Silk fibroin hemostatic sponge loaded with polyhydroxy phenolic compound and preparation method of silk fibroin hemostatic sponge

Abstract

The invention provides a silk fibroin hemostatic sponge loaded with polyhydroxy phenolic compounds and a preparation method of the silk fibroin hemostatic sponge, aiming at the defect that hemostatic materials have poor effects on large wounds and serious bleeding, and the silk fibroin hemostatic sponge loaded with the polyhydroxy phenolic compounds is prepared from silk fibroin compounds loaded with the polyhydroxy phenolic compounds, wherein the silk fibroin compound loaded with the polyhydroxy phenolic compound is prepared from the polyhydroxy phenolic compound and silk fibroin according to the feeding mass ratio of 1t; the material is prepared in a 1: 1 self-assembly mode. The polyhydroxy phenolic compound is preferably insoluble and most preferably ellagic acid, and the feeding ratio of the polyhydroxy phenolic compound to the silk fibroin is preferably 1: 2-1: 16 and more preferably 1: 4. The hemostatic capacity of the silk fibroin compound loaded with the polyhydroxy phenolic compound is remarkably superior to that of silk fibroin, Yunnan Baiyao, tranexamic acid, chitosan hemostatic powder and the like, and the silk fibroin compound is free of cytotoxicity, free of hemolysis, capable of being self-degraded and high in safety. The silk fibroin hemostatic sponge loaded with the polyhydroxy phenolic compound has excellent hemostatic performance, is easy to peel, does not cause secondary damage, and can be self-degraded.

Description

Technical Field

[0001] The present invention belongs to the technical field of materials, and particularly relates to a silk fibroin hemostatic sponge loaded with polyhydroxyphenolic compounds and a preparation method thereof. Background Art

[0002] Bleeding is a sudden and severe blood leakage caused by blood vessel rupture and is one of the most common causes of death in the world. Surgical bleeding deaths and bleeding deaths caused by wars are the main situations. According to statistics, severe bleeding accounts for more than 35% of pre-hospital deaths and more than 40% of deaths within 24 hours after injury. The massive blood loss caused by modern wars has increased significantly, and the deaths within 30 minutes after war injuries account for 80% of the total war injury deaths. On the battlefield, the first hour after a casualty is injured is called the "golden hour" of rescue. Therefore, the cost and technical research of hemostatic materials not only bring a huge burden to the global economy, but also are an urgent problem to be solved in any first-aid scenario.

[0003] Currently, the hemostatic market is mainly dominated by absorbable hemostatic materials, including gauze, sponges, sprays, hemostatic powders, etc. Their main components are mostly natural polymers such as cellulose, chitosan, and fibrin. Their main advantages include short hemostasis time, biodegradability, relatively mild foreign body reactions, no need for secondary surgery to remove, and avoidance of secondary injuries to patients. However, their high costs, inconsistent absorption times, limited adaptability, and poor effects on large wounds and severe bleeding limit their clinical applications. For example, oxidized regenerated cellulose hemostatic materials contain a large number of acidic groups. After being fully saturated with water, the pH reaches 1.7. In an acidic environment, although it has a certain antibacterial effect, it may also cause strong inflammatory reactions at the wound site with the remaining materials and hinder the regeneration of bone tissue, and even form granulomas, abscesses, etc. Therefore, it is necessary to develop an absorbable hemostatic material that is fast, efficient, economical, and has no toxic side effects.

[0004] Silk fibroin, a natural polymer protein extracted from silk, has excellent in vivo degradability, biocompatibility, and anti-inflammatory properties. In terms of the hemostatic mechanism, silk fibroin activates coagulation factor XII, thereby triggering the intrinsic coagulation cascade reaction. It is a biological macromolecule composed of 5509 amino acids, consisting of a heavy chain (391 kDa) and a light chain (26 kDa), where the heavy chain and the light chain are connected by disulfide bonds. When silk fibroin dissolved in LiBr solution or a ternary solution is poured into an excess of polar organic solvents such as alcohol, a white suspension of protein complexes immediately appears. When silk fibroin is kept in solution, due to the multiple interactions between the side chains of fibroin, salt ions, and water molecules, the peptide chains exist in a random coil conformation, that is, the α-helix, which is also the natural conformational state of silk fibroin. In this state, silk fibroin does not have the ability to self-assemble with polyhydroxyphenolic compounds because the phenyl rings of amino acid residues have large steric hindrance, and it is difficult for polyhydroxyphenolic compounds to integrate into the α-helix structure, thus being exposed on the outer side of silk fibroin molecules. When using an anti-solvent such as ethanol, the peptide chains will be induced to rapidly fold towards a more stable β-sheet assembly. The β-sheet conformation will expose hydrophobic amino acid residues, which can enhance the encapsulation of hydrophobic drugs through hydrophobic interactions and π-π stacking, facilitating subsequent self-assembly with polyhydroxyphenolic compounds.

[0005] Currently, the silk fibroin hemostatic drugs on the market cannot solve the obstacle of the blood interface hydration layer, have a poor adhesion rate to the bleeding wound surface, and cannot meet the tissue strength adhesion requirements in a wet environment. Polyhydroxyphenolic compounds can form a secondary cross-linked structure with silk fibroin, thereby recruiting more platelets, solving the possibility of silk fibroin being washed away by blood. At the same time, polyhydroxyphenolic compounds and silk fibroin can also show a synergistic effect to promote endogenous hemostasis. Summary of the Invention

[0006] The purpose of the present invention is to solve the pain points existing in the prior art, such as poor effects on large wounds and severe bleeding, insufficient blood absorption and oozing ability, and inability to handle irregular wounds. The present invention provides a silk fibroin hemostatic sponge loaded with polyhydroxyphenolic compounds, its preparation method and application. The technical solution is as follows:

[0007] The present invention provides a silk fibroin hemostatic sponge loaded with polyhydroxyphenolic compounds, which is prepared into a hemostatic sponge from a silk fibroin complex loaded with polyhydroxyphenolic compounds. The silk fibroin complex loaded with polyhydroxyphenolic compounds is prepared by self-assembly of polyhydroxyphenolic compounds and silk fibroin. Among them, silk fibroin is processed by a β-sheet conformational transformation process, and the feeding ratio of polyhydroxyphenolic compounds to silk fibroin is a mass ratio of <1:1; the hemostatic sponge is prepared by one of physical cross-linking method, chemical cross-linking method, sol-gel method, electrospinning method, freeze-drying method, phase separation method, 3D printing technology, and hydrogel method.

[0008] As an improvement of the present invention, the polyhydroxyphenolic compound is selected from one or more of resveratrol, quercetin, baicalein, kaempferol, lignans, curcumin, ellagic acid, chlorogenic acid, ferulic acid, caffeic acid, syringic acid, sinapic acid, rutin, myricetin, magnolol, fisetin, puerarin, rhein, lonicerin, proanthocyanidins, p-coumaric acid, vanillic acid, hesperetin, naringenin, luteolin, genistein, emodin, oleanolic acid, epigallocatechin gallate, robinin, tea polyphenols, epigallocatechin, salvianolic acid, gallic acid, pyrogallic acid, catechin, tannic acid.

[0009] As a further improvement, the polyhydroxyphenolic compound is a poorly soluble polyhydroxyphenolic compound, preferably selected from one or more of resveratrol, quercetin, baicalein, kaempferol, lignans, curcumin, ellagic acid, chlorogenic acid, ferulic acid, caffeic acid, syringic acid, sinapic acid, rutin, myricetin, magnolol, fisetin, puerarin, rhein, lonicerin, proanthocyanidins, p-coumaric acid, vanillic acid, hesperetin, naringenin, luteolin, genistein, emodin, oleanolic acid, epigallocatechin gallate, and more preferably ellagic acid.

