Silk fibroin complex loaded with polyhydroxy phenolic compound, preparation method therefor, and use thereof

Abstract

Aiming at the inadequacy of existing hemostatic materials for large-scale trauma and serious bleeding, the present invention provides a silk fibroin complex loaded with a polyhydroxy phenolic compound and a preparation method therefor. The complex is prepared by self-assembly of the polyhydroxy phenolic compound and the silk fibroin. The silk fibroin is subjected to a β-sheet conformation transformation process. The polyhydroxy phenolic compound is preferably an indissoluble compound, most preferably ellagic acid. The feeding ratio of the polyhydroxy phenolic compound to the silk fibroin is a mass ratio of ˂ 1:1, preferably 1:2 to 1:16, more preferably 1:4. The hemostatic ability of the complex is significantly superior to that of the polyhydroxy phenolic compound, the silk fibroin, Yunnan Baiyao, tranexamic acid, chitosan hemostatic powder, and the like. Meanwhile, the complex has a larger particle size and mass than the silk fibroin, and can rapidly settle to a bleeding site in blood. The complex exhibits no cytotoxicity, is blood-insoluble, and is capable of self-degradation in vivo, demonstrating high safety. Meanwhile, the complex has the characteristics of wound healing, antibiosis, infection resistance, adhesion resistance, and the like, and can be applied to the preparation of corresponding formulations.

Description

A polyhydroxyphenol compound-loaded silk fibroin composite and its preparation method and application Technical Field

[0001] The present invention belongs to the field of material technology, and in particular relates to a silk fibroin composite loaded with polyhydroxyphenol compounds, and a preparation method and application thereof. Background Art

[0002] Hemorrhage, the sudden and severe loss of blood due to a ruptured blood vessel, is one of the most common causes of death worldwide. Deaths from surgical bleeding and war-related hemorrhage are the most common. According to statistics, severe hemorrhage accounts for over 35% of pre-hospital deaths and over 40% of deaths within 24 hours of injury. Extensive blood loss in modern warfare has increased significantly, with deaths within 30 minutes of injury accounting for 80% of all combat fatalities. On the battlefield, the first hour after a casualty's injury is known as the "golden hour" of emergency care. Consequently, the cost and technological research of hemostatic materials not only place a significant burden on the global economy but also present a pressing challenge in any emergency scenario.

[0003] The current hemostatic market is dominated by absorbable hemostatic materials, including gauze, sponges, sprays, and hemostatic powders. Their primary components are natural polymers such as cellulose, chitosan, and fibrin. Their main advantages include short hemostatic times, biodegradability, relatively mild foreign body reactions, the elimination of secondary surgical removal, and the avoidance of secondary injury to the patient. However, their high cost, variable absorption times, limited adaptability, and poor efficacy against major trauma and severe bleeding limit their clinical application. For example, oxidized regenerated cellulose hemostatic materials contain numerous acidic groups, reaching a pH of 1.7 after being fully saturated with water. While these materials exhibit some antibacterial properties in acidic environments, residual material in the wound can cause a strong inflammatory response, hinder bone regeneration, and even form granulomas and abscesses. Therefore, the development of a fast, efficient, cost-effective, and non-toxic absorbable hemostatic material is essential.

[0004] Silk fibroin, a natural high-molecular-weight protein extracted from silk, possesses excellent in vivo degradability, biocompatibility, and anti-inflammatory properties. In its hemostatic mechanism, silk fibroin activates coagulation factor XII, thereby triggering the endogenous coagulation cascade. It is a biomacromolecule composed of 5509 amino acids, consisting of a heavy chain (391 kDa) and a light chain (26 kDa), linked by disulfide bonds. When silk fibroin dissolved in a LiBr solution or a ternary solution is added to an excess of a polar organic solvent, such as alcohol, a white suspension of protein complexes immediately forms. While silk fibroin is maintained in solution, due to multiple interactions between its side chains and salt ions and water molecules, the peptide chains exist in a random coil conformation, known as an α-helix. This state is also the natural conformation of silk fibroin. In this state, silk fibroin lacks the ability to self-assemble with polyhydroxyphenolic compounds. This is because the steric hindrance of the benzene rings of the amino acid residues makes it difficult for polyhydroxyphenolic compounds to integrate into the α-helical structure, resulting in their exposure on the outside of the silk fibroin molecule. When an antisolvent such as ethanol is used, the peptide chain will be induced to rapidly fold toward 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 polyhydroxyphenol compounds.

[0005] Currently available silk fibroin hemostatic drugs cannot overcome the obstruction of the blood interface hydration layer, have poor adhesion to bleeding wounds, and cannot meet the tissue strength adhesion requirements in a wet environment. However, polyhydroxyphenol compounds can form a secondary cross-linked structure with silk fibroin, thereby recruiting more platelets and solving the problem of silk fibroin being washed away by blood. At the same time, polyhydroxyphenol compounds and silk fibroin can also show a synergistic effect, promoting endogenous hemostasis.

[0006] Summary of the Invention

[0007] The purpose of the present invention is to solve the pain points of the existing technology, such as poor effect on large trauma and severe bleeding, insufficient blood absorption and blood seepage ability, and inability to treat irregular wounds. A silk fibroin complex loaded with polyhydroxyphenol compounds and its preparation method and application are provided. The technical solution is as follows:

[0008] The present invention provides a silk fibroin composite loaded with a polyhydroxyphenol compound, which is prepared by self-assembly of the polyhydroxyphenol compound and the silk fibroin; wherein the silk fibroin is treated by a β-folding conformational transformation process, and the feeding ratio of the polyhydroxyphenol compound to the silk fibroin is a mass ratio of less than 1:1, preferably 1:2 to 1:16, and most preferably 1:4.

