Metal ion cross-linked alkali-soluble pachymaran hydrogel bead as well as preparation method and application of metal ion cross-linked alkali-soluble pachymaran hydrogel bead

Abstract

The invention belongs to the field of biopolymer materials, and particularly relates to a metal ion cross-linked alkali-soluble pachymaran hydrogel bead as well as a preparation method and application of the metal ion cross-linked alkali-soluble pachymaran hydrogel bead. The alkali-soluble pachymaran solution is dropwise added into the metal ion solution, the pachymaran hydrogel beads cross-linked with various metal ions can be obtained through cross-linking, the pachymaran hydrogel beads cross-linked with the metal ions are good in coagulation performance, red blood cells can be coagulated on the surface of a material, and coagulation hindering caused by excessive dispersion of blood is avoided; no extra cross-linking agent needs to be added in the preparation of the hydrogel bead, the toxicity of a chemical cross-linking agent is avoided, and the hydrogel bead has good biocompatibility and excellent antibacterial performance. As a novel polysaccharide medical material, the pachymaran hydrogel bead has a good application prospect.

Description

Technical Field

[0001] The present application belongs to the field of biopolymer materials, and more specifically, relates to a metal ion cross-linked alkali-soluble Poria cocos polysaccharide hydrogel bead and its preparation method and application. Background Art

[0002] Rapid and effective hemostatic methods can significantly improve the survival rate of patients with traumatic bleeding. Therefore, finding an effective hemostatic material is a clinical problem that needs to be solved urgently. Commonly used hemostatic materials in clinical practice usually have the following problems: the structure is relatively loose, making it difficult to fit tightly to the irregular wound surface, and excessive swelling in a moist environment causes structural collapse; the operation is complicated, and coagulation relies on physical compression and lacks active coagulation function, which is disadvantageous when dealing with patients with coagulation dysfunction; the lack of antibacterial effect and poor biocompatibility make it easy to develop secondary infection in the later stage, affecting wound recovery. Natural polysaccharide hemostatic materials have attracted widespread attention due to their unique bioactivity, biocompatibility, and low cost and green characteristics.

[0003] Poria cocos is a traditional Chinese medicine with both medicinal and edible properties, with a long history of use in my country. Poria cocos polysaccharides, comprising 70%-90% of the dry weight of the sclerotium, are the primary component and active substance in the plant. Based on solubility, Poria cocos polysaccharides can be divided into two categories: water-soluble and alkali-soluble. Poria cocos alkali-soluble polysaccharides (PCAPs) account for over 80% of the total Poria cocos polysaccharide content. In the preparation of traditional Chinese medicine, Poria cocos is typically decocted with water as a solvent, which allows the utilization of water-soluble polysaccharides while leaving a significant amount of PCAP in the medicinal residue and being discarded. However, in Poria cocos research and industrial applications, a widespread focus on water-soluble polysaccharides and a neglect of alkali-soluble polysaccharides is common, leading to significant waste of PCAPs. Summary of the Invention

[0004] In response to the defects of the prior art, the purpose of this application is to provide a metal ion cross-linked alkali-soluble Poria cocos polysaccharide hydrogel beads and a preparation method and application thereof. By adding an alkali-soluble Poria cocos polysaccharide solution dropwise to a metal ion solution, Poria cocos polysaccharide hydrogel beads cross-linked with multiple metal ions can be cross-linked to obtain the PCAP hydrogel beads referred to in the present invention. The PCAP hydrogel beads have good coagulation properties and can cause red blood cells to coagulate on the surface of the material, avoiding the obstruction of coagulation caused by excessive dispersion of blood. The preparation of the hydrogel beads of the present invention does not require the addition of additional cross-linking agents, avoiding the toxicity of chemical cross-linking agents, and has good biocompatibility and excellent antibacterial properties.

[0005] To achieve the above objectives, in a first aspect, the present application provides a method for preparing metal ion cross-linked alkali-soluble Pachymaran hydrogel beads, comprising the following steps: (1) dissolving alkali-soluble Poria cocos polysaccharide in alkali solution to obtain an alkali-soluble Poria cocos polysaccharide solution; (2) adding the alkali-soluble pachymaran solution dropwise to an aqueous solution of a soluble metal salt, allowing it to stand for stable solidification to obtain hydrogel beads, and then washing to remove free metal ions on the surface of the hydrogel beads, thereby obtaining the metal ion-crosslinked alkali-soluble pachymaran hydrogel beads; The concentration of pachymaran in the alkali-soluble pachymaran solution is 150-200 g / L, and the mass volume concentration of the metal salt in the aqueous solution of the soluble metal salt is 2-25% (w / v).

[0006] Preferably, the preparation method of the alkali-soluble Pachymaran comprises the following steps: Extracting the Poria cocos powder with ethanol, filtering and drying the residue to obtain the primary Poria cocos residue; boiling the primary Poria cocos residue with water, filtering and drying the residue to obtain secondary Poria cocos residue; The secondary Poria residue is extracted with alkali solution, the residue is removed by filtration, the obtained filtrate is acidified to adjust the pH, and the filtrate is allowed to stand to obtain a precipitate; The precipitate is desalted with pure water and then dried to obtain alkali-soluble Pachymaran.