[0010] The present invention also provides a method for preparing the above-mentioned silk fibroin hemostatic sponge, which comprises three steps: silk fibroin conformation transition, self-assembly with polyhydroxyphenolic compounds, and preparation of the hemostatic sponge. Step (1): A silk fibroin solution is prepared by a process of converting silk fibroin from an α-helix to a β-sheet conformation. The conformation change can be achieved by chemical methods or physical methods. Chemical methods include promoting the transition through polyols, polylactic acid, metal ions, pH, or hydroxypropyl methylcellulose. Physical methods include promoting the transition through high temperature, hydrostatic pressure, ultra-low temperature storage, freeze-drying, shear force, ultrasonic waves, eddy currents, laser irradiation, high-pressure carbon dioxide treatment. Step (2): After dissolving or suspending the polyhydroxyphenolic compound, it is mixed and stirred with the silk fibroin solution obtained in step (1) to cause self-assembly, thereby obtaining a silk fibroin complex loaded with polyhydroxyphenolic compounds. Step (3): The silk fibroin complex loaded with polyhydroxyphenolic compounds obtained in step (2) is prepared into a silk fibroin hemostatic sponge loaded with polyhydroxyphenolic compounds by one of physical cross-linking methods, chemical cross-linking methods, sol-gel methods, electrospinning methods, freeze-drying methods, phase separation methods, 3D printing techniques, and hydrogel methods.

[0011] As an improvement, in step (1), a polyol is used to promote the transition of silk fibroin to the β-sheet conformation, and ethanol is preferably used.

[0012] As an improvement of the preparation method, the polyhydroxyphenolic compound is a poorly soluble polyhydroxyphenolic compound. In step (2), the poorly soluble polyhydroxyphenolic compound is dissolved or suspended in an organic solvent, and the organic solvent is selected from methanol, ethanol, propanol, propylene glycol, glycerol, n-butanol, and isobutanol.

[0013] As the most preferred ellagic acid, the feeding ratio of ellagic acid to silk fibroin is preferably 1:2 to 1:16 in mass ratio, more preferably 1:4 in mass ratio. In step (2), the solvent of the ellagic acid suspension is preferably propylene glycol. After the ellagic acid suspension and silk fibroin are co-incubated, the final concentration of propylene glycol in the obtained solution is 5-20%, and the most preferred final concentration of propylene glycol is 10%.

[0014] Beneficial effects: The silk fibroin complex loaded with polyhydroxyphenolic compounds developed in the present invention, as an effective hemostatic agent, has significantly better hemostatic and coagulation abilities than polyhydroxyphenolic compounds and silk fibroin itself, indicating that these two materials have a synergistic effect. At the same time, the hemostatic effect of this hemostatic agent is significantly better than that of many commercially available hemostatic products, such as Yunnan Baiyao, tranexamic acid, chitosan hemostatic powder, etc.; at the same time, this silk fibroin complex hemostatic agent has no cytotoxicity, does not hemolyze, can self-degrade in vivo, and has high safety. The silk fibroin hemostatic sponge loaded with polyhydroxyphenolic compounds provided by the present invention has excellent hemostatic performance, is easy to peel off, will not cause secondary damage, and can self-degrade. Description of the Drawings

[0015] The present invention will be further described below with reference to the drawings:

[0016] Figure 1 Fourier transform infrared spectroscopy (FTIR).

[0017] Figures 2 to 4 Blood viscosity comparison chart (n = 3). In each figure, figure A: *, compared with the blank control group, *P < 0.05, **P < 0.01, ***P < 0.001; #, compared with the β-SF-EA group, #P < 0.05, ##P < 0.01, P < 0.001. Compared with the β-SF-polyhydroxyphenolic compound group, *P < 0.05, **P < 0.01, ***P < 0.001. For example, compared with the β-SF-EA group, *P < 0.05, **P < 0.01, ***P < 0.001.

[0018] Figure 5 Coagulation time comparison chart (n = 3), *, compared with the β-SF-EA group, *P < 0.05, **P < 0.01, ***P < 0.001.

[0019] Figure 6Drug loading comparison chart (n = 3). *, compared with the β-SF-EA group, *P < 0.05, **P < 0.01, ***P < 0.001; ND indicates not detected, Not Detected.

[0020] Figures 7 to 8 Drug loading comparison chart (n = 3). *, compared with the water group, *P < 0.05, **P < 0.01, ***P < 0.001.

[0021] Figure 9 Fourier transform infrared spectroscopy (FTIR), in which EA and β-SF are physically mixed at a ratio of 1:1 (w / w).

[0022] Figure 10 Molecular docking diagram. L74 represents Leu (leucine) at position 74, and D27 represents Asp (aspartic acid) at position 27.

[0023] Figure 11 Characterization: (A) FTIR spectrum, (B) XRD, (C) TG, (D) 1H NMR.

[0024] Figure 12 Blood adsorption experiment. Among them, a is the blank, b is β-SF, c is EA, d is Yunnan Baiyao, e is tranexamic acid, f is β-SF-EA, Figure 13 Same as above.

[0025] Figure 13 Test tube tilting blood coagulation experiment diagram.

[0026] Figure 14 Blood coagulation index BCI diagram (n = 3).

[0027] Figure 15 Rat tail amputation experiment. Among them, Figure A is the bleeding diagram of the rat tail after immediately sprinkling the drug powder, and Figure B is the hemostasis time diagram after tail amputation; *, compared with the β-SF-EA group, *P < 0.05, **P < 0.01, ***P < 0.001.

[0028] Figure 16 Rat liver and femoral artery bleeding time diagram (n = 3). *, compared with the β-SF-EA group, *P < 0.05, **P < 0.01,

[0029] ***P < 0.001.

[0030] Figure 17 Relative cell proliferation rate diagram of LO2 cells and L929 cells after co-incubation with drugs for 48 h (n = 6).

[0031] Figure 18 β-SF-EA in vitro degradation diagram (n = 3).

[0032] Figure 19 Wound peeling force, *P < 0.05, **P < 0.01, ***P < 0.001.

[0033] Figure 20 Blood coagulation index BCI graph (n = 3).

[0034] Figure 21 Rat tail amputation hemostasis experiment.

[0035] Figure 22 Rat femoral artery injury model experiment.

[0036] Figure 23 Rat liver injury model experiment.

[0037] Figures 21 to 23 Among them, Control is the gauze control, Getatin is the positive control commercially available gelatin sponge, Figure A is the hemostasis time graph after injury, and Figure B is the blood loss after injury; *, compared with the β-SF-EA hemostatic sponge group, *P < 0.05, **P < 0.01, ***P < 0.001.

[0038] Figure 24 β-SF-EA hemostatic sponge in vitro degradation graph (n = 3). Detailed implementation manner

[0039] Example 1 Preparation of silk fibroin complex loaded with polyhydroxyphenolic compounds (β-SF - polyhydroxyphenolic compounds)

[0040] Step (1) Transformation of the β-sheet conformation of silk fibroin: Dilute silk fibroin (SF) 5 - 10 times with water and slowly pour it into an excessive amount of vigorously stirred organic solvent, stir for 2 - 4 h to complete the transformation of the β-sheet conformation, remove the liquid part by mechanical separation methods such as filtration, centrifugation, and membrane filtration, wash the remaining part repeatedly and then freeze-dry to obtain β-sheet silk fibroin (β-SF), wherein the organic solvent is selected from methanol, ethanol, propylene glycol, butylene glycol, etc., preferably ethanol; the mechanical separation method is preferably high-speed centrifugation method, such as high-speed centrifugation at 10000 rpm for 15 - 20 min.