[0009] As a further improvement, the polyhydroxyphenol compound is selected from one or more of resveratrol, quercetin, baicalein, kaempferol, lignans, curcumin, ellagic acid, chlorogenic acid, ferulic acid, caffeic acid, syringic acid, sinapinic acid, rutin, myricetin, magnolic acid, fisetin, puerarin, rhein, honeysuckle, proanthocyanidins, p-coumaric acid, vanillic acid, hesperidin, naringenin, luteolin, genistein, emodin, oleanolic acid, epigallocatechin gallate, acaciaside, tea polyphenols, epigallocatechin, salvianolic acid, gallic acid, pyrogallol, catechin, and tannic acid.

[0010] As a further improvement, the polyhydroxyphenol compound is a poorly soluble polyhydroxyphenol compound, preferably one or more of resveratrol, quercetin, baicalein, kaempferol, lignans, curcumin, ellagic acid, chlorogenic acid, ferulic acid, caffeic acid, syringic acid, sinapinic acid, rutin, myricetin, magnolic acid, fisetin, puerarin, rhein, honeysuckle, proanthocyanidins, p-coumaric acid, vanillic acid, hesperetin, naringenin, luteolin, genistein, emodin, oleanolic acid, and epigallocatechin gallate, most preferably ellagic acid.

[0011] As the most preferred ellagic acid, the feed ratio of ellagic acid to silk fibroin is preferably 1:2 to 1:16 by mass, more preferably 1:4 by mass.

[0012] The present invention also provides a method for preparing the above-mentioned silk fibroin complex, comprising two steps: silk fibroin conformational transformation and self-assembly with a polyhydroxyphenol compound: step (1): preparing a silk fibroin solution by a process of transforming the silk fibroin from an α-helix to a β-sheet conformation, wherein the conformational transformation can be achieved by chemical or physical methods, wherein the chemical method comprises promoting the transformation by polyols, polylactic acid, metal ions, pH or hydroxypropyl methylcellulose, and the physical method comprises promoting the transformation by high temperature, hydration and pressure, ultralow temperature placement, freeze drying, shear force, ultrasound, eddy current, laser irradiation, and high-pressure carbon dioxide treatment; step (2): dissolving or suspending the polyhydroxyphenol compound, and then mixing and stirring the polyhydroxyphenol compound with the silk fibroin solution obtained in step (1) to cause self-assembly, thereby obtaining a silk fibroin complex loaded with the polyhydroxyphenol compound. In step (1), preferably, a polyol is used to promote the transformation of the β-sheet conformation of the silk fibroin, and more preferably, the polyol is ethanol.

[0013] As an improvement to the preparation method, the polyhydroxyphenol compound is a poorly soluble polyhydroxyphenol compound, and in step (2), the poorly soluble polyhydroxyphenol 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.

[0014] When the poorly soluble polyhydroxyphenol compound is ellagic acid, the preferred organic solvent is propylene glycol. After the ellagic acid suspension is incubated with silk fibroin in step (2), the final concentration of propylene glycol in the resulting solution is 5-20%, and the most preferred final concentration of propylene glycol is 10%.

[0015] The present invention provides the use of the silk fibroin complex in preparing a preparation having hemostatic efficacy, comprising the silk fibroin complex and a pharmaceutically acceptable excipient. The preparation can be in the form of gauze, sponge, spinning agent, spray, powder, granule, gel, sealant, ointment, film, patch, or embolic agent.

[0016] The present invention also provides the use of the silk fibroin complex in preparing a preparation having wound healing, and / or anti-infection, and / or anti-adhesion effects, comprising the silk fibroin complex and a pharmaceutically acceptable excipient.

[0017] The beneficial effects of the present invention are:

[0018] The silk fibroin protein complex loaded with polyhydroxyphenol compounds developed by the present invention is an effective hemostatic agent. Its hemostatic and coagulation ability is significantly better than that of polyhydroxyphenol compounds and silk fibroin itself, indicating that the two materials have a synergistic effect. At the same time, the hemostatic effect of the hemostatic agent is significantly better than that of a variety of hemostatic products on the market, such as Yunnan Baiyao, tranexamic acid, chitosan hemostatic powder, etc. The silk fibroin protein complex provided by the present invention has a larger particle size, heavier texture, more stable mechanical strength and strong wet tissue adhesion than its silk fibroin. Compared with other drugs, it has a faster sedimentation rate in the blood and is easy to quickly settle to the bleeding site. The powder texture of Yunnan Baiyao, tranexamic acid, chitosan hemostatic powder and silk fibroin is lighter and more likely to float on the surface of whole blood and be washed away by the blood flow, thereby affecting its hemostatic effect. The silk fibroin protein complex hemostatic agent is non-cytotoxic, non-hemolytic, self-degradable in the body, and highly safe. At the same time, the silk fibroin protein complex also has wound healing, antibacterial, anti-infection, anti-adhesion and other effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present invention will be further described below with reference to the accompanying drawings:

[0020] Figure 1 Fourier transform infrared spectroscopy (FTIR).

