above embodiments demonstrate that the preparation process described in this invention successfully disguises Astragalus polysaccharides as monosaccharide / oligosaccharide structures. In in vitro experiments, this product exhibits significantly enhanced bioactivity in inducing cancer cell apoptosis, activating dendritic cells, and reversing cancer cell drug resistance, validating the beneficial effects and industrial application potential of this invention.
[0046] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.
Claims
1. A process for preparing Astragalus polysaccharides disguised as monosaccharides, characterized in that, Includes the following steps: S1. Extraction: Astragalus polysaccharides were extracted from Astragalus raw materials using supercritical fluid extraction technology; S2. Purification: The Astragalus polysaccharide obtained in step one is purified to meet the purity standard for intravenous injection. S3. Degradation and Disguise: The purified Astragalus polysaccharide from step two is dissolved in a solvent and subjected to ultrasonic degradation treatment to degrade specific glucan structural units in the polysaccharide chain and disguise them as monosaccharide or oligosaccharide structures. S4. Separation and product acquisition: The solution after step three is separated and dried to obtain the Astragalus polysaccharide disguised as a monosaccharide product.
2. The preparation process of Astragalus polysaccharide disguised as monosaccharide according to claim 1, characterized in that, In step one, the supercritical fluid extraction technology uses supercritical carbon dioxide as the extraction fluid and a mixed solution of ethanol and water as the entrainer.
3. The preparation process of Astragalus polysaccharide disguised as monosaccharide according to claim 1, characterized in that, In step two, the purity of the purified Astragalus polysaccharide is not less than 99.99%, and the total impurity content is not higher than 100 ppm.
4. The preparation process of Astragalus polysaccharide disguised as monosaccharide according to claim 1, characterized in that, In step three, the solvent is water for injection or phosphate buffer.
5. The preparation process of Astragalus polysaccharide disguised as monosaccharide according to claim 1, characterized in that, In step three, the ultrasonic degradation treatment has a power of 100 W to 1000 W, a frequency of 20 kHz to 100 kHz, a treatment temperature of 20℃ to 50℃, and a treatment time of 10 minutes to 120 minutes.
6. The preparation process of Astragalus polysaccharide disguised as monosaccharide according to claim 1, characterized in that, In step three, the specific dextran structural unit includes glucose units linked by α-(1→4) glycosidic bonds, glucose units linked by α-(1→6) glycosidic bonds, and segments composed of glucose units linked by both types of glycosidic bonds. The ultrasonic degradation treatment can target and break the α-(1→4) and α-(1→6) glycosidic bonds in the segments, exposing the terminal α-D-glucopyran ring structure, thereby achieving structural camouflage from polysaccharide to monosaccharide or oligosaccharide.
7. The preparation process of Astragalus polysaccharide disguised as monosaccharide according to claim 1, characterized in that, In step four, the separation method includes at least one of membrane filtration, chromatographic separation, or alcohol precipitation.
8. The preparation process of Astragalus polysaccharide disguised as monosaccharide according to claim 1, characterized in that, The products are mainly oligosaccharide or monosaccharide fragments composed of α-D-glucose pyran rings, with an average degree of polymerization between 1 and 10.
9. A pharmaceutical composition, characterized in that, It comprises the Astragalus polysaccharide masquerading as a monosaccharide product as described in claim 8, and a pharmaceutically acceptable carrier or excipient.
10. The use of the Astragalus polysaccharide masquerading as a monosaccharide product according to claim 8 or the pharmaceutical composition according to claim 9 in the preparation of a medicament for the following purposes includes, but is not limited to: inducing apoptosis of cancer cells, activating immune cells, reversing drug resistance of cancer cells, and serving as a dendritic cell trainer to enable dendritic cells to target and recognize cancer cells with stress responses and to block the nutrient supply channels of cancer cells.