[0041] Fourier transform infrared spectroscopy (FTIR), mix 5% of the sample with 95% KBr and grind it into a fine powder, perform transmission scanning with an FTIR spectrometer (Nicolet is5), each spectrum is obtained in transmission mode (ten scans), and the resolution is 4 cm -1 , and the spectral range is 4000 - 500 cm -1 . The FTIR spectrum of a relatively high level of β-sheet structure is at 1615 - 1640 cm -1, 1510 - 1525 cm -1 showed sharp peaks at, while the FTIR spectrum of SF in its natural state showed sharp peaks at 1640 - 1660 cm -1 , 1535 - 1542 cm -1 , namely α - SF. From the measurement results in Figure 1 , it can be seen that the characteristic peaks of natural α - SF are 1642 cm -1 and 1535 cm -1 . The characteristic peaks of SF after being treated in step (1) are 1620 cm -1 and 1517 cm -1 , indicating that SF has completed the conformational transition of β - sheet and become β - SF.

[0042] Step (2) Self - assembly of polyhydroxy phenols and silk fibroin: The β - SF obtained in step (1) is prepared into a suspension with water and placed in a magnetic stirrer at 500 - 2000 rpm; polyhydroxy phenols are dissolved or suspended with solvents. For water - soluble polyhydroxy phenols, water is used as the solvent, and for poorly soluble polyhydroxy phenols, organic solvents such as water, ethanol, propylene glycol, and butylene glycol are used as the solvent. The prepared solution or suspension is added dropwise to the β - SF suspension, and mixed and stirred for 12 - 48 h to obtain a silk fibroin complex loaded with polyhydroxy phenols.

[0043] Among them, the polyhydroxyphenolic compounds are selected from water-insoluble polyhydroxyphenolic compounds, including ellagic acid (EA), resveratrol (RES), quercetin (QUE), baicalein (BAE), kaempferol (KAE), lignan, curcumin (CUR), chlorogenic acid (CGA), ferulic acid (FA), caffeic acid (CFA), syringic acid (SA), sinapic acid, rutin (RUT), myricetin (MYR), magnolol acid (MA), fisetin (FIS), puerarin (PUE), rhein (RHE), lonicerin (Lon), proanthocyanidins (PC), p-coumaric acid (p-CA), vanillic acid (VA), hesperetin (Hes), naringenin (NAR), luteolin (Lut), genistein (Gen), emodin (Emo), oleanolic acid (Ola), danshensu (DS), epigallocatechin gallate (EGCG); water-soluble polyhydroxyphenolic compounds, including gallic acid (GA), pyrogallic acid (PA), catechin (C), tannic acid (TA), tea polyphenols (TP), acaciin, epigallocatechin (EGC), etc.

[0044] Example 2 Preparation of silk fibroin complex loaded with polyhydroxyphenolic compounds

[0045] Different from Example 1, in step (1), ethanol is selected as the organic solvent, and the mechanical separation method is high-speed centrifugation at 10,000 rpm for 15 - 20 min. In step (2), an equal amount of polyhydroxy phenolic compounds and silk fibroin are added. In the preparation of the polyhydroxy phenolic compound suspension, water is used as the solvent for water-soluble polyhydroxy phenolic compounds, and water or an organic solvent is used as the solvent for poorly water-soluble polyhydroxy phenolic compounds. The mechanical separation method is to take the precipitate part after high-speed or ultra-high-speed centrifugation. Step (3) of drying into powder is added: the solution obtained in step (2) is dried into powder after removing the liquid by mechanical separation methods such as suction filtration, centrifugation, membrane filtration, sedimentation, etc., or dried into powder in one step using a drying device such as a spray dryer, and the silk fibroin complex loaded with polyhydroxy phenolic compounds is obtained; the mechanical separation method preferably takes the precipitate part after high-speed or ultra-high-speed centrifugation.

[0046] Using insoluble polyhydroxy phenolic compounds as raw materials, ellagic acid-silk fibroin complex (β-SF-EA), resveratrol-silk fibroin complex (β-SF-RES), quercetin-silk fibroin complex (β-SF-QUE), baicalein-silk fibroin complex (β-SF-BAE), kaempferol-silk fibroin complex (β-SF-KAE), lignan-silk fibroin complex (β-SF-Lignan), curcumin-silk fibroin complex (β-SF-CUR), chlorogenic acid-silk fibroin complex (β-SF-CGA), ferulic acid-silk fibroin complex (β-SF-FA), caffeic acid-silk fibroin complex (β-SF-CFA), syringic acid-silk fibroin complex (β-SF-SA), sinapic acid-silk fibroin complex (β-SF-Sinapic Acid), rutin-silk fibroin complex (β-SF-RUT), myricetin-silk fibroin complex (β-SF-MYR), magnolol-silk fibroin complex (β-SF-MA), fisetin-silk fibroin complex (β-SF-FIS), puerarin-silk fibroin complex (β-SF-PUE), rhein-silk fibroin complex (β-SF-RHE), lonicerin-silk fibroin complex (β-SF-Lon), procyanidin-silk fibroin complex (β-SF-PC), p-coumaric acid-silk fibroin complex (β-SF-p-CA), vanillic acid-silk fibroin complex (β-SF-VA), hesperetin-silk fibroin complex (β-SF-Hes), naringenin-silk fibroin complex (β-SF-NAR), luteolin-silk fibroin complex (β-SF-Lut), genistein-silk fibroin complex (β-SF-Gen), emodin-silk fibroin complex (β-SF-Emo), oleanolic acid-silk fibroin complex (β-SF-Ola), salvianolic acid-silk fibroin complex (β-SF-DS), epigallocatechin gallate-silk fibroin complex (β-SF-EGCG) were prepared; using water-soluble polyhydroxy phenolic compounds as raw materials, gallic acid-silk fibroin complex (β-SF-GA), pyrogallic acid-silk fibroin complex (β-SF-PA), catechin-silk fibroin complex (β-SF-C), tannic acid-silk fibroin complex (β-SF-TA), robinin-silk fibroin complex (β-SF-Acaciin), epigallocatechin-silk fibroin complex (β-SF-EGC), tea polyphenol-silk fibroin complex (β-SF-TP) were prepared.

[0047] Example 3 Investigation of Coagulation Effect

[0048] The obtained β-SF-polyhydroxyphenol compounds were prepared into a 2 mg / mL test solution with physiological saline for the investigation of blood coagulation effect: Blood was taken from the abdominal aorta of New Zealand white rabbits and placed in a heparin sodium anticoagulation tube. 2 mL of the test solution was added to the tube. Using physiological saline as a control, the mixture was evenly mixed and incubated at 37 °C for 15 min, and then the whole blood viscosity was detected with a full-automatic blood rheometer (all operations were completed within 2 h after blood collection).

[0049] The whole blood viscosity is the result of the friction between blood cells and plasma protein molecules during blood flow and is the most important indicator in hemorheology. When the blood viscosity increases, it indicates that blood flow is blocked. At the same time, the deformability of red blood cells decreases and their aggregation increases, and the blood flow into small blood vessels and capillaries decreases and the passing ability decreases. Figures 2 to 4 The results showed that the silk fibroin complex loaded with polyhydroxyphenol compounds (β-SF-polyhydroxyphenol compounds) could significantly increase the whole blood viscosity compared with the blank group (P<0.05), and was significantly better than the effects of β-SF and the original compounds themselves (P<0.05), indicating that the formation of the complex could significantly enhance the blood coagulation effect; and overall, the effect of the complex formed by the self-assembly of silk fibroin and poorly soluble polyhydroxyphenol compounds was better than that of the self-assembly complex with water-soluble polyhydroxyphenol compounds.

[0050] Weigh 0.1 g of the drug powder to be tested and place it in a 1.5 mL centrifuge tube. At the same time, measure 0.5 mL of anticoagulated blood and add it to the centrifuge tubes containing different drugs. Place them vertically on the experimental workbench and start timing. After 30 s, rotate and invert the centrifuge tube to observe whether the blood flows. Repeat the operation until the anticoagulated blood cannot flow, stop timing, and record it as the coagulation time. Figure 5 The results in Figures 2 to 4 showed a similar trend to the results in

[0051] Among all the complexes, the blood coagulation effect of β-SF-EA was the best. This may be related to the multiple hydrogen bonds and planar structure of EA. On the one hand, the existence of multiple hydrogen bonds increases the synergistic effect between hydrogen bonds and further improves the stability of hydrogen bonds; on the other hand, ellagic acid has a symmetric catechol structure and a planar structure, which can form intramolecular and intermolecular hydrogen bonds, making the rigidity of the complex molecules enhanced. This stable molecular structure can reduce the molecular dynamic changes, thereby indirectly improving the stability of intermolecular hydrogen bonds.

[0052] Example 4 Preparation of polyhydroxyphenol compound-silk fibroin complex (α-SF-polyhydroxyphenol compound)

[0053] Different from Example 2, step (1) is deleted. In step (2), β-SF is replaced with untreated SF, that is, SF in its natural α-helical conformation. In step (2), an equal amount of polyhydroxyphenolic compounds and silk fibroin are fed to prepare a polyhydroxyphenolic compound-α-silk fibroin complex, called α-SF-polyhydroxyphenolic compound.

[0054] Example 5 Preparation of Polyhydroxyphenolic Compound-Silk Fibroin Complex (SH-SF-Polyhydroxyphenolic Compound)

[0055] Different from Example 2, in step (1), the following method is selected: Dissolve silk fibroin in an aqueous solution containing tris(2-carboxyethyl)phosphine hydrochloride, and place it at room temperature for a reduction reaction treatment for 5 minutes to obtain a SF solution with a concentration of 1.0 - 5.0 wt% that has undergone a reduction reaction treatment. The rest is the same as the method in Example 2. In step (2), an equal amount of polyhydroxyphenolic compounds and silk fibroin are fed, and the resulting polyhydroxyphenolic compound-silk fibroin complex is called SH-SF-polyhydroxyphenolic compound. This method uses a method of disrupting disulfide bonds to expose hydrophobic amino acids to bind polyhydroxyphenolic compounds such as ellagic acid.

[0056] Example 6 Investigation of Self-Assembly Efficiency

[0057] Ellagic acid is used as a model drug for polyhydroxyphenolic compounds. Three ellagic acid-silk fibroin complexes, namely β-SF-EA, α-SF-EA, and SH-SF-EA, are prepared according to the steps in Examples 2, 4, and 5. The solvent for EA is water. The assembly efficiency is evaluated by the drug loading and encapsulation rate, which are calculated using formulas (1) and (2) respectively. The "amount of EA in the silk fibroin complex" in the numerator is calculated by the difference method, that is, subtracting the free amount of EA after co-incubation with SF from the EA feeding amount. The content of EA is analyzed by high-performance liquid chromatography (HPLC). The chromatographic column used is a Hedera C18 column, the mobile phase is acetonitrile:water = 20:80 (v / v), the flow rate is 1.0 mL / min, the detection wavelength is 254 nm, the injection volume is 10 μL, and the chromatogram acquisition time is 15 min. The drug loading and encapsulation rate are calculated using the following formulas:

[0058] Drug loading = Amount of EA in silk fibroin complex / Total feeding amount of SF and EA Formula (1)

[0059] Encapsulation rate = Amount of EA in silk fibroin complex / Feeding amount of EA Formula (2)

[0060] By Figure 6As can be seen from the results, the drug loading capacity: β-SF-EA > SH-SF-EA >> α-SF-EA. Among them, the drug loading capacity of β-SF-EA reached 9.6%, while EA was hardly detectable in α-SF-EA, indicating that the efficiency of self-assembly of α-helical structure fibroin and ellagic acid is very low, highlighting the necessity of conformational inversion of fibroin; SH-SF-EA uses the destruction of disulfide bonds in fibroin molecules to expose their hydrophobic amino acids, thereby completing the self-assembly with ellagic acid, but the drug loading capacity is only 4.5%, far lower than that of β-SF-EA (P < 0.001).

[0061] Example 7 Investigation of the Solvent on the Drug Loading Capacity of EA

[0062] Using ellagic acid as the model drug for polyhydroxy phenolic compounds, β-SF-EA was prepared by the steps in Example 2. In step (2), water, ethanol, propylene glycol, butylene glycol, etc. were used as solvents to prepare the suspension of polyhydroxy phenolic compounds. If an organic solvent was used as the suspension solvent, the final concentration of the organic solvent in the solution obtained after co-incubation of the polyhydroxy phenolic compound suspension and β-SF was 10%; and the method in Example 6 was used to investigate their drug loading capacities. Figure 7 The results in the table show that the addition of organic solvents effectively increased the drug loading capacity of ellagic acid. Among them, propylene glycol > butylene glycol > ethanol > water, and the drug loading capacity was the highest when propylene glycol was used, reaching 19.57 ± 1.01%.

[0063] Next, the effect of propylene glycol concentration on the drug loading capacity of EA was investigated. In step (2), propylene glycol was used as the solvent to prepare the EA suspension, and the final concentrations of propylene glycol in the solution obtained after co-incubation with β-SF were 0%, 5%, 10%, 15%, and 20% respectively. Figure 8 The results in the table show that the drug loading capacities of the propylene glycol aqueous solution groups were all better than that of the water group, and the 10% propylene glycol aqueous solution group had the best effect, with the drug loading capacity reaching 19.57 ± 1.01%.

[0064] Example 8 Investigation of Different EA:SF Feeding Ratios

[0065] Using ellagic acid as the model drug for polyhydroxy phenolic compounds, β-SF-EA was prepared by the steps in Example 2. Among them, in step (2), propylene glycol was used as the solvent to prepare the EA suspension, and the final concentration of propylene glycol in the solution obtained after co-incubation with β-SF was 10%; at the same time, the feeding ratio of EA to β-SF was controlled as EA:β-SF = 1:0.5, 1:1, 1:2, 1:4, 1:8, 1:12, 1:16 (w / w). As can be seen from Table 1, almost no drug loading capacity and encapsulation efficiency of EA could be observed for EA:β-SF = 1:0.5 and 1:1, while they could be observed in other groups; at the same time, Fourier transform infrared spectroscopy was performed on the samples of each group using the method in Example 1 ( Figure 9), it can be seen that the disappearance of the EA hydroxyl peak (3471 cm -1 ) was observed in the groups with EA:β-SF = 1:2, 1:4, 1:8, 1:12, 1:16 (w / w), indicating that at this time, hydrogen bond binding between EA and β-SF has been completed and self-assembly was successful. However, the EA hydroxyl peak still exists in the groups with EA:β-SF = 1:0.5, 1:1 and their physical mixture groups, and the self-assembly of the two molecules cannot be achieved. The above results comprehensively show that only when the feeding ratio EA:β-SF < 1:1 (w / w) can the self-assembly of β-SF and EA be achieved. Among them, EA:β-SF = 1:4 can achieve the maximum drug loading and encapsulation efficiency of EA (Table 1), which is called the optimal feeding ratio.

[0066] Furthermore, the method in Example 3 was used to investigate the effect of different EA:β-SF feeding ratios on whole blood viscosity. The results in Table 2 show that compared with the normal saline group, all groups with EA:β-SF < 1:1 can significantly increase the whole blood viscosity at low, medium, and high shear rates (P < 0.5), and are higher than the EA and β-SF groups (P < 0.5), indicating that β-SF-EA prepared with an EA:β-SF < 1:1 feeding ratio has a significant blood coagulation effect, and EA and β-SF have a synergistic effect; among them, EA:β-SF = 1:4 has the best blood coagulation effect.