[0007] Preferably, the concentration of the alkali solution in step (1) is 0.3-1 mol / L.

[0008] Further preferably, in step (1), the alkali-soluble pachymaran is dissolved in an alkali solution, and bubbles are removed by centrifugal separation to obtain an alkali-soluble pachymaran solution.

[0009] Preferably, the metal ions contained in the aqueous solution of the soluble metal salt are Ca 2+ Mg 2+ 、Fe 3+ 、Mn 2+ and Cu 2+ One or more of .

[0010] Preferably, the dripping speed in step (2) is 0.4-1 mL / min, and the dripping height is 6-12 cm.

[0011] Preferably, the step (2) is to allow the mixture to stand for stable solidification, and the standing time is 10 to 60 minutes.

[0012] According to another aspect of the present invention, provided are metal ion cross-linked alkali-soluble Pachymaran hydrogel beads prepared by the preparation method.

[0013] According to another aspect of the present invention, there is provided a use of the metal ion cross-linked alkali-soluble Pachymaran hydrogel beads as or in the preparation of a hemostatic product.

[0014] In general, the above technical solutions conceived by this application have the following beneficial effects compared with the existing technologies: (1) The present invention proposes to prepare polysaccharide hydrogel beads using natural polysaccharide alkali-soluble Poria cocos polysaccharide PCAP as raw material to cross-link metal ions. Such PCAP hydrogel beads have good swelling properties, excellent in vitro and in vivo coagulation properties, and good cell adhesion.

[0015] (2) The preparation of the metal ion cross-linked alkali-soluble Poria cocos polysaccharide hydrogel beads of the present invention does not require chemical modification, the addition of additional cross-linking agents, or the compounding of other matrices, and the preparation process is simple and efficient. The hydrogel beads have significant coagulation and hemostasis effects. Using a mouse liver injury model, it was found that the amount of bleeding of the alkali-soluble Poria cocos polysaccharide hydrogel beads cross-linked with calcium ions, magnesium ions, and zinc ions was significantly less than that of the commercially available Celox hemostatic powder. Antibacterial experiments showed that the PCAP hydrogel beads have excellent anti-Escherichia coli and Staphylococcus aureus effects. Therefore, the metal ion cross-linked alkali-soluble Poria cocos polysaccharide hydrogel beads have good application prospects as a new type of polysaccharide medical material.

[0016] (3) The preparation method of the metal ion-crosslinked Poria cocos polysaccharide hydrogel beads of the present invention is simple, environmentally friendly, and has low production cost. It can be prepared in large quantities by injection via a syringe pump and is expected to be used in the field of pharmaceutical biomaterials. This invention provides a new approach to the resource utilization of Poria cocos residues in traditional Chinese medicine, while promoting the innovative development of natural polysaccharide-based biomaterials. It has far-reaching significance in the field of natural polysaccharide hemostatic materials and recycling and environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work. The significance of the differences between the groups was tested by one-way analysis of variance (ANOVA) and Tukey's multiple comparison test (** p <0.05), and the same letters indicate no significant difference.

[0018] Figure 1 Macroscopic photographs of PCAP hydrogel beads in Examples 1 to 6 of the present invention; Figure 1 (Content a-Content f) Macroscopic photographs of PCAP-Ca, PCAP-Mg, PCAP-Zn, PCAP-Fe, PCAP-Mn, and PCAP-Cu hydrogel beads, respectively.

[0019] Figure 2 1 is a scanning electron micrograph of the PCAP hydrogel beads in Examples 1 to 6 of the present invention; Figure 2(Content a-Content f) are scanning electron microscope images of PCAP-Ca, PCAP-Mg, PCAP-Zn, PCAP-Fe, PCAP-Mn, and PCAP-Cu, respectively.

[0020] Figure 3 FTIR spectra of the PCAP hydrogel beads in Examples 1 to 6 are shown.

[0021] Figure 4 The swelling properties of the PCAP hydrogel beads in Examples 1 to 6 are shown.

[0022] Figure 5 The coagulation index graphs of the PCAP hydrogel beads in Examples 1 to 6 are shown, wherein the gauze group did not coagulate over time due to excessive blood absorption by the gauze.

[0023] Figure 6 The hemostatic effects of the PCAP hydrogel beads in Examples 1 to 6 in the mouse liver bleeding model are demonstrated.

[0024] Figure 7 Graphs showing the hemostasis data of the PCAP hydrogel beads in Examples 1 to 6 in a mouse liver bleeding model are shown. Item a is the time required for different PCAP hydrogel beads to stop bleeding in the model, and item b is the amount of bleeding when different PCAP hydrogel beads stop bleeding in the model. Figure 8 The effects of PCAP hydrogel beads on erythrocyte adhesion in Examples 1 to 6 are demonstrated.

[0025] Figure 9 The cytotoxicity test results of the PCAP hydrogel beads in Examples 1 to 6 are shown.

[0026] Figure 10 The antibacterial experimental results of the PCAP hydrogel beads in Examples 1 to 6 are shown. Content a is the anti-Escherichia coli effect of the PCAP hydrogel beads after 24 hours of co-culture, and content b is the anti-Staphylococcus aureus effect of the PCAP hydrogel beads after 24 hours of co-culture.

[0027] Figure 11 These are photos of the product forms obtained in Comparative Examples 1 to 5, content a is Comparative Example 1, content b is Comparative Example 2, content c is Comparative Example 3, content d is Comparative Example 4, and content e is Comparative Example 5.

[0028] Figure 12 Comparative Examples 6 to 11 respectively use an aqueous solution of Inonotus obliquus polysaccharide, an aqueous solution of Poria cocos water-soluble polysaccharide, and an alkaline solution of Lentinan to dropwise add to ferric chloride and calcium chloride solutions, to obtain product photos. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solutions and advantages of this application more clear, the following further describes this application in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit this application.

[0030] The present invention provides a method for preparing metal ion cross-linked alkali-soluble Pachymaran hydrogel beads, comprising the following steps: (1) dissolving alkali-soluble Poria cocos polysaccharide in alkali solution to obtain an alkali-soluble Poria cocos polysaccharide solution; (2) The alkali-soluble tuckahoe polysaccharide solution is added dropwise to an aqueous solution of a soluble metal salt, and allowed to stand for stable solidification to obtain hydrogel beads, which are then washed to remove free metal ions on the surface of the hydrogel beads, thereby obtaining the metal ion-crosslinked alkali-soluble tuckahoe polysaccharide hydrogel beads.

[0031] Among them, the concentration of Poria cocos polysaccharide in the alkali-soluble Poria cocos polysaccharide solution is 150~200 g / L, and the concentration of metal salt in the aqueous solution of soluble metal salt is 2~25% (w / v), indicating that each 100 mL of solution contains 2~25 g of metal salt, preferably 5~20% (w / v).

[0032] In some embodiments of the present invention, PCAP is prepared using the method described in patent application number CN113861449A , wherein the extracted PCAP is dissolved in an alkaline solution, then injected into a metal ion solution via a syringe pump. After stabilization, the excess metal ions are washed away and dried. In some embodiments, deionized water is used to wash away excess metal ions from the surface of the gel beads.

[0033] In some embodiments, the method for preparing the alkali-soluble Pachymaran comprises the following steps: Extracting the Poria cocos powder with ethanol, filtering and drying the residue to obtain the primary Poria cocos residue; boiling the primary Poria cocos residue with water, filtering and drying the residue to obtain secondary Poria cocos residue; The secondary Poria residue is extracted with alkali solution, the residue is removed by filtration, the obtained filtrate is acidified to adjust the pH, and the filtrate is allowed to stand to obtain a precipitate; The precipitate is desalted with pure water and then dried to obtain alkali-soluble Poria cocos polysaccharide (abbreviated as PCAP).

[0034] More specifically, in some embodiments of the present invention, the steps for extracting alkali-soluble Poria cocos polysaccharide from Poria cocos medicinal materials are as follows: (1) Grind the Poria cocos, pass it through a 40-mesh sieve, soak it in 100% ethanol for 12 hours, and then dry the filter residue to obtain the primary Poria cocos residue; boil the primary Poria cocos residue in 20 times the amount of water for 2 hours, filter and discard the filtrate, repeat this process three times, and dry the filter residue to obtain the secondary Poria cocos residue; (2) The secondary Poria residue was dispersed in 1 mol / L sodium hydroxide solution at a material-liquid weight ratio of 1:30, stirred at room temperature, filtered to remove the residue, and the filtrate was added with 1 mol / L hydrochloric acid to adjust the pH to 7. After standing for 1 hour, the precipitate was desalted with pure water and dried to obtain alkali-soluble Poria polysaccharide.

[0035] In some embodiments, the concentration of the alkali solution used to dissolve the alkali-soluble tuckahoe polysaccharide is 0.3 to 1 mol / L. Step (1) dissolves the alkali-soluble tuckahoe polysaccharide in the alkali solution and removes bubbles by centrifugation to obtain an alkali-soluble tuckahoe polysaccharide solution. The purpose of removing bubbles is to obtain hydrogel beads with better molding quality.

[0036] The soluble metal salt of the present invention refers to a metal salt that is easily soluble in water. The metal ions contained in the aqueous solution of the soluble metal salt include but are not limited to Ca 2+ Mg 2+ 、Fe 3+ 、Mn 2+ and Cu 2+ The soluble metal salt may be one or more of soluble metal chlorides, soluble metal sulfates, soluble metal nitrates and soluble metal acetates; soluble metal chlorides include calcium chloride, magnesium chloride, zinc chloride, ferric chloride, manganese chloride and copper chloride; soluble metal sulfates include ferric sulfate, zinc sulfate, manganese sulfate and copper sulfate; soluble metal nitrates include calcium nitrate, magnesium nitrate, ferric nitrate, zinc nitrate, manganese nitrate and copper nitrate; soluble metal acetates include calcium acetate, magnesium acetate, ferric acetate, zinc acetate, manganese acetate and copper acetate.

[0037] In some embodiments, in order to obtain hydrogel beads with better forming quality, the dripping speed in step (2) is 0.4-1 mL / min, and the dripping height is 6-12 cm. In step (2) of the present invention, a syringe pump, a peristaltic pump, etc. can be used to drip the alkali-soluble Poria cocos polysaccharide solution into the aqueous solution of the metal chloride. During the experiment, it was found that the concentration of the alkali-soluble Poria cocos polysaccharide solution, the concentration of the metal ion solution, the dripping speed of the alkali-soluble Poria cocos polysaccharide solution, and the dripping height (or dripping height) all need to be controlled within an appropriate range, otherwise hydrogel beads cannot be obtained.