[0021] Figures 2 to 4 show comparisons of blood viscosity (n=3). In each figure, Panel 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-polyhydroxyphenol 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.

[0022] Figure 5 Comparison of coagulation time (n=3), *, compared with the β-SF-EA group, *P<0.05, **P<0.01, ***P<0.001.

[0023] Figure 6 Comparison of drug loading (n=3). *, compared with the β-SF-EA group, *P<0.05, **P<0.01, ***P<0.001; ND means not detected.

[0024] Figures 7 and 8 show comparison of drug loading (n=3). *, compared with the water group, *P<0.05, **P<0.01, ***P<0.001.

[0025] Figure 9 Fourier transform infrared spectroscopy (FTIR) analysis, where EA and β-SF are physically mixed in a 1:1 (w / w) ratio.

[0026] Figure 10 Molecular docking diagram. L74 represents Leu (leucine) at position 74, and D27 represents Asp (aspartic acid) at position 27.

[0027] Figure 11 Characterization: (A) FTIR spectrum, (B) XRD, (C) TG, (D) 1H NMR.

[0028] Figure 12. Blood adsorption experiment. In the experiment, a is blank, b is β-SF, c is EA, d is Yunnan Baiyao, e is tranexamic acid, and f is β-SF-EA. The same as in Figure 13.

[0029] Figure 13: Diagram of the test tube tilt coagulation experiment.

[0030] Figure 14 shows blood coagulation index (BCI) (n=3).

[0031] Figure 15: Rat tail amputation experiment. Panel A shows bleeding after drug powder application immediately after tail amputation, and Panel B shows the time it takes for bleeding to stop after tail amputation. *, compared with the β-SF-EA group, *P < 0.05, **P < 0.01, ***P < 0.001.

[0032] Figure 16 shows the bleeding time of rat liver and femoral artery (n=3). *, compared with the β-SF-EA group, *P<0.05, **P<0.01, ***P<0.001.

[0033] Figure 17 Relative cell proliferation rate of LO2 cells and L929 cells after co-incubation with drugs for 48 hours (n=6).

[0034] FIG18 shows the in vitro degradation of β-SF-EA (n=3).

[0035] FIG19 Characterization of ellagic acid-loaded silk fibroin nanoparticle hemostatic powder (β-SFN-EA).

[0036] Figure 20: Bleeding time of the auricular artery, liver and femoral artery of rabbits (n=6).

[0037] Figure 21 shows the amount of bleeding in the auricular artery, liver, and femoral artery of rabbits (n=6).

[0038] Figure 22 In vivo biocompatibility: H&E and Masson's trichrome staining of surrounding tissues isolated 7, 15, and 30 days after subcutaneous implantation of the material.

[0039] Figure 23 Comparison of wound closure rates (n=3), *, 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.

[0040] Figure 24 H&E staining of wounds.

[0041] Figure 25 H&E staining of rabbit femoral artery wound.

[0042] FIG26 Comparison of liver healing rates (n=3), *, compared with the β-SF-EA group, *P<0.05, **P<0.01, ***P<0.001. DETAILED DESCRIPTION

[0043] Example 1 Preparation of polyhydroxyphenol compound-loaded silk fibroin complex (β-SF-polyhydroxyphenol compound)

[0044] Step (1) Transformation of the β-fold conformation of silk fibroin: Dilute silk fibroin (SF) 5 to 10 times with water and then slowly pour it into a vigorously stirred excess organic solvent, stirring for 2 to 4 hours to complete the transformation of the β-fold conformation, remove the liquid portion by mechanical separation methods such as filtration, centrifugation, and membrane filtration, and repeatedly wash the remaining portion and then freeze-dry to obtain β-folded 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, such as high-speed centrifugation at 10,000 rpm for 15 to 20 minutes.

[0045] Fourier transform infrared spectroscopy (FTIR) analysis: 5% of the sample was mixed with 95% KBr and ground into a fine powder. The sample was then scanned using a Nicolet is5 spectrometer in transmission mode. Each spectrum was obtained in transmission mode (ten scans) with a resolution of 4 cm -1 , the spectral range is 4000-500cm -1 The FTIR spectrum of the relatively high level of β-sheet structure is at 1615-1640 cm -1 、1510-1525cm -1 The FTIR spectrum of SF in its natural state shows a sharp peak at 1640-1660 cm -1 、1535-1542cm -1 The characteristic peak of natural α-SF is 1642 cm -1 and 1535cm -1 The characteristic peak of SF after treatment in step (1) is 1620 cm -1 and 1517cm -1 , it can be seen that SF has completed the conformational transition of β-folding and become β-SF.

[0046] Step (2) Self-assembly of polyhydroxyphenol compounds 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; the polyhydroxyphenol compound is dissolved or suspended with a solvent, wherein water is used as a solvent for water-soluble polyhydroxyphenol compounds and water or an organic solvent such as ethanol, propylene glycol, or butylene glycol is used as a solvent for poorly soluble polyhydroxyphenol compounds. The prepared solution or suspension is added dropwise to the β-SF suspension, and the mixture is stirred for 12-48 hours to obtain a silk fibroin complex loaded with polyhydroxyphenol compounds.