[0067] To sum up, the preparation process of β-SF-EA is to use β-sheet SF for preparation, with a feeding ratio of EA:β-SF < 1:1 (w / w). The specific steps are shown in Example 1; among them, the optimal process is EA:β-SF = 1:4 (w / w). Ellagic acid in step (2) is used to prepare a suspension with propylene glycol as the solvent, and the final concentration of propylene glycol in the solution obtained after co-incubation with SF is 10%. Failure to transform the β-sheet conformation of SF or a feeding ratio of EA:β-SF ≥ 1:1 cannot achieve the self-assembly of EA and β-SF. When EA:β-SF = 1:2 (w / w), both the drug loading and encapsulation efficiency are relatively low, indicating that although the self-assembly of EA and β-SF can be achieved at this time, the loss is relatively large.

[0068] Table 1 Effects of the feeding ratio of β-SF to EA on drug loading and encapsulation efficiency (n = 3)

[0069]

[0070] ND indicates not detected.

[0071] Table 2 Effects of different EA:β-SF feeding ratios on whole blood viscosity (n = 3)

[0072]

[0073] *: Compared with the normal saline group, *P<0.05, **P<0.01, ***P<0.001; #: Compared with the EA:β-SF = 1:4 group, #P<0.05, ##P<0.01, P<0.001.

[0074] Example 9 Molecular Docking of Silk Fibroin Complex

[0075] Computer simulation studies were carried out on the binding mode of ellagic acid and silk fibroin. The crystal structure of the heavy chain of silk fibroin (PDB ID: 3UA0) was obtained from the PDB bank. Since there were few previous studies on the binding mode, we reasonably predicted the binding site through the SiteMap module in 2018. After hydrogenation of the silk fibroin crystal structure and removal of irrelevant ions, the small molecule of ellagic acid was converted into a 3D conformation via the LigPrep module, and up to 32 isomers were output. Finally, 20 binding poses were output by Glide extra precision (XP). As Figure 10 shown, after the two phenolic hydroxyl groups in the ellagic acid small molecule form hydrogen bonds with L74, they also need to form hydrogen bonds with D27 on another heavy chain, indicating that continued hydrogen bonding with the other two highly symmetric hydroxyl groups in ellagic acid is required to achieve stable binding. In addition, since the heavy chains in silk fibroin maintain a highly similar β-sheet secondary structure, there may not be enough hydrophobic interactions between the ellagic acid small molecule and two heavy chain proteins to maintain stability. It is reasonably speculated that the ellagic acid small molecule will bind and wrap at least three to four heavy chain proteins to achieve stable binding. The molecular docking results explain the molecular mechanism of the experimental result that "EA:β-SF < 1:1 (w / w) is required to achieve the self-assembly of β-SF and EA" in Example 8.

[0076] In Examples 10 to 14, the tested samples were β-SF-EA prepared under the optimal process.

[0077] Example 10 Characterization of Silk Fibroin Complex Loaded with Polyhydroxyphenolic Compounds

[0078] FT-IR proved the self-assembly of EA and β-SF ( Figure 10 A): The disappearance of the hydroxyl peak of EA (3471 cm -1 ) indicated that EA and β-SF might self-assemble through intermolecular hydrogen bonds. X-ray diffraction (XRD) ( Figure 10B) The characteristic peaks of EA in (B) weakened or disappeared after binding to silk fibroin, indicating that this was not a simple physical mixture but a new crystal phase formed through interactions, confirming the FT-IR results; at the same time, a change in crystal form was seen. β-SF showed broad diffraction peaks, indicating that they were amorphous macromolecules; EA showed many strong diffraction peaks, indicating a crystalline state; when β-SF and EA self-assembled, the peak intensity of EA decreased significantly, and it could be judged that β-SF-EA was mainly amorphous macromolecules.

[0079] Thermogravimetric (TG) curves were evaluated and divided into three stages ( Figure 11 C). In the first stage, from room temperature to 120 °C, the weight loss was approximately 6.1%, mainly due to the evaporation and desorption of adsorbed water; in the second stage, the temperature was between 150 °C and 450 °C, corresponding to the decomposition of silk fibroin molecular chains, the decomposition of amino acid residue side chains, and the breakage of peptide bonds. Among them, the silk I crystal structure (α-helix and random coil) degraded at 250 °C, and the silk II crystal structure (β-sheet) degraded at 260 °C; β-SF showed a main degradation peak at 260 °C, forming a stable β-sheet conformation on the surface; interestingly, the main degradation peak of β-SF-EA shifted to 280 °C, indicating that the silk II crystal structure was more stable, which might be due to the strong interaction between EA and the protein during the self-assembly process, thus improving the stability of the complex. To determine the optimal amount of EA incorporated into β-SF, proton nuclear magnetic resonance (1H NMR) was performed ( Figure 11 D): In β-SF-EA, the peaks related to EA were significantly weakened (<0.007), confirming that EA bound to β-SF through catechol hydrogen, further proving their binding.

[0080] Example 11 Investigation of Coagulation Effect

[0081] (1) Plasma sample treatment

[0082] New Zealand white rabbits were adaptively fed for one week and fasted the night before the experiment. Anesthesia was induced by intraperitoneal injection of 1% sodium pentobarbital at a dose of 3 mL·kg -1 . After anesthesia, blood was collected by abdominal aortic puncture. The blood was collected into a 5 mL heparin sodium anticoagulant tube. After blood collection, the blood and anticoagulant were thoroughly mixed and set aside for use.

[0083] (2) In vitro blood adsorption capacity experiment

[0084] Weigh 0.1 g of the drug powder to be tested and place it in a 1.5 mL centrifuge tube. At the same time, measure 0.5 mL of anticoagulated blood and add it to the centrifuge tube containing different drugs. Place it vertically on the experimental bench and start timing. After 30 s, rotate and invert the centrifuge tube to observe whether the blood flows. Repeat the operation until the anticoagulated blood no longer flows, and then stop timing. Figure 12As shown in the results, β-SF-EA achieved complete adsorption of blood within two minutes, while other groups could not achieve this effect, indicating that the blood adsorption ability of β-SF-EA was significantly better than that of the positive controls Yunnan Baiyao and tranexamic acid, and also significantly better than that of the monomers β-SF and EA, suggesting that β-SF and EA could synergistically enhance the effect.

[0085] (3) Tube tilting coagulation experiment

[0086] Accurately weigh 5.0 mg of the drug powder to be tested and place it in a 10 mL test tube, spreading the powder as much as possible at the bottom of the test tube. Respectively add 1 mL of anticoagulated blood into the test tube, then add 25 μL of 0.2 mol·L -1 CaCl2 solution, and immediately mix well repeatedly and start timing immediately. First, let it stand for 1 min, tilt the test tube every 30 s until the blood coagulates and does not flow, and then stop timing. Figure 13 It was observed that the complete blood coagulation time of the β-SF-EA group (2 min) was much less than that of the Yunnan Baiyao group (5 min), the tranexamic acid group (4 min 30 s), the β-SF group (5 min), and the EA group (>5 min), indicating that β-SF and EA could synergistically enhance the effect. In this experiment, it was observed that β-SF-EA had a faster blood coagulation ability compared to other drugs.

[0087] (4) Hemorheology determination

[0088] Whole blood viscosity is the result of the friction between blood cells and plasma protein molecules during blood flow and is the most important indicator in hemorheology. When the blood viscosity increases, it indicates that blood flow is blocked, and at the same time, the deformability of red blood cells decreases and their aggregation increases, reducing the blood flow into small blood vessels and capillaries and decreasing the passing ability. After β-SF-EA acts on blood, the whole blood viscosity at shear rates of 1·s -1 、30·s -1 、100·s -1 、150·s -1 、200·s -1 was measured by the method in Example 3. The results in Table 3 showed that regardless of the shear rate, β-SF-EA significantly increased the whole blood viscosity (P<0.05), and was significantly better than the commercially available chitosan hemostatic powder group (P<0.05). The control group of commercially available chitosan hemostatic powder was purchased from the instantaneous composite microporous polysaccharide hemostatic powder of Saikesaisi Biotechnology Co., Ltd., and its main active ingredient was chitosan.

[0089] Table 3 Comparison of whole blood viscosity between β-SF-EA and chitosan (n = 3)

[0090]

[0091] *: Compared with the blank group, *P < 0.05, **P < 0.01, ***P < 0.001; #: Compared with the commercially available chitosan hemostatic powder group, #P < 0.05, ##P < 0.01, P < 0.001.

[0092] (4) Blood coagulation index BCI

[0093] Prepare β-SF-EA with different masses (10, 20, 30, 40, 50 mg), evenly place them in several 50 mL centrifuge tubes, gently add 0.1 mL of anticoagulant dropwise onto the samples, and then immediately add 0.02 mL of 0.2 mol / L CaCl2 solution. After 5 min, gently add 25 mL of deionized water to the beaker, centrifuge at 300 rpm for 5 min, then take out the solution and measure its Abs value with an enzyme-labeling instrument at a wavelength of 540 nm. Set up a control: Add 0.1 mL of anticoagulant to the beaker, then add 25 mL of deionized water, and assume the Abs value measured at the same wavelength is 100 as the reference value. Then the blood coagulation index BCI is: BCI = 100 × A 样品 / A 对照 . The BCI index is an indicator reflecting the blood coagulation effect. The smaller the index, the better the blood coagulation effect. Figure 14 It shows that increasing the dosage (increasing the concentration in the same volume) will significantly reduce the blood coagulation index (BCI), indicating that the blood coagulation effect is positively correlated with the dosage.

[0094] Investigation of the hemostatic effect in Example 12

[0095] Male SD rats (about 250.0 g) were used to evaluate the hemostatic ability of β-SF-EA.

[0096] Rat tail amputation hemostasis experiment: SD rats were anesthetized by intraperitoneal injection with 1% pentobarbital sodium. Measure 6 cm from the end of the rat's tail, cut off the rat's tail with surgical scissors, and immediately sprinkle 20 mg of sample powder (β-SF-EA, Yunnan Baiyao, tranexamic acid) on the wound after bleeding, and start timing from the beginning of bleeding.

[0097] Hemostasis experiment on the liver wound surface of rats: SD rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital. The anesthetized rats were fixed supine on the dissection table, and a longitudinal incision was made along the midline of the abdomen to enter the abdominal cavity and expose the right lobe of the rat liver. The abdominal fluid was blotted dry with a clean gauze, and parallel wound surfaces about 2.0 cm long and about 0.5 cm deep were made on the right lobe of the liver with a scalpel. After the wound surface bled, it was immediately blotted dry with pre-weighed cotton, and then 20 mg of sample powder (EA, β-SF, β-SF-EA, Yunnan Baiyao, tranexamic acid) was respectively sprinkled on the wound surface, and the timing started. The wound surface was first compressed with cotton for 30 s to observe whether there was bleeding. If there was continuous oozing of blood, it was compressed with cotton for another 30 s and observed again. This was repeated until there was no obvious blood stain on the cotton surface, which was considered successful hemostasis, and the timing was stopped and the hemostasis time was recorded.

[0098] Hemostasis experiment on the thigh muscle wound of rats: SD rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital. The anesthetized rats were fixed supine on the dissection table, and the leg hair was shaved off with a rat hair clipper. After disinfecting the inner thigh with alcohol, a muscle wound about 3 cm long and about 1 cm deep was made with a scalpel. The timing started when the wound bled. After 5 s of bleeding, 20 mg of sample powder (EA, β-SF, β-SF-EA, Yunnan Baiyao, tranexamic acid) was respectively sprinkled, the hemostasis effect was observed, and the hemostasis time was recorded.

[0099] Figure 15 It is a diagram of the rat tail amputation experiment. When β-SF-EA was placed on the wound, the blood loss stopped quickly, and almost no blood stain was left on the filter paper. It could significantly promote hemostasis within 30 seconds; in the blank group, the bleeding exceeded five minutes, losing statistical significance and not listed in Figure B. As a control, the tranexamic acid group and the Yunnan Baiyao group required more than 4 times the hemostasis time, and the blood loss was significantly higher than that of the β-SF-EA group (P<0.05).

[0100] Figure 16 It is a diagram of the bleeding time of the rat liver and femoral artery. In the blank group, the bleeding of the femoral artery exceeded five minutes, losing statistical significance and not listed in the figure. Figure 16 It shows that β-SF-EA can significantly shorten the hemostasis time of the two models, and its hemostatic efficacy is significantly better than that of Yunnan Baiyao (P<0.05) and tranexamic acid (P<0.05), and is also significantly better than that of the EA group (P<0.05) and the β-SF group (P<0.05), indicating that silk fibroin and ellagic acid have a synergistic effect. The above results are consistent with the hemorheology and test tube tilt coagulation results in Example 11. In both of these rat bleeding models, it was observed that β-SF-EA had a faster sedimentation speed in the blood compared to other drugs and was easily quickly sedimented to the bleeding site, while the textures of Yunnan Baiyao, tranexamic acid, and β-SF powders were lighter and more likely to float on the surface of whole blood and be washed away by the blood flow, thus affecting their hemostatic effects.

[0101] Example 13 Safety Investigation

[0102] (1) Cell Resuscitation

[0103] Take out the cryopreserved LO2 cells and L929 cells, and gently shake them in a 37°C water bath until they are completely melted. In the laminar flow hood, transfer the cell suspension in the cryopreservation tube to a cell culture flask, add 4 mL of DMEM medium containing 10% fetal bovine serum to the culture flask, and gently pipette to disperse the cells evenly in the medium. Then place them in a 5% CO2, 37°C cell culture incubator for culture. After 6 hours of culture, disinfect the culture flask with alcohol and place it in the laminar flow hood. Aspirate the old medium, wash it, and then add 4 mL of medium again, and continue to culture in the cell culture incubator.

[0104] (2) CCK8 Quantification Method

[0105] Take 0.2 mg of β-SF-EA and EA respectively and dissolve them in 1 mL of DMSO, and set up gradient concentrations respectively.

[0106] Take cells in the logarithmic growth phase, wash them with PBS solution, add trypsin digestion solution to make the cells into a suspended state, calculate the cell density with a cell counting chamber, and then inoculate 1×10 5 cells / mL cells per well into a 96-well plate and place it in a cell culture incubator for 24 hours. After the cells adhere to the wall, aspirate the culture medium and wash twice with PBS. For the experimental groups: the extracts of β-SF-EA and EA, the blank group: DMEM medium containing 10% fetal bovine serum, and the positive group: the extract of paclitaxel, take 100 μL each and add them to the 96-well plate inoculated with cells. Set six parallel samples for each group, and then place them in a 37°C, 5% CO2 cell culture incubator for 48 hours. Aspirate the culture medium, wash twice with PBS, add 100 μL of CCK8 solution (90 μL of DMEM medium + 10 μL of CCK8), incubate in the dark in the incubator for 30 minutes, and then detect the absorbance (OD) at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Repeat 6 parallel samples for each time point. Calculate the relative cell proliferation rate according to the following formula: Relative proliferation rate (RGR)% = OD 实验组 / OD 空白组 *100%. Determine the cytotoxicity level according to the cytotoxicity grade in Table 4, where the positive control should not be lower than grade 3. The cell grade of 0-1 is qualified.