[0038] In some embodiments, the step (2) allows the material to stand for stable solidification, and the standing time is 10 to 60 minutes.

[0039] The metal ion cross-linked alkali-soluble Poria cocos polysaccharide hydrogel beads prepared in some embodiments of the present invention have a wet bead particle size of 1.5 to 3 mm and a dry bead particle size of 0.5 to 1 mm. The metal ion cross-linked alkali-soluble Poria cocos polysaccharide hydrogel beads provided by the present invention can be used as a hemostatic product, or can be compounded with other ingredients to prepare a hemostatic product. The hydrogel beads themselves have a good coagulation effect and a good antibacterial effect. Therefore, the hemostatic product prepared using the gel beads of the present invention can quickly stop bleeding and also has an antibacterial protection effect.

[0040] The inventors discovered that PCAP can uniquely crosslink with metal ions to form hydrogel beads (PCAP hydrogel beads), which exhibit excellent hemostatic effects. They combine the biocompatibility of natural polysaccharides with the excellent antimicrobial properties and biological characteristics of metal ions. PCAP hydrogel beads are quick and easy to prepare, exhibit excellent coagulation properties, and possess a certain mechanical strength, enabling red blood cells to aggregate on the surface, preventing clotting caused by excessive blood dispersion. The hydrogel beads of the present invention do not require the addition of additional crosslinkers, avoiding the associated toxicity of chemical crosslinkers. They exhibit good biocompatibility and excellent antimicrobial properties. These Poria cocos polysaccharide hydrogel beads hold great promise as a novel polysaccharide medical material.

[0041] The alkali solution described in the above technical solution is an alkali metal hydroxide, and sodium hydroxide solution is used in the following examples.

[0042] The metal ion solutions described in the above technical solutions are all chloride solutions of the corresponding metals.

[0043] The PCAP alkaline solution described in the above technical solution should be maintained within a certain concentration. When the concentration is too low, the PCAP hydrogel beads are difficult to form, and when the concentration is too high, the PCAP hydrogel beads are prone to tailing.

[0044] The embodiments of the present invention are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and processes are given. However, the protection scope of the present invention is not limited to the following embodiments. The process parameters in the following embodiments that do not specify specific conditions are generally based on conventional conditions.

[0045] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to form one or more new numerical ranges, and these numerical ranges should be considered to be specifically disclosed in the present invention.

[0046] The process parameters in the following examples where no specific conditions are specified are generally based on conventional conditions.

[0047] The embodiments of the present application are described below in conjunction with the drawings in the embodiments of the present application.

[0048] Example 1 Preparation of PCAP-Fe hydrogel beads.

[0049] (1) Dissolve PCAP in a 0.5 mol / L sodium hydroxide aqueous solution at room temperature and vortex thoroughly until fully dissolved to obtain a 190 g / L PCAP base solution (i.e., the concentration of PCAP in the PCAP base solution is 190 g / L). Centrifuge the PCAP base solution at 1000 rpm for 2 min to remove bubbles, then load it into a syringe and replace the needle for later use.

[0050] (2) Take ferric chloride, place it in a beaker, and add pure water to dissolve it. The concentration of ferric chloride in the resulting metal ion solution is 10% (w / v).

[0051] (3) The PCAP alkaline solution was dripped into the metal ion solution using a syringe pump at a dripping rate of 0.6 mL / min and a dripping height (i.e., the vertical distance between the syringe needle tip and the surface of the metal ion solution) of 8 cm. The solution was then allowed to stand for 30 minutes and the PCAP-Fe hydrogel beads were obtained after washing.

[0052] Example 2 Preparation of PCAP-Ca hydrogel beads.

[0053] PCAP-Ca hydrogel beads were prepared in the same manner except that the ferric chloride in Example 1 was replaced with calcium chloride.

[0054] Example 3 Preparation of PCAP-Mg hydrogel beads.

[0055] PCAP-Mg hydrogel beads were prepared in the same manner except that the ferric chloride in Example 1 was replaced by magnesium chloride.

[0056] Example 4 Preparation of PCAP-Zn hydrogel beads.

[0057] PCAP-Zn hydrogel beads were prepared in the same manner except that the ferric chloride in Example 1 was replaced by zinc chloride.

[0058] Example 5 Preparation of PCAP-Mn hydrogel beads.

[0059] PCAP-Mn hydrogel beads were prepared in the same manner except that the ferric chloride in Example 1 was replaced with manganese chloride.

[0060] Example 6 Preparation of PCAP-Cu hydrogel beads.

[0061] PCAP-Cu hydrogel beads were prepared in the same manner except that the ferric chloride in Example 1 was replaced by copper chloride.

[0062] Use a digital camera to record the appearance of the PCAP hydrogel beads in the above example. Its appearance features are as follows: Figure 1 (af) shown. Figure 1 Contents a, b, c, d, e, and f are macroscopic images of PCAP hydrogel beads corresponding to different metal ions in Examples 1 to 6, respectively. PCAP hydrogel beads cross-linked with different metal ions exhibit distinct colors and appearances. Testing revealed wet bead diameters ranging from 1.5 to 3 mm, while freeze-dried dry beads have a diameter of 0.5 to 1 mm.

[0063] SEM scanning was performed on the PCAP hydrogel beads corresponding to different metal ions in Examples 1 to 6. The prepared PCAP hydrogel beads were placed on a stage and carbon-coated using a Cressington 108 carbon / A sample preparation station. The vacuum was less than 0.05 mbar, the excitation voltage was 5.3 V, and the sample coating thickness was 20 nm. The coated samples were then placed in a scanning electron microscope (SEM) to observe their micromorphology at an accelerating voltage of 10 kV.

[0064] Figure 2 Content a, content b, content c, content d, content e and content f are SEM scanning microstructure images of the PCAP hydrogel beads corresponding to different metal ions in Examples 1 to 6, respectively. It can be seen that a dense network structure can be observed in the PCAP hydrogel beads after being magnified by an electron microscope, which provides conditions for the aggregation and agglomeration of red blood cells.

[0065] ATR-FTIR tests were performed on the PCAP hydrogel beads corresponding to different metal ions in Examples 1 to 6. The test method was as follows: the prepared PCAP hydrogel beads were placed on the ATR sample stage, a layer of tin foil was lightly pressed on the PCAP hydrogel beads, and then the stage was pressed tightly. No special treatment was required for the sample. The test conditions were: scanning range 4000-600 cm -1 , the number of scans is 16 times, and the resolution is 2cm -1 . Figure 3 ATR-FTIR scan of PCAP hydrogel beads prepared with a 10% (w / v) metal ion concentration. It can be seen that the infrared peaks of PCAP change after complexation, either by shifting the peak position or generating new peaks, indicating that the metal ions are not simply adsorbed and embedded in PCAP.

[0066] Swelling tests were performed on the PCAP hydrogel beads corresponding to different metal ions in Examples 1 to 6. The test method was as follows: PCAP hydrogel beads were placed in a 37°C water bath using a PBS solution with a pH of 7.4 as the swelling medium. 10 mg of PCAP hydrogel beads were placed in a 5 mL EP tube and 1.5 mL of PBS solution was added. After a predetermined time, the PBS solution was aspirated, and any remaining PBS solution on the surface of the PCAP hydrogel beads was gently absorbed with filter paper. The beads were weighed and the swelling ratio was calculated. Figure 4 The figure shows the swelling curves of PCAP hydrogel beads corresponding to different metal ions. Within 20 minutes, some groups of PCAP hydrogel beads can reach a swelling rate of 300~400%, indicating that PCAP hydrogel has the ability to quickly absorb a large amount of liquid, can make red blood cells cluster, and promote coagulation. In addition, Figure 4 It can also be seen that the hydrogel beads corresponding to different metal ions have different swelling properties under the same conditions, indicating that the type of metal ions affects the cross-linking structure of the hydrogel and its swelling behavior.

[0067] In vitro coagulation test. The test method involves adding 300 μL of 0.15 mol / L calcium chloride solution to 1 mL of anticoagulated rabbit whole blood in a 37°C water bath. The sample is checked every 10 seconds. The blood clotting time is recorded, based on the standard of remaining still when the glass tube is tilted 180° and inverted. This represents the self-clotting time of the blank rabbit blood sample without any coagulation material. 5 mg of PCAP hydrogel beads corresponding to different metal ions are transferred to a glass tube. After adding 1 mL of anticoagulated whole blood, 300 μL of 0.15 mol / L calcium chloride solution is immediately added to initiate coagulation. The blood clotting time is recorded, representing the clotting time of the PCAP hydrogel bead group. The test results are shown in Table 1.

[0068] Table 1 Test results of coagulation time of different hydrogel bead samples

[0069] Table 1 shows that the coagulation time of the blank group was approximately 592 s. Except for the PCAP-Cu hydrogel beads, whose coagulation time was approximately 577 s, the hydrogel beads containing other ions all showed a significant effect of promoting coagulation. According to the literature, copper ions usually exhibit an anticoagulant effect, but the network structure within the hydrogel beads can offset the anticoagulant effect of copper ions to a certain extent, so that they still have a certain coagulation effect compared with the blank group.

[0070] Blood clotting index (BCI) experiment. The test method is as follows: 10 mg of PCAP hydrogel beads corresponding to different metal ions are placed in 15 mL centrifuge tubes, and the anticoagulated rabbit blood and calcium chloride solution are preheated at 37 ° C for 5 minutes. Then, 100 μL of anticoagulated whole blood is added to the sample, followed by 30 μL of 0.15 mol / L calcium chloride solution, and finally it is incubated at 37 ° C for 6 minutes. 10 mL of deionized water is slowly added to the blood clot, and the action is gentle, so as not to break the blood clot. Then, 100 μL of the supernatant is taken to a 96-well plate, and its absorbance is measured at 540 nm by a microplate reader. The BCI value (Blood clotting index) is calculated according to the formula:

[0071] Figure 5 Figure 2 shows the BCI results of different PCAP hydrogel beads. The larger the BCI value, the worse the coagulation effect. It can be seen that the hydrogel beads in each group of the examples showed significant coagulation effects compared to the control group, among which PCAP-Zn, PCAP-Fe and PCAP-Ca had the best coagulation effects.

[0072] Hemolysis test. The test method is as follows: immerse 20 mg of PCAP hydrogel beads in 10 mL of PBS and incubate at 37°C for 24 h to obtain an extract. Take 1 mL of the extract and place it in a 1.5 mL centrifuge tube, add 100 μL of fresh anticoagulated blood, and mix thoroughly. After incubation at 37°C for 1 h, centrifuge the mixture of anticoagulated blood and the extract (3000 rpm, 10 min), and take 200 μL of the supernatant to a 96-well plate for testing. Measure the absorbance at 540 nm using an enzyme reader. PBS is used as a negative control, and water is selected as a positive control. The clinical approval of medical materials requires that the hemolysis rate does not exceed 5%.

[0073] 00% The experimental results are as follows Figure 6 As shown, the results showed that the positive control group had a bright red blood color, and the added blood sample was completely hemolyzed, with a hemolysis rate of 100%. The bottom of the PCAP hydrogel bead centrifuge tubes prepared in Examples 1 to 6 were all non-hemolyzed red blood cells, and the supernatant was transparent. The hemolysis rate was less than 5%, meeting the clinical licensing requirements for medical materials.

[0074] Animal model experiment. The experimental method is as follows: mice are anesthetized by intraperitoneal injection of 20% urethane at a dose of 0.6 mL / 100 g, their abdominal hair is trimmed, the abdomen is exposed and disinfected with alcohol cotton balls. The abdominal cavity is opened and the mouse liver lobe is exposed. The original weight of the sterile filter paper is recorded, and then it is placed under the open exposed liver. A 1 cm bleeding wound is made on the liver lobe with a scalpel. 20 mg of PCAP hydrogel beads are taken to cover the wound and press it with appropriate pressure. The bleeding situation is observed every 5-15 seconds until the bleeding stops, and pre-weighed sterile filter paper is used to absorb the uncoagulated blood. The pre-weighed sterile filter paper is measured to calculate the amount of bleeding, the clotting time is recorded, and it is recorded with a camera. Figure 7 The effect of mouse liver bleeding model, Figure 7 Content a is the time required for different PCAP hydrogel beads to stop bleeding in the model, and content b is the amount of bleeding when different PCAP hydrogel beads stop bleeding in the model.

[0075] Red blood cell adhesion test: Blood was dripped onto the surface of the PCAP hydrogel beads. After complete coagulation, the excess blood clot was rinsed with PBS. The red blood cells on the sample were fixed with paraformaldehyde overnight. The excess paraformaldehyde was then washed with PBS. The sample was further dehydrated with 70%, 85%, and 100% ethanol solutions for 10 minutes each. After air drying, the sample was carbonized and observed according to the above-mentioned SEM conditions. Figure 8 The SEM image of red blood cells adhered to PCAP hydrogel beads. Figure 8 It can be seen that the morphology of red blood cells on the surfaces of PCAP-Fe, PCAP-Ca, PCAP-Mg, and PCAP-Zn is normal without obvious deformation.

[0076] Cytotoxicity Assay. The CCK-8 assay was used to evaluate the cytotoxicity of each PCAP hydrogel bead against L929 cells. Sterilized PCAP hydrogel beads were immersed in L929 culture medium (MEM + 10% FBS + 1% P / S) and incubated at 37°C for 24 hours. The extract concentration was 1 mg / mL. L929 cells were then seeded at a density of 10,000 cells per well in a 96-well plate and incubated at 37°C overnight. The culture medium was then replaced with the prepared hydrogel bead extract and incubated again at 37 ± 1°C for 24 hours. Subsequently, CCK-8 solution was added to each well and incubated at 37 ± 1°C for 3 hours. Cell viability was assessed by measuring the OD value of the co-culture supernatant at 450 nm using a microplate reader. Figure 9 Figure 2 is the cytotoxicity result of PCAP hydrogel beads. It can be seen that PCAP-Fe, PCAP-Ca, and PCAP-Mg have less cytotoxicity.

[0077] Antibacterial test. The experimental method is: Escherichia coli ( E. coli ) and Staphylococcus aureus ( S. aureus) were inoculated into LB liquid medium and cultured at 37°C with shaking until the logarithmic growth phase. Then, completely sterilized PCAP hydrogel beads were immersed in 2 mL of liquid culture medium at a concentration of 35 mg / mL. 20 μL of bacterial culture solution was then added and co-cultured at 37°C with shaking for 24 hours. The BLANK group remained unincubated, while the POSITIVE group received ampicillin and Celox, a commercially available hemostatic powder. The 24-hour co-culture solution was evenly spread onto solid culture medium and incubated at 37°C (40% humidity). After 12 hours, the bacterial colonies on the culture medium were observed for growth. Figure 10 Content a shows the anti-E. coli effect of PCAP hydrogel beads after 24 hours of co-culture, and content b shows the anti-Staphylococcus aureus effect of PCAP hydrogel beads after 24 hours of co-culture. It can be seen that the anti-E. coli effect of PCAP hydrogel beads in each group of the examples is better than that of the commercially available Celox hemostatic powder.

[0078] Comparative Example 1 Other aspects are the same as in Example 2, except that the concentration of the PCAP alkaline solution is 10 g / L.

[0079] Comparative Example 2 Other aspects are the same as in Example 2, except that the concentration of the PCAP alkaline solution is 210 g / L.

[0080] Comparative Example 3 The other aspects are the same as those of Example 2, except that the concentration of calcium chloride is 1% (w / v).

[0081] Comparative Example 4 The other aspects are the same as those of Example 2, except that the concentration of calcium chloride is 30% (w / v).

[0082] Comparative Example 5 The other steps were the same as those in Example 2, except that the dripping rate was 1.5 mL / min.

[0083] Use a digital camera to record photos of the product forms obtained in Comparative Examples 1 to 5, such as Figure 11 From left to right, these correspond to Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, and Comparative Example 5. It can be seen that no hydrogel beads were obtained in Comparative Examples 1 to 5. This indicates that complete hydrogel beads cannot be obtained if the concentration of the PCAP alkaline solution during instillation is too high or too low, or the concentration of the metal ion solution is too high or too low, or the instillation speed is too fast.

[0084] Comparative Example 6 The other steps were the same as in Example 1, except that step (1) was as follows: at room temperature, Inonotus obliquus polysaccharide was dissolved in deionized water and vortexed thoroughly until fully dissolved to obtain a 200 g / L aqueous solution of Inonotus obliquus polysaccharide. The aqueous solution of Inonotus obliquus polysaccharide was centrifuged at 1000 rpm for 2 min to remove bubbles, and then loaded into a syringe, and the needle was replaced for later use.

[0085] Comparative Example 7 The other steps were the same as those in Example 2, except that step (1) was as follows: at room temperature, Inonotus obliquus polysaccharide was dissolved in deionized water and vortexed thoroughly until fully dissolved to obtain a 200 g / L aqueous solution of Inonotus obliquus polysaccharide. The aqueous solution of Inonotus obliquus polysaccharide was centrifuged at 1000 rpm for 2 min to remove bubbles, and then loaded into a syringe, and the needle was replaced for later use.

[0086] Comparative Example 8 The other steps were the same as those in Example 1, except that step (1) was as follows: at room temperature, the water-soluble polysaccharide of Poria cocos was dissolved in deionized water and vortexed thoroughly until fully dissolved to obtain a 200 g / L aqueous solution of the water-soluble polysaccharide of Poria cocos. The aqueous solution of the water-soluble polysaccharide of Poria cocos was centrifuged at 1000 rpm for 2 min to remove bubbles, and then loaded into a syringe, and the needle was replaced for later use.

[0087] Comparative Example 9 The other steps were the same as those in Example 2, except that step (1) was as follows: at room temperature, the water-soluble polysaccharide of Poria cocos was dissolved in deionized water and vortexed thoroughly until fully dissolved to obtain a 200 g / L aqueous solution of the water-soluble polysaccharide of Poria cocos. The aqueous solution of the water-soluble polysaccharide of Poria cocos was centrifuged at 1000 rpm for 2 min to remove bubbles, and then loaded into a syringe, and the needle was replaced for later use.

[0088] In Comparative Examples 8 and 9, the water-soluble Poria cocos polysaccharide was prepared as follows: Poria cocos was soaked in 8 times the weight of ethanol, condensed and refluxed for 1 hour, impurities were removed, the Poria cocos was recovered, 8 times the weight of water was added, condensed and refluxed for 1 hour, repeated three times, and the reflux solution was collected. The reflux solution was concentrated and freeze-dried to obtain the polysaccharide.

[0089] Comparative Example 10 The other procedures were the same as in Example 1, except that step (1) was as follows: at room temperature, lentinan (commercially available, source leaf, CAS# 37339-90-5) was dissolved in a 0.5 mol / L sodium hydroxide aqueous solution and vortexed thoroughly until fully dissolved to obtain a 200 g / L lentinan alkaline solution. The lentinan alkaline solution was centrifuged at 1000 rpm for 2 min to remove bubbles, then loaded into a syringe, and the needle was replaced for later use.

[0090] Comparative Example 11 The other procedures were the same as those in Example 2, except that step (1) was as follows: at room temperature, lentinan (commercially available, source leaf, CAS# 37339-90-5) was dissolved in a 0.5 mol / L sodium hydroxide aqueous solution and vortexed thoroughly until fully dissolved to obtain a 200 g / L lentinan alkaline solution. The lentinan alkaline solution was centrifuged at 1000 rpm for 2 min to remove bubbles, then loaded into a syringe, and the needle was replaced for later use.

[0091] Comparative Examples 6 and 7 are taken as a group, Comparative Examples 8 and 9 are taken as a group, and Comparative Examples 10 and 11 are taken as a group. Inonotus obliquus polysaccharide aqueous solution, Poria cocos water-soluble polysaccharide aqueous solution and Lentinan edodes polysaccharide alkaline solution are respectively added dropwise to ferric chloride and calcium chloride solutions. The obtained product photos are shown in FIG. Figure 12 As shown. It can be seen that by using the same method, alkali-soluble Poria cocos polysaccharide is replaced by water-soluble Inonotus obliquus polysaccharide, water-soluble Poria cocos polysaccharide and alkali-soluble Lentinan, no hydrogel beads can be obtained. In the experiment, for the above-mentioned polysaccharide categories, other feasible preparation conditions were also tried to explore, but no hydrogel beads could be obtained. In the embodiment of the present invention, an alkali-soluble Poria cocos polysaccharide solution was added dropwise to a soluble metal salt solution to obtain hydrogel beads. The possible mechanism is that PCAP is dissolved in a NaOH solution to open the PCAP triple helix chain, and metal ions enter the chain gap, and cross-linking occurs through complexation to form a stable structure. No additional additives are required to form hydrogel beads of good quality, which is not available in other polysaccharide types. In addition, from the appearance, the viscosity of the alkali-soluble Poria polysaccharide solution in the embodiment of the present invention is significantly greater than that of other polysaccharide types at the same concentration. This may also be because the molecular weight, chain length, branching, etc. of the alkali-soluble Poria polysaccharide are quite different from those of other polysaccharide types, which makes the viscosity of the alkali-soluble Poria polysaccharide solution higher and more prone to cross-linking and entanglement between chains, thereby forming a more stable gel bead structure.

[0092] Example 7 The other steps were the same as in Example 1, except that the concentration of PCAP in the PCAP alkaline solution was 150 g / L and the concentration of ferric chloride in step (2) was 20% (w / v). Hydrogel beads were also prepared using the same method as in Example 1.

[0093] Example 8 The other steps were the same as in Example 1, except that the concentration of the sodium hydroxide aqueous solution was 1 mol / L, the concentration of the PCAP alkaline solution was 180 g / L, and the concentration of ferric chloride in step (2) was 5% (w / v). Hydrogel beads were also prepared using the same method as in Example 1.

[0094] Example 9 The other steps are the same as those in Example 1, except that ferric chloride is replaced by copper sulfate. Hydrogel beads can also be prepared using the same method as in Example 1.

[0095] Example 10 The other steps are the same as those in Example 1, except that ferric chloride is replaced by ferric nitrate. Hydrogel beads can also be prepared using the same method as in Example 1.

[0096] Comparative Example 12 Other steps were the same as in Example 1, except that the concentration of the PCAP alkaline solution was 100 g / L. Gel beads were obtained by the same method as in Example 1, but with irregular morphology.

[0097] The above description is merely a specific embodiment of the present application, but the scope of protection of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in this application should be included in the scope of protection of this application. Therefore, the scope of protection of this application should be based on the scope of protection of the claims.

Claims

1. A method for preparing metal ion cross-linked alkali-soluble Pachymaran hydrogel beads, characterized in that: The steps include: (1) dissolving alkali-soluble Poria cocos polysaccharide in alkali solution to obtain an alkali-soluble Poria cocos polysaccharide solution; (2) adding the alkali-soluble pachymaran solution dropwise to an aqueous solution of a soluble metal salt, allowing it to stand for stable solidification to obtain hydrogel beads, and then washing to remove free metal ions on the surface of the hydrogel beads, thereby obtaining the metal ion-crosslinked alkali-soluble pachymaran hydrogel beads; The concentration of pachymaran in the alkali-soluble pachymaran solution is 150-200 g / L, and the mass volume concentration of the metal salt in the aqueous solution of the soluble metal salt is 2-25%.

2. The preparation method according to claim 1, wherein The preparation method of the alkali-soluble Poria cocos polysaccharide comprises the following steps: Extracting the Poria cocos powder with ethanol, filtering and drying the residue to obtain the primary Poria cocos residue; boiling the primary Poria cocos residue with water, filtering and drying the residue to obtain secondary Poria cocos residue; The secondary Poria residue is extracted with alkali solution, the residue is removed by filtration, the obtained filtrate is acidified to adjust the pH, and the filtrate is allowed to stand to obtain a precipitate; The precipitate is desalted with pure water and then dried to obtain alkali-soluble Pachymaran.

3. The preparation method according to claim 1, wherein The concentration of the alkali solution in step (1) is 0.3~1 mol / L.

4. The preparation method according to claim 1, wherein Step (1) dissolving the alkali-soluble pachymaran in alkali solution and removing bubbles by centrifugal separation to obtain an alkali-soluble pachymaran solution.

5. The preparation method according to claim 1, wherein The metal ions contained in the aqueous solution of the soluble metal salt are Ca 2+ Mg 2+ 、Fe 3+ 、Mn 2+ and Cu 2+ One or more of .

6. The preparation method according to claim 1, wherein The dripping speed in step (2) is 0.4-1 mL / min, and the dripping height is 6-12 cm.

7. The preparation method according to claim 1, wherein The step (2) is to allow the mixture to stand for stable solidification, and the standing time is 10 to 60 minutes.

8. Metal ion cross-linked alkali-soluble Pachymaran hydrogel beads prepared by the preparation method according to any one of claims 1 to 7.

9. The metal ion cross-linked alkali-soluble Pachymaran hydrogel beads according to claim 8, characterized in that: The wet bead size is 1.5~3mm, and the dry bead size is 0.5~1mm.

10. Use of the metal ion cross-linked alkali-soluble Pachymaran hydrogel beads according to claim 8 in the use or preparation of hemostatic products.

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