Phosphorylated bald calvatia polysaccharide-starch hemostatic microspheres as well as preparation method and application thereof
By preparing phosphorylated puffball polysaccharide-starch hemostatic microspheres, the problems of slow onset and poor efficacy of existing drugs for treating postpartum hemorrhage have been solved, achieving rapid hemostasis and promoting healing, and the material is safe and has no toxic side effects.
Patent Information
- Application Number
- CN202511556619.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-29
- Publication Date
- 2026-01-13
AI Technical Summary
Existing medications for treating postpartum hemorrhage have a long onset time and are not very effective for those with weak or insensitive uterine contractions. Existing strategies for improving coagulation function are costly or have limited effectiveness.
Phosphorylated puffball polysaccharide-starch hemostatic microspheres were prepared. By phosphorylation modification, puffball polysaccharide binds to starch microspheres, forming a physical barrier and activating coagulation factors to achieve rapid hemostasis.
It achieves rapid hemostasis, shortening the complete hemostasis time to 10-12 hours, and has an immediate hemostatic effect. It also has antioxidant, anti-inflammatory and antibacterial activities, good biocompatibility and high safety.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to a composite hemostatic material combining a polysaccharide-modified product and starch microspheres, particularly to a phosphorylated puffball polysaccharide-starch hemostatic microsphere, its preparation method, and its application in the preparation of drugs for treating postpartum hemorrhage. Background Technology
[0002] Postpartum hemorrhage (PPH) is the leading cause of maternal death worldwide. Currently, first-line treatments for PPH primarily involve drugs that promote uterine contractions, such as oxytocin, misoprostol, and carboprost tromethamine. These drugs achieve hemostasis by enhancing the contraction of uterine smooth muscle to compress blood vessels.
[0003] However, these medications have limitations in terms of onset time. Even the fastest-acting carbetocin takes about 2 minutes to take effect. Before the medication takes effect, the mother remains at risk of continuous heavy bleeding. Furthermore, the effectiveness of these medications is significantly reduced in mothers with weak uterine contractions or those who are insensitive to uterine contraction drugs.
[0004] In recent years, hemostasis strategies aimed at improving coagulation function have attracted attention. For example, lyophilized fibrinogen is used to supplement coagulation factors, and absorbable hemostatic microspheres (such as Hemospray) achieve hemostasis by forming a physical barrier and activating coagulation cascade. These methods provide new ideas for the treatment of PPH, but problems such as high cost, limited applicability, or need to be improved still exist.
[0005] *Colombia baldata* is a natural fungus whose polysaccharide components possess certain biological activities. Starch microspheres are biocompatible biomaterials that can rapidly absorb water and swell. However, there are currently no reports on the phosphorylation modification of *Colombia baldata* polysaccharides and their combination with starch microspheres for the preparation of hemostatic drugs for postpartum hemorrhage. Summary of the Invention
[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide phosphorylated puffball polysaccharide-starch hemostatic microspheres that have rapid onset of action, good hemostatic effect, and high biocompatibility. Another purpose of this invention is to provide a method for preparing the above-mentioned hemostatic microspheres. Yet another purpose of this invention is to provide the application of the above-mentioned hemostatic microspheres in the preparation of drugs for treating postpartum hemorrhage.
[0007] To achieve the above-mentioned technical objectives, the technical solution of the present invention is as follows:
[0008] In a first aspect, the present invention provides a method for preparing phosphorylated puffball polysaccharide-starch hemostatic microspheres, comprising the following steps:
[0009] S1. Phosphorylation modification of puffball polysaccharide resembling baldness:
[0010] The polysaccharide of puffball was dissolved in N,N-dimethylformamide (DMF), sodium tripolyphosphate (STMP) was added, the pH was adjusted to 8.0-10.0 with sodium hydroxide solution, and the reaction was carried out at 60-80℃. After the reaction was completed, the pH was neutralized to 7.0 with hydrochloric acid, and phosphorylated puffball polysaccharide was obtained by dialysis, ethanol precipitation, centrifugation and freeze drying.
[0011] Preferably, the molar ratio of sodium tripolyphosphate to puffball polysaccharide is 1:1 to 5:1.
[0012] S2. Activation of starch microspheres:
[0013] The carboxyl groups on the surface of moderately cross-linked starch microspheres were activated using a mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS).
[0014] S3. Coupling of phosphorylated puffball polysaccharide with activated starch microspheres:
[0015] The phosphorylated puffball polysaccharide obtained in step S1 and the starch microspheres activated in step S2 were mixed in a buffer solution of pH 10-12, reacted at 40-50℃, centrifuged, and washed with phosphate buffered saline (PBS) to obtain phosphorylated puffball polysaccharide-starch hemostatic microspheres.
[0016] Preferably, the mass ratio of the phosphorylated puffball polysaccharide to the starch microspheres is 1:1 to 1:5.
[0017] Secondly, the present invention provides phosphorylated puffball polysaccharide-starch hemostatic microspheres prepared by the above-described preparation method.
[0018] Thirdly, the present invention provides the application of the above-mentioned phosphorylated puffball polysaccharide-starch hemostatic microspheres in the preparation of hemostatic drugs.
[0019] Preferably, the hemostatic drug is used to treat postpartum hemorrhage.
[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0021] 1. This invention significantly enhances the negative charge of puffball polysaccharide by phosphorylation, thereby more effectively activating platelet aggregation and coagulation factors, resulting in strong intrinsic hemostatic activity. When combined with starch microspheres, the microspheres rapidly absorb water and swell, forming a physical barrier and generating local pressure on the bleeding wound. Simultaneously, the highly active phosphorylated polysaccharide is enriched at the bleeding site, achieving a synergistic effect of physical and biochemical hemostasis.
[0022] 2. Animal experiments have shown that the hemostatic microspheres have an immediate hemostatic effect, and the time to complete hemostasis can be shortened to 10-12 hours. Compared with the waiting time before the traditional uterine contraction agent takes effect, bleeding can be controlled earlier.
[0023] 3. Experiments have shown that the phosphorylated puffball polysaccharide of the present invention not only has excellent hemostatic function, but also has antioxidant, anti-inflammatory and antibacterial activities, which is of positive significance for preventing postpartum infection and promoting uterine wound healing.
[0024] 4. Both puffballs and starch are natural sources with good biocompatibility. Their degradation products have no toxic side effects, making them highly safe for clinical application. Detailed Implementation
[0025] Example 1: Preparation of phosphorylated puffball polysaccharide
[0026] Weigh 1.0 g of puffball polysaccharide and dissolve it in 100 mL of N,N-dimethylformamide, stirring until completely dissolved. Add sodium tripolyphosphate (STMP) at a molar ratio of 3:1, and adjust the pH to 9.0 with 2 mol / L sodium hydroxide solution. Place the reaction system in a 70°C water bath and stir for 12 hours. After the reaction is complete, neutralize to pH 7.0 with 1 mol / L hydrochloric acid. Transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyze with deionized water for 48 hours to remove small molecule impurities. After dialysis, add anhydrous ethanol to the solution to precipitate, and collect the precipitate by centrifugation (8000 rpm, 10 min). Freeze-dry the precipitate to obtain white flocculent phosphorylated puffball polysaccharide. The degree of phosphorylation substitution (DS) was determined to be 0.25.
[0027] Example 2: Activity determination of phosphorylated puffball polysaccharide
[0028] (1) Hemostatic activity assay:
[0029] The effects of different concentrations (0, 0.1, 0.5, 1.0, 2.0 mg / mL) of raw puffball polysaccharide and phosphorylated puffball polysaccharide prepared in Example 1 on rabbit plasma clotting time were determined using APTT (activated partial thromboplastin time) and PT (prothrombin time) kits. Heparin sodium (0.1 U / mL) was used as a positive control.
[0030] The hemostatic ability data of puffball polysaccharides are shown in the table below:
[0031] The hemostatic ability data of phosphorylated puffball polysaccharides are shown in the table below:
[0032] The results showed that phosphorylation modification prolonged the APTT of puffball polysaccharide, and the effect was better than that of unmodified polysaccharide, demonstrating its enhanced coagulation activity.
[0033] (2) Antibacterial activity assay:
[0034] The inhibitory effects of raw puffball polysaccharide and phosphorylated puffball polysaccharide on Staphylococcus aureus and Escherichia coli were detected using the Oxford cup method.
[0035] The antibacterial activity data of puffball polysaccharides are shown in the table below:
[0036] The antibacterial activity data of phosphorylated puffball polysaccharides are shown in the table below:
[0037] The results showed that phosphorylation modification increased the diameter of the inhibition zone of puffball polysaccharide against both bacteria, indicating that its antibacterial activity was enhanced.
[0038] Example 3: Preparation of phosphorylated puffball polysaccharide-starch hemostatic microspheres
[0039] Weigh 100 mg of moderately cross-linked starch microspheres and add them to 2-morpholinoethanesulfonic acid (pH 6.0) containing 50 mg of EDC and 30 mg of NHS. Activate the microspheres at room temperature in the dark for 1 hour. Centrifuge and discard the supernatant. Wash twice with phosphate buffer solution (pH 7.4) to obtain activated starch microspheres.
[0040] The phosphorylated puffball polysaccharide (33 mg) prepared in Example 1 was dissolved in phosphate buffer solution (pH 11.0) and mixed with the activated starch microspheres described above. The mixture was placed in a 50°C water bath and gently stirred for 1.0 hour. After the reaction, the microspheres were collected by centrifugation and washed three times with phosphate buffer solution to remove uncoupled polysaccharides. The residual polysaccharide content in the supernatant was determined by the phenol-sulfuric acid method, and the binding rate of phosphorylated puffball polysaccharide to starch microspheres was calculated to be 85.4%. The phosphorylated puffball polysaccharide-starch hemostatic microspheres were finally obtained.
[0041] Example 4: Animal experimental efficacy verification of hemostatic microspheres
[0042] (1) Select healthy adult pregnant rabbits, and after they give birth, create a standard trauma model in the uterine wall by surgery or inject oxytocin antagonists intravenously to induce a stable postpartum hemorrhage state.
[0043] (2) Pregnant rabbits were randomly divided into three groups: Experimental group: hemostatic microspheres prepared in Example 3 were sprayed onto the uterine wound. Positive control group: commercially available hemostatic gauze was packed onto the wound. Blank control group: an equal amount of physiological saline was sprayed onto the wound.
[0044] (3) Indicator detection:
[0045] Immediate bleeding volume: Bleeding volume was recorded within the first 2 minutes after treatment began. Bleeding volume in the experimental group was significantly lower than that in the two control groups.
[0046] Time to complete hemostasis: The time from the start of treatment to the complete cessation of active bleeding from the wound was recorded. The time to complete hemostasis in the experimental group was 10-12 hours, significantly shorter than that in the positive control group and the blank control group.
[0047] Coagulation function: Blood was collected before and after hemostasis to measure PT and APTT values. In the experimental group, APTT was expected to be prolonged after drug administration (indicating that systemic coagulation activity was activated) and returned to normal more quickly.
[0048] Pathological examination: Uterine tissue was taken for pathological examination 24 hours after hemostasis. The experimental group showed mild inflammatory reaction, no obvious foreign body rejection reaction or tissue necrosis, and new granulation tissue was visible.
[0049] (4) Conclusion: The phosphorylated puffball polysaccharide-starch hemostatic microspheres of the present invention showed rapid, safe and effective hemostatic effect in the postpartum hemorrhage model of pregnant rabbits, and had certain potential to promote healing.
[0050] The above embodiments do not limit the present invention in any way. All technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.
Claims
1. A phosphorylcholine-sclerotherapy microspheres of Calvatia phalloides, characterized in that, The hemostatic microspheres are formed by coupling the phosphorylated Calvatia lilacina polysaccharide and activated starch microspheres through a covalent bond.
2. A process for the preparation of phosphorylcholine pycnocomorphic Scleroderma polysaccharide-starch hemostatic microspheres as claimed in claim 1, characterized by, The method comprises the following steps: S1, dissolving the Calvatia lilacina polysaccharide in N,N-dimethylformamide, adding sodium tripolyphosphate, and reacting under alkaline conditions at 60-80 DEG C to obtain the phosphorylated Calvatia lilacina polysaccharide after purification; S2, activating the carboxyl groups on the surface of the starch microspheres by using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide; S3, mixing the phosphorylated Calvatia lilacina polysaccharide obtained in step S1 and the activated starch microspheres in an alkaline buffer solution, and reacting at 40-50 DEG C to obtain the hemostatic microspheres after washing.
3. The production method according to claim 2, characterized by, In step S1, the molar ratio of sodium tripolyphosphate to Calvatia lilacina polysaccharide is 1:1-5:
1.
4. The production method according to claim 2, characterized by, In step S3, the mass ratio of the phosphorylated Calvatia lilacina polysaccharide to the starch microspheres is 1:1-1:
5.
5. Use of the phosphorylated Calvatia lilacina polysaccharide-starch hemostatic microspheres according to claim 1 in the preparation of a hemostatic medicament.
6. Use according to claim 5, characterized in that, The hemostatic medicament is used for treating postpartum hemorrhage.