[0107] Table 4 Classification Criteria for Cell Proliferation Response

[0108] Relative cell proliferation / % ≥100 75-99 50-74 25-49 1-24 Level 0 1 2 3 4

[0109] We studied the toxicity of β-SF-EA and EA on L929 cells and LO2 cells through CCK8 experiments. Figure 17 As shown, the relative proliferation rates of all groups of β-SF-EA were higher than 85%. According to the toxicity grading standard of ISO10993-1, it indicated that the cytotoxicity of β-SF-EA was at level 0-1. Meanwhile, the addition of silk fibroin effectively improved the biocompatibility of ellagic acid. In summary, β-SF-EA did not affect cell proliferation and had good cell compatibility.

[0110] (2) Hemolysis test

[0111] Dilute 8 mL of fresh rabbit blood with 10 mL of normal saline. Put 20 mg of β-SF-EA into a 50 mL centrifuge tube, add 10 mL of normal saline, incubate in a water bath at 37 °C for 10 min, then add 0.2 mL of diluted rabbit blood, shake gently, after incubating in a water bath for 60 min, centrifuge at 1000 rpm for 5 min, take the supernatant, and measure the absorbance at 540 nm. The positive control group uses 10 mL of distilled water plus 0.2 mL of rabbit blood, and the negative control group uses 10 mL of normal saline plus 0.2 mL of rabbit blood, and the operation method is the same. Each group has three parallels. The hemolysis rate is calculated by the following formula: Hemolysis rate (%) = (Absorbance of test sample - Absorbance of negative control) / (Absorbance of positive control - Absorbance of negative control) * 100%.

[0112] When different materials come into direct contact with blood, it is possible to cause hemolysis due to the rupture of red blood cells. The hemolysis test evaluates the hemolysis situation of materials by checking the hemoglobin concentration. After calculation, the hemolysis rate of β-SF-EA was 1.13 ± 0.02% (Table 5). According to the provisions of GB / T4233.2, a hemolysis rate lower than 5% is considered qualified. Therefore, the prepared β-SF-EA meets international standards.

[0113] Table 5 Hemolysis rate (n = 3)

[0114]

[0115] Example 14 Investigation of degradation performance

[0116] Weigh a certain mass of β-SF-EA and place it in a PBS solution containing 0.1 μg / mL protease XIV, incubate at 37 °C, and use the PBS solution without protease XIV as the blank sample. Incubate the samples (n = 3) in PBS solution and PBS solution containing 0.1 μg / mL protease XIV for 5, 10, 15, 20, 25, 30 d. All degraded samples replace the fresh solution at a fixed time every day. Dry the degradation products at 60 °C, weigh the samples until they reach a constant weight, and the remaining mass retention rate R M is calculated by the following formula: R M / % = M dt / Mi *100%, where M i is the initial mass, M dt is the mass remaining after t days, in mg.

[0117] As a hemostatic preparation applied in vitro and in vivo, it is required to have good biodegradability itself. In an ideal state, at the initial stage of wound healing, the hemostatic powder can firmly bond tissues together to avoid cracking. Preferably, it degrades slowly. As the wound heals, the drug gradually degrades, and finally the remaining drug components can be completely degraded after the wound heals, absorbed by the human body or excreted from the body through metabolism. The degradation effect of β-SF-EA at 37 °C is as Figure 18 shown. It can be found that the degradation rate of β-SF-EA in PBS is relatively slow, and the remaining mass at the 30th day is 90.11 ± 3.51%. In the PBS solution containing protease XIV, the degradation effect of β-SF-EA can be roughly divided into two stages: the first stage (0 - 15 days), in this stage the degradation rate is relatively slow, and the remaining mass at 15 days is 67.83 ± 2.25%; the second stage (15 - 30 days), the degradation rate begins to increase significantly, and the remaining mass at 30 days is 24.50 ± 0.70%, and most of it has degraded, indicating that it has good degradability.

[0118] Preparation of silk fibroin hemostatic sponge loaded with polyhydroxyphenolic compounds in Example 15

[0119] Based on Example 1 or 2, add step (3): The silk fibroin complex loaded with polyhydroxyphenolic compounds obtained in step (2) is prepared by one of physical cross-linking method, chemical cross-linking method, sol-gel method, electrospinning method, freeze-drying method, phase separation method, 3D printing technology, and hydrogel method. The following are some examples of the preparation methods:

[0120] (1) Load the silk fibroin complex loaded with polyhydroxyphenolic compounds into a commercially available porous sponge (such as gelatin sponge, collagen sponge, etc.);

[0121] (2) Load the silk fibroin complex loaded with polyhydroxyphenolic compounds into a wood cellulose sponge;

[0122] (3) Prepared by adding a cross-linking agent to the silk fibroin complex loaded with polyhydroxyphenolic compounds;

[0123] (4) Prepared by using the silk fibroin complex loaded with polyhydroxyphenolic compounds through aerogel technology and electrospun nanofiber sponge technology;

[0124] (5) Prepared by adding a thickening agent and a plasticizer to the silk fibroin complex loaded with polyhydroxyphenolic compounds and then freeze-drying;

[0125] (6) The silk fibroin complex loaded with polyhydroxy phenolic compounds is prepared by freeze-drying method. (7)

[0127] Determination of the liquid absorption ratio of the completely liquid-absorbed in Example 16

[0128] In this example, the hemostatic sponge uses ellagic acid as the model drug, and the silk fibroin hemostatic sponge loaded with ellagic acid is prepared by chemical cross-linking method. The cross-linking agents selected are glutaraldehyde, genipin, transglutaminase and 1,4-butanediol diglycidyl ether. Weigh 5 g of the prepared hemostatic sponge and put it into anticoagulated whole blood. After the sponge absorbs the liquid and swells completely, take it out and weigh it after no liquid drips. Calculate the liquid absorption ratio A = (W w - W d ) / W d × 100%, where W w and W d are the wet weight and dry weight of the hemostatic sponge respectively. The results are shown in Table 6. After measurement, the pore sizes of the four hemostatic sponges are different, but the liquid absorption ratios are relatively high. Among them, the hemostatic sponge with a pore size in the range of 300 - 600 μm added with 0.5% transglutaminase performs the best. In Examples 17 - 19, the test samples are prepared by using the method in this example and the optimal cross-linking agent transglutaminase, and still labeled with β-SF-EA.

[0129] Table 6 Pore size distribution, liquid absorption ratio and liquid absorption rate against anticoagulant

[0130] Crosslinker type <![CDATA[1 , 4-butylene glycol diglycidyl ether]]> Genipin Glutaraldehyde Transglutaminase Pore size distribution / μm 300-750 400-800 350-700 300-600 Absorbing liquid magnification for anticoagulated whole blood / % 1082 1003 1095 1235

[0131] Anti-adhesion property test in Example 17

[0132] Use a medical wound sealant as a control. The anti-adhesion property is evaluated by using a rat back adhesion model. First, shave the back hair of the anesthetized rats (250 - 280 g, 8 weeks old) with a razor and use Veet hair removal cream for hair removal, then disinfect the back skin with 75% (by volume) ethanol. Make two 1-cm-long incisions on the back of the rats with a scalpel. Apply three materials (1 × 1.5 × 0.2 cubic centimeters), namely the medical wound sealant SF hemostatic sponge, β-SF-EA hemostatic sponge, to the wound and gently press to ensure that the wound fits well with the sponge for 2 hours to allow the blood clot to fully mature and solidify. Gently press the dressing to ensure good contact between the wound and the dressing. Fix the sponge at the lower edge of the tensiometer and vertically peel the sponge along the wound to measure the maximum peeling force during the process of peeling the sponge from the wound.

[0133] After 2 hours, the wound sealant When slightly peeled from the wound, it can be observed that it adheres to the tissue. Without peeling the material, the tissue is pulled up by more than 1 cm. After peeling the material, the wound is severely torn and secondary bleeding is observed. The SF hemostatic sponge can also be observed to adhere to the tissue and is not easily peeled from the wound. After peeling the material, secondary bleeding can be observed, but the degree of tearing is not deep. In contrast, the β-SF-EA hemostatic sponge can be easily peeled from the wound, and the wound is tightly closed without secondary bleeding.

[0134] Figure 19 The results in it show that the β-SF-EA hemostatic sponge shows the lowest wound peeling force of 234 mN, while the wound peeling force of the SF hemostatic sponge is 411 mN. Medical wound sealant closes the wound well, but its wound peeling force is 1081 mN, with strong adhesiveness, resulting in severe tearing and secondary bleeding of the wound during the peeling process. Therefore, the β-SF-EA hemostatic sponge significantly reduces the adhesion to tissues, facilitates easy peeling, and does not cause secondary damage.

[0135] Example 18 Determination of in vitro blood coagulation promotion index

[0136] The blood coagulation index BCI of the β-SF-EA hemostatic sponge was measured by the method in Example 11(4), Figure 20 and the results in it show that increasing the administration dose (increasing the concentration in the same volume) will cause a significant decrease in BCI, indicating that the blood coagulation effect is positively correlated with the administration dose.

[0137] Example 19 Investigation of hemostatic effect

[0138] The hemostatic effect of the β-SF-EA hemostatic sponge was measured by the method in Example 12, Figure 21 showing the results of the rat tail amputation hemostasis experiment. The Control control group (410 ± 3 s) had the longest hemostasis time, and the β-SF-EA hemostatic sponge group had the shortest hemostasis time (46.66 ± 5.37 s). The results of blood loss corresponded to this. The Control group (434 ± 2.23 mg) had the most blood loss, and the β-SF-EA group had the least (23.76 ± 3.05 mg). It is worth noting that compared with the β-SF sponge and the commercially available gelatin sponge, the β-SF-EA hemostatic sponge significantly had a shorter hemostasis time and less blood loss. Figure 22 in the rat femoral artery injury model experiment and Figure 23 the liver bleeding model also showed the shortest coagulation time and the least blood loss in the β-SF-EA hemostatic sponge group. In summary, it is proved that the β-SF-EA hemostatic sponge has excellent hemostatic ability.

[0139] Example 20 Investigation of degradation performance

[0140] Clinically, hemostatic materials are often used to treat wounds or incisions in the human body. Once the hemostatic effect is achieved, they need to be removed from the human body and processed. Therefore, their in vitro biodegradability needs to be considered. The in vitro degradation performance of the β-SF-EA hemostatic sponge was measured by the method in Example 14. The incubation time was changed to 2, 4, 6, 8, 10, and 12 d. After the degradation products were freeze-dried for 36 h, they were weighed, and the other conditions remained unchanged. Figure 24 The results showed that the β-SF-EA hemostatic sponge was similar to β-SF-EA at 37°C. It had a slow degradation rate in PBS, and the remaining mass at the 12th day was 91.61 ± 3.51%. In the PBS solution containing protease XIV, the degradation effect of the β-SF-EA hemostatic sponge could be roughly divided into two stages: the first stage (0-6 d), in which the degradation rate was relatively slow, and the remaining mass at 6 d was 71.96 ± 2.25%; the second stage (6-12 d), in which the degradation rate began to increase significantly, and the remaining mass at 12 d was 26.25 ± 0.60%, indicating that most of it had been degraded, which showed that it had good degradability.

Claims

1. A silk fibroin hemostatic sponge loaded with polyhydroxyphenolic compounds, characterized in that: A hemostatic sponge is prepared from a silk fibroin complex loaded with a polyhydroxy phenolic compound. The silk fibroin complex loaded with the polyhydroxy phenolic compound is prepared by self-assembly of the polyhydroxy phenolic compound and silk fibroin, wherein the silk fibroin is treated by a β-sheet conformational transition process, and the feeding ratio of the polyhydroxy phenolic compound to silk fibroin is a mass ratio of <1:1; the hemostatic sponge is prepared by one of physical cross-linking method, chemical cross-linking method, sol-gel method, electrospinning method, freeze-drying method, phase separation method, 3D printing technology, and hydrogel method.

2. The silk fibroin hemostatic sponge as described in claim 1, wherein: The polyhydroxy phenolic compound is selected from one or more of resveratrol, quercetin, baicalein, kaempferol, lignan, curcumin, ellagic acid, chlorogenic acid, ferulic acid, caffeic acid, syringic acid, sinapic acid, rutin, myricetin, magnolol, fisetin, puerarin, rhein, lonicerin, procyanidin, p-coumaric acid, vanillic acid, hesperetin, naringenin, luteolin, genistein, emodin, oleanolic acid, epigallocatechin gallate, robinin, tea polyphenols, epigallocatechin, salvianol, gallic acid, pyrogallic acid, catechin, tannic acid.

3. The fibroin hemostatic sponge as described in claim 2, characterized in that: The polyhydroxy phenolic compound is a poorly soluble polyhydroxy phenolic compound.

4. The fibroin hemostatic sponge as described in claim 3, characterized in that: The poorly soluble polyhydroxy phenolic compound is selected from one or more of resveratrol, quercetin, baicalein, kaempferol, lignan, curcumin, ellagic acid, chlorogenic acid, ferulic acid, caffeic acid, syringic acid, sinapic acid, rutin, myricetin, magnolol, fisetin, puerarin, rhein, lonicerin, procyanidin, p-coumaric acid, vanillic acid, hesperetin, naringenin, luteolin, genistein, emodin, oleanolic acid, epigallocatechin gallate.

5. The fibroin hemostatic sponge according to claim 4, wherein: The poorly soluble polyhydroxy phenolic compound is ellagic acid.

6. The fibroin hemostatic sponge according to claim 5, wherein: The feeding ratio of ellagic acid to silk fibroin is a mass ratio of 1:2 to 1:

16.

7. The fibroin hemostatic sponge according to claim 6, wherein: The feeding ratio of ellagic acid to silk fibroin is a mass ratio of 1:

4.

8. The preparation method of the silk fibroin hemostatic sponge according to any one of claims 1 to 7 comprises three steps: silk fibroin conformational transition, self-assembly with polyhydroxyphenolic compounds, and preparation of the hemostatic sponge: Step (1): A silk fibroin solution is prepared by a process of transforming silk fibroin from an α-helix to a β-sheet conformation. The conformational change can be achieved by chemical methods or physical methods. Chemical methods include promoting the transition through polyols, polylactic acid, metal ions, pH, or hydroxypropyl methylcellulose. Physical methods include promoting the transition through high temperature, hydrostatic pressure, ultra-low temperature storage, freeze-drying, shear force, ultrasonic waves, eddy current, laser irradiation, or high-pressure carbon dioxide treatment; Step (2): After dissolving or suspending the polyhydroxyphenolic compound, it is mixed and stirred with the silk fibroin solution obtained in Step (1) to cause self-assembly, thereby obtaining a silk fibroin complex loaded with the polyhydroxyphenolic compound; Step (3): The silk fibroin complex loaded with the polyhydroxyphenolic compound obtained in Step (2) is prepared into a silk fibroin hemostatic sponge loaded with the polyhydroxyphenolic compound by one of physical cross-linking method, chemical cross-linking method, sol-gel method, electrospinning method, freeze-drying method, phase separation method, 3D printing technology, or hydrogel method.

9. The preparation method according to claim 8, characterized in that: In Step (1), polyols are used to promote the transition of silk fibroin to the β-sheet conformation.

10. The preparation method according to claim 9, characterized in that: The polyol is ethanol.

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