[0047] The polyhydroxyphenol compound is selected from the group consisting of water-insoluble polyhydroxyphenol compounds, including ellagic acid (EA), resveratrol (RES), quercetin (QUE), baicalein (BAE), kaempferol (KAE), lignans (Lignan), curcumin (CUR), chlorogenic acid (CGA), ferulic acid (FA), caffeic acid (CFA), syringic acid (SA), sinapic acid (Sinapic Acid), rutin (RUT), myricetin (MYR), magnolic acid (MAG), chrysanthemum acid (CH), linalool (FL), linalool (FL), linalool (FL), linalool (FL), linalool (FL), linalool (FL), linalool (FL), linalool (FL), linalool (FL), linalool (FL), linalool (FL), linalool (FL), linalool (FL), linalool (FL), linalool (FL), linalool (FL), linalool (FL), linalool (FL), linalool (FL), linalool (FL), linalool (FL), linalool (FL), linalool (FL), linalool (FL), linalool (FL), linalool (FL), linalool 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 polyhydroxyphenol compounds, including Gallic Acid (GA), Pyrogallic Acid (PGA), Acid, PA), catechin (C), tannic acid (TA), tea polyphenols (TP), acaciaside (Acaciin), epigallocatechin (EGC), etc.

[0048] Example 2 Preparation of Silk Fibroin Composites Loaded with Polyhydroxyphenol Compounds

[0049] The difference from Example 1 is that the organic solvent in step (1) is ethanol, and the mechanical separation method is high-speed centrifugation at 10,000 rpm for 15 to 20 minutes. In step (2), equal amounts of polyhydroxyphenol compounds and silk fibroin are added. In the preparation of the polyhydroxyphenol compound suspension, water is used as the solvent for the water-soluble polyhydroxyphenol compound, and water or an organic solvent is used as the solvent for the poorly soluble polyhydroxyphenol compound. The mechanical separation method is to take the precipitate after high-speed or ultra-high-speed centrifugation. Step (3) of drying to powder is added: the solution obtained in step (2) is dried to powder after removing the liquid by mechanical separation methods such as suction filtration, centrifugation, membrane filtration, sedimentation, or a spray dryer or other drying equipment is used to pulverize the solution in one step, thereby obtaining a silk fibroin complex loaded with polyhydroxyphenol compounds; the mechanical separation method is preferably to take the precipitate after high-speed or ultra-high-speed centrifugation.

[0050] Using poorly soluble polyhydroxyphenol 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), sinapinic acid-silk fibroin complex (β-SF-Sinapic acid-silk fibroin complex) were prepared. Acid), rutin-fibroin complex (β-SF-RUT), myricetin-fibroin complex (β-SF-MYR), magnolonic acid-fibroin complex (β-SF-MA), fisetin-fibroin complex (β-SF-FIS), puerarin-fibroin complex (β-SF-PUE), rhein-fibroin complex (β-SF-RHE), honeysuckle-fibroin complex (β-SF-Lon), proanthocyanidin-fibroin complex (β-SF-PC), p-coumaric acid-fibroin complex (β-SF-p-CA), vanillic acid-fibroin complex (β-SF-VA), hesperidin-fibroin complex (β-SF-Hes), naringenin-fibroin complex (β-SF-NAR), luteolin-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), salvianol-silk fibroin complex (β-SF-DS), epigallocatechin gallate-silk fibroin complex (β-SF-EGCG); using water-soluble polyhydroxyphenol compounds as raw materials, gallic acid-silk fibroin complex (β-SF-GA), pyrogallol-silk fibroin complex (β-SF-PA), catechin-silk fibroin complex (β-SF-C), tannic acid-silk fibroin complex (β-SF-TA), acaciaside-silk fibroin complex (β-SF-Acaciin), epigallocatechin-silk fibroin complex (β-SF-EGC), and tea polyphenol-silk fibroin complex (β-SF-TP) were prepared.

[0051] Example 3 Investigation of coagulation effect

[0052] The obtained β-SF-polyhydroxyphenol compounds were prepared into 2 mg / mL test solution with normal saline for investigation of coagulation effect: blood was collected from the abdominal aorta of New Zealand white rabbits and placed in a sodium heparin anticoagulant tube. 2 mL of the test solution was added to the tube, with normal saline as the control. The solution was mixed and incubated at 37°C for 15 minutes. After that, the whole blood viscosity was detected with an automatic blood rheometer (all operations were completed within 2 hours after the blood was collected).

[0053] Whole blood viscosity is the result of friction between blood cells and plasma protein molecules during blood flow and is the most important indicator in hemorheology. Increased blood viscosity indicates obstructed blood flow, decreased red blood cell deformability, and increased aggregation, reducing blood flow into microvessels and capillaries and decreasing blood flow capacity. The results in Figures 2-4 show that the polyhydroxyphenol-loaded silk fibroin complex (β-SF-polyhydroxyphenol) significantly increased whole blood viscosity compared to the blank control (P < 0.05), and significantly outperformed the effect of β-SF combined with the original compound alone (P < 0.05), indicating that the complex formation significantly enhances coagulation efficacy. Overall, the self-assembled complex of silk fibroin and poorly soluble polyhydroxyphenols was superior to the self-assembled complex of silk fibroin and water-soluble polyhydroxyphenols.

[0054] Weigh 0.1g of the drug powder to be tested into a 1.5mL centrifuge tube. Simultaneously, measure 0.5mL of anticoagulated blood and add it to the centrifuge tubes containing the different drugs. Place the tubes vertically on the laboratory bench and start the timer. After 30 seconds, rotate and invert the tubes to observe whether the blood flows. Repeat this process until the anticoagulated blood stops flowing. Stop the timer and record the clotting time. The results in Figure 5 show a similar trend to those in Figures 2 to 4.

[0055] Of all the complexes, β-SF-EA exhibited the best coagulation effect. This may be due to the multiple hydrogen bonds and planar structure of EA. On the one hand, the presence of multiple hydrogen bonds increases the synergistic effect between hydrogen bonds, further enhancing their stability. On the other hand, ellagic acid possesses a symmetrical catechol structure and a planar structure, which can form intra- and intermolecular hydrogen bonds, enhancing the rigidity of the complex molecules. This stable molecular structure reduces molecular dynamics, thereby indirectly enhancing the stability of intermolecular hydrogen bonds.

[0056] Example 4 Preparation of Polyhydroxyphenol Compound-Silk Fibroin Complex (α-SF-Polyhydroxyphenol Compound)

[0057] The difference from Example 2 is that step (1) is deleted, and in step (2), β-SF is replaced by untreated SF, that is, SF with a natural α-helical conformation. In step (2), equal amounts of polyhydroxyphenol compound and silk fibroin are added to prepare a polyhydroxyphenol compound-α-silk fibroin complex, which is called α-SF-polyhydroxyphenol compound.

[0058] Example 5 Preparation of Polyhydroxyphenol Compound-Silk Fibroin Complex (SH-SF-Polyhydroxyphenol Compound)

[0059] The difference from Example 2 is that the following method is used in step (1): dissolving silk fibroin in a solution containing tri(2- The SF solution is placed in an aqueous solution of (acid)-hydroxyethyl)phosphine hydrochloride at room temperature for a reduction reaction for 5 minutes to obtain a 1.0-5.0 wt% SF solution after the reduction reaction. The remaining steps are the same as those in Example 2, except that equal amounts of polyhydroxyphenol compound and silk fibroin are added in step (2). The resulting polyhydroxyphenol compound-silk fibroin complex is referred to as SH-SF-polyhydroxyphenol compound. This method utilizes a method of breaking disulfide bonds to expose hydrophobic amino acids to bind polyhydroxyphenol compounds such as ellagic acid.

[0060] Example 6 Self-assembly performance investigation

[0061] Using ellagic acid as a model drug for polyhydroxyphenol compounds, three ellagic acid-silk fibroin complexes were prepared according to the procedures of Examples 2, 4, and 5. Water was used as the solvent for EA, namely β-SF-EA, α-SF-EA, and SH-SF-EA. The assembly efficiency was evaluated by drug loading and encapsulation efficiency, calculated using Formulas 1 and 2, respectively. The "amount of EA in the silk fibroin complex" was calculated using the difference method: the amount of EA added minus the amount released after incubation with SF. EA content was analyzed by high-performance liquid chromatography (HPLC) using a Hedera C18 column, a mobile phase of acetonitrile:water (20:80 v / v), a flow rate of 1.0 mL / min, a detection wavelength of 254 nm, an injection volume of 10 μL, and a spectral acquisition time of 15 min. The drug loading and encapsulation efficiency were calculated using the following formulas: Drug loading = amount of EA in the silk fibroin complex / total amount of SF and EA (Formula 1) Encapsulation efficiency = amount of EA in the silk fibroin complex / amount of EA (Formula 2)

[0062] As shown in the results in Figure 6, the drug loading capacity is: β-SF-EA>SH-SF-EA>>α-SF-EA, among which the drug loading capacity of β-SF-EA reaches 9.6%, while EA is almost undetectable in α-SF-EA, indicating that the efficiency of self-assembly of α-helical silk protein with ellagic acid is very low, highlighting the necessity of conformational flipping of silk protein; SH-SF-EA uses the destruction of disulfide bonds in silk protein molecules to expose its hydrophobic amino acids, thereby completing self-assembly with ellagic acid, but the drug loading capacity is only 4.5%, much lower than that of β-SF-EA (P<0.001).

[0063] Example 7 Investigation of the effect of solvent on EA drug loading

[0064] Ellagic acid was used as a model drug for polyhydroxyphenol compounds. β-SF-EA was prepared using the steps in Example 2. In step (2), a suspension of the polyhydroxyphenol compound was prepared using water, ethanol, propylene glycol, butylene glycol, and other solvents. If an organic solvent was used as the suspension solvent, the final concentration of the organic solvent in the resulting solution after incubation of the polyhydroxyphenol compound suspension with β-SF was 10%. The drug loading capacity of these solutions was then investigated using the method in Example 6. The results in Figure 7 show that the addition of organic solvents effectively increased the drug loading capacity of ellagic acid, with the order of propylene glycol > butylene glycol > ethanol > water. The highest drug loading capacity was achieved when propylene glycol was used, reaching 19.57±1.01%.

[0065] Next, the effect of propylene glycol concentration on EA drug loading was investigated. In step (2), propylene glycol was used as the solvent to prepare the EA suspension. After incubation with β-SF, the final propylene glycol concentrations in the resulting solutions were 0%, 5%, 10%, 15%, and 20%, respectively. The results in Figure 8 demonstrate that the drug loading in the propylene glycol aqueous solution group was superior to that in the water group, with the 10% propylene glycol aqueous solution group achieving the best effect, reaching a drug loading of 19.57±1.01%.

[0066] Example 8 Investigation of different EA:SF feed ratios

[0067] Ellagic acid was used as a model drug for polyhydroxyphenol compounds, and β-SF-EA was prepared using the steps in Example 2, wherein propylene glycol was used as a solvent to prepare an EA suspension in step (2), and the final concentration of propylene glycol in the solution obtained after co-incubation with β-SF was 10%; at the same time, the feed ratio of EA to β-SF was controlled to be EA:β-SF = 1:0.5, 1:1, 1:2, 1:4, 1:8, 1:12, 1:16 (w / w). As shown in Table 1, the drug loading and encapsulation efficiency of EA were almost not observed in the EA:β-SF = 1:0.5 and 1:1 groups, while they were observed in the other groups; at the same time, Fourier transform infrared spectroscopy analysis of each group of samples was performed using the method in Example 1 (Figure 9), and it was found that the EA hydroxyl peak (3471cm) was observed in the EA:β-SF = 1:2, 1:4, 1:8, 1:12, 1:16 (w / w) groups. -1 ), indicating that EA and β-SF have completed hydrogen bonding and self-assembled successfully. However, the EA hydroxyl peak still exists in the EA:β-SF = 1:0.5 and 1:1 groups and their physical mixtures, indicating that self-assembly of the two molecules cannot be achieved. The above results show that the self-assembly of β-SF and EA can only be achieved when the feed ratio EA:β-SF is less than 1:1 (w / w). Among them, EA:β-SF = 1:4 can achieve the maximum EA drug loading and encapsulation efficiency (Table 1), which is called the optimal feed ratio.

[0068] The method in Example 3 was further used to investigate the effects of different EA:β-SF feed ratios on whole blood viscosity. The results in Table 2 showed that the EA:β-SF <1:1 groups significantly increased whole blood viscosity at low, medium, and high shear rates compared with the normal saline group (P < 0.5), and were higher than those of the EA and β-SF groups (P < 0.5), indicating that the β-SF-EA prepared with an EA:β-SF <1:1 feed ratio had significant coagulation efficacy, and that EA and β-SF enhanced the effect synergistically; among them, EA:β-SF = 1:4 had the best coagulation effect.

[0069] In summary, the preparation process of β-SF-EA is to use β-folded SF for preparation, with a feed ratio of EA:β-SF <1:1 (w / w), and the specific steps are shown in Example 1; among which the optimal process is EA:β-SF = 1:4 (w / w), and the ellagic acid in step (2) is prepared as a suspension using propylene glycol as a solvent, and the final concentration of propylene glycol in the resulting solution after incubation with SF is 10%. Without the transformation of the β-folded conformation of SF or the feed ratio EA:β-SF ≥ 1:1, the self-assembly of EA and β-SF cannot be achieved. When EA:β-SF = 1:2 (w / w), the drug loading capacity and encapsulation efficiency are both low, indicating that although the self-assembly of EA and β-SF can be achieved at this time, the loss is large.

[0070] Table 1 Effect of the feed ratio of β-SF to EA on drug loading and encapsulation efficiency (n=3) ND stands for Not Detected.

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

[0072] Example 9 Molecular docking of silk fibroin complex

[0073] Computer simulation study of the binding mode of ellagic acid and silk fibroin was conducted. After obtaining the silk fibroin heavy chain crystal structure (PDB ID: 3UA0) from PDB bank, due to the lack of previous studies on the binding mode, we In 2018, the SiteMap module was used to predict binding sites. After hydrogenation of the silk fibroin crystal structure and removal of irrelevant ions, the ellagic acid small molecule was converted to a 3D conformation using the LigPrep module, outputting up to 32 isomers. Finally, Glide extra precision (XP) was used to output 20 binding poses. As shown in Figure 10, after the two phenolic hydroxyl groups in the ellagic acid small molecule form a hydrogen bond with L74, they also need to form a hydrogen bond with D27 on the other heavy chain, indicating that further hydrogen bonding with the other two highly symmetrical hydroxyl groups in ellagic acid is required to achieve stable binding. Furthermore, since the heavy chains in silk fibroin maintain a highly similar β-sheet secondary structure, the ellagic acid small molecule may not have sufficient hydrophobic interactions with the two heavy chains to maintain stability. It is reasonable to speculate that the ellagic acid small molecule will bind to and encapsulate at least three to four heavy chains to achieve stable binding. These molecular docking results explain the molecular mechanism underlying the experimental results in Example 8 that "self-assembly of β-SF and EA is only possible when the ratio of EA:β-SF is less than 1:1 (w / w)."

[0074] The tested samples in Examples 10 to 14 and 22 to 24 were prepared using the optimal process to obtain β-SF-EA.

[0075] Example 10 Characterization of Silk Fibroin Composites Loaded with Polyhydroxyphenol Compounds

[0076] FT-IR confirmed the self-assembly of EA and β-SF (Figure 11A): EA hydroxyl peak (3471 cm -1) indicates that EA and β-SF may self-assemble through intermolecular hydrogen bonds. The characteristic peaks of EA in X-ray diffraction (XRD) (Figure 11B) weaken or disappear after binding to silk fibroin, indicating that this is not a simple physical mixture, but a new crystalline phase formed through interaction, confirming the FT-IR results. At the same time, changes in the crystal form are observed. β-SF displays broad diffraction peaks, indicating that they are amorphous macromolecules, while EA displays many strong diffraction peaks, indicating that it is in a crystalline state. After β-SF and EA self-assemble, the EA peak intensity decreases significantly, indicating that β-SF-EA is mainly amorphous macromolecules.

[0077] Thermogravimetric (TG) curves were evaluated and divided into three stages (Figure 11C). 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 weight loss was between 150°C and 450°C, corresponding to the decomposition of the silk protein molecular chain, the decomposition of the amino acid residue side chain, and the cleavage of the peptide bond. 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 major 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 structure was more stable, which may be due to the strong interaction between EA and protein during self-assembly, thereby 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 11D): in β-SF-EA, the peak associated with EA was significantly weakened (<0.007), confirming that EA was bound to β-SF through catechol hydrogen, further demonstrating their association.

[0078] Example 11 Investigation of blood coagulation effect

[0079] (1) Plasma sample processing

[0080] New Zealand white rabbits were fed for one week and fasted the night before the experiment. 1% sodium pentobarbital was used at a concentration of 3 mL·kg -1 The anesthetic dose was injected intraperitoneally. After anesthesia, blood was collected from the abdominal aorta using a 5 mL sodium heparin anticoagulant tube. After the blood was collected, the blood and anticoagulant were thoroughly mixed and set aside.

[0081] (2) In vitro blood adsorption ability test

[0082] Weigh 0.1g of the drug powder to be tested and place it in a 1.5mL centrifuge tube. At the same time, measure 0.5mL of anticoagulant blood, add it to the centrifuge tubes containing different drugs, place it vertically on the experimental workbench, and start timing. After 30s, rotate the inverted centrifuge tube to observe whether the blood flows. Repeat the operation until the anticoagulant blood cannot flow and stop timing. As shown in Figure 12, β-SF-EA achieved complete adsorption of blood within two minutes, while other groups could not achieve this effect, indicating that β-SF-EA's ability to adsorb blood is significantly better than the positive control Yunnan Baiyao and tranexamic acid, and is also significantly better than β-SF and EA monomers, indicating that β-SF and EA can synergistically enhance their effectiveness.

[0083] (3) Test tube tilt coagulation test

[0084] Accurately weigh 5.0 mg of the drug powder to be tested and place it in a 10 mL test tube. Spread the powder as much as possible on the bottom of the test tube. Take 1 mL of anticoagulant blood and add it to the test tube, then add 0.2 mol·L -1 25μL of CaCl2 solution was added and mixed repeatedly immediately, and the timing was started immediately. Let it stand for 1 minute, tilt the test tube every 30 seconds, and stop timing when the blood coagulates and stops flowing. As shown in Figure 13, the complete coagulation time of the blood in the β-SF-EA group (2 minutes) was much shorter than that of the Yunnan Baiyao group (5 minutes), tranexamic acid group (4 minutes 30 seconds), β-SF (5 minutes) and EA (>5 minutes), indicating that β-SF and EA can synergistically enhance their effects. This experiment observed that β-SF-EA has a faster coagulation ability than other drugs.

[0085] (4) Hemorheology measurement

[0086] Whole blood viscosity is the result of friction between blood cells and plasma protein molecules when blood flows, and is the most important indicator in blood rheology. When blood viscosity increases, it means that blood flow is obstructed, and at the same time, the deformability of red blood cells decreases and aggregation increases, the flow rate of blood into microvessels and capillaries decreases, and the flow capacity decreases. After β-SF-EA acts on blood, the viscosity is measured by the method in Example 3. -1 、30·s -1 , 100·s -1 , 150·s -1 , 200·s -1 The results in Table 3 show that β-SF-EA significantly increased whole blood viscosity under varying shear rates (P < 0.05), and was significantly superior to the commercial chitosan hemostatic powder group (P < 0.05). The commercial chitosan hemostatic powder in the control group was purchased from Saikesaisi Biotechnology Co., Ltd., a transient composite microporous polysaccharide hemostatic powder whose main active ingredient is chitosan.

[0087] Table 3 Comparison of whole blood viscosity between β-SF-EA and chitosan (n=3) *: Compared with the blank group, *P<0.05, **P<0.01, ***P<0.001; #: Compared with the commercial chitosan hemostatic powder group, #P<0.05, ##P<0.01, ###P<0.001.

[0088] (4) Blood coagulation index (BCI)

[0089] Prepare different masses of β-SF-EA (10, 20, 30, 40, 50 mg) and place them flat in several 50 mL centrifuge tubes. Take 0.1 mL of anticoagulated blood and gently drip it onto the sample. Immediately add 0.02 mL of 0.2 mol / L CaCl2 solution. After 5 minutes, gently add 25 mL of deionized water to the beaker. Centrifuge at 300 rpm for 5 minutes. Then remove the solution and measure its Abs value with a microplate reader at a wavelength of 540 nm. Set up a control: add 0.1 mL of anticoagulated blood to a beaker, then add 25 mL of deionized water. The Abs value measured at the same wavelength is assumed to be 100 as a reference value. The coagulation index BCI is: BCI = 100 × A 样品 / A 对照 The BCI index is an indicator of coagulation efficacy; the smaller the index, the better the coagulation effect. Figure 14 shows that increasing the dosage (increasing the concentration in the same volume) significantly reduces the coagulation index (BCI), indicating a positive correlation between coagulation efficacy and dosage.

[0090] Example 12 Investigation of hemostatic effect

[0091] Male SD rats (about 250.0 g) were used to evaluate the hemostatic ability of β-SF-EA.

[0092] Rat tail amputation hemostasis assay: SD rats were anesthetized with an intraperitoneal injection of 1% sodium pentobarbital. A 6 cm section was measured from the end of the rat's tail and cut with surgical scissors. Immediately after bleeding, 20 mg of sample powder (β-SF-EA, Yunnan Baiyao, or tranexamic acid) was sprinkled onto the wound. The timer was timed from the start of bleeding.

[0093] Rat liver wound hemostasis assay: SD rats were anesthetized with an intraperitoneal injection of 1% sodium pentobarbital. The anesthetized rats were placed in a supine position on a dissecting table. A longitudinal incision was made along the midline of the abdomen to access the peritoneal cavity and expose the right lobe of the liver. Peritoneal fluid was blotted with clean gauze. A parallel wound approximately 2.0 cm long and 0.5 cm deep was made in the right lobe of the liver using a razor blade. Any bleeding from the wound was immediately blotted with pre-weighed cotton. 20 mg of sample powder (EA, β-SF, β-SF-EA, Yunnan Baiyao, or tranexamic acid) was then sprinkled on the wound surface, and a timer was started. The wound surface was first pressed with cotton for 30 seconds and observed for bleeding. If bleeding continued, the wound was pressed again with cotton for 30 seconds and observed again. This cycle was repeated until no visible blood appeared on the cotton surface. Hemostasis was considered successful. The timer was stopped and the hemostasis time was recorded.

[0094] Rat thigh muscle trauma hemostasis assay: SD rats were anesthetized with an intraperitoneal injection of 1% sodium pentobarbital and immobilized in a supine position on a dissecting table. Leg hair was shaved with a rat hair scraper. After disinfecting the inner thigh with alcohol, a scalpel was used to create a muscle wound approximately 3 cm long and 1 cm deep. Bleeding was timed, and after 5 seconds of bleeding, 20 mg of each sample powder (EA, β-SF, β-SF-EA, Yunnan Baiyao, and tranexamic acid) was applied. The hemostatic effect was observed and the hemostasis time was recorded.

[0095] Figure 15 shows a rat tail amputation experiment. When β-SF-EA was applied to the wound, bleeding stopped rapidly, leaving virtually no blood on the filter paper. It significantly promoted hemostasis within 30 seconds. In the blank group, bleeding lasted for over five minutes, losing statistical significance and not shown in Figure B. As controls, the tranexamic acid and Yunnan Baiyao groups required four times longer to achieve hemostasis, and the amount of bleeding was significantly higher than in the β-SF-EA group (P < 0.05).

[0096] Figure 16 is a graph of the bleeding time of the rat liver and femoral artery. The femoral artery bleeding in the blank group exceeded five minutes, lost statistical significance, and was not listed in the figure. Figure 16 shows that β-SF-EA can significantly shorten the hemostasis time of the two models, and the hemostatic efficacy is significantly better than Yunnan Baiyao (P < 0.05) and tranexamic acid (P < 0.05), and is also significantly better than EA (P < 0.05) and β-SF group (P < 0.05), indicating that silk fibroin and ellagic acid have a synergistic effect. The above results are consistent with the blood rheology and test tube tilt coagulation results in Example 11. In both rat bleeding models, it was observed that β-SF-EA has a faster sedimentation rate in the blood than other drugs and is easy to quickly settle to the bleeding site, while Yunnan Baiyao, tranexamic acid, and β-SF powders are lighter in texture and more likely to float on the surface of the whole blood and be washed away by the blood flow, thereby affecting their hemostatic effect.

[0097] Example 13 Safety Investigation

[0098] (1) Cell recovery

[0099] Remove frozen LO2 and L929 cells and gently shake them in a 37°C water bath until completely thawed. In a clean bench, transfer the cell suspension from the cryotube to a cell culture flask and add 4 mL of DMEM medium containing 10% fetal bovine serum. Gently pipette and stir to evenly disperse the cells in the medium. Then, incubate the flask in a 5% CO2, 37°C cell incubator. After 6 hours of incubation, disinfect the flask with alcohol and place it in the clean bench. Remove the old medium, rinse, and refill with 4 mL of medium before continuing to incubate in the cell incubator.

[0100] (2) CCK8 quantification

[0101] 0.2 mg of β-SF-EA and EA were dissolved in 1 mL of DMSO, and gradient concentrations were set up.

[0102] Take cells in the logarithmic growth cycle, wash them with PBS solution, add trypsin digestion solution to treat the cells into a suspended state, calculate the cell density with a cell counting plate, and calculate 1×10 per well. 5 After cells / mL were seeded into 96-well plates, they were placed in a cell culture incubator and cultured for 24 hours. After the cells adhered, the culture medium was aspirated and washed twice with PBS. 100 μL of the experimental group: β-SF-EA and EA extracts, the blank group: DMEM culture medium containing 10% fetal bovine serum, and the positive group: paclitaxel extract were taken and added to the 96-well plates seeded with cells. Six parallel samples were set up for each group, and then placed in a 37°C, 5% CO2 cell culture incubator for 48 hours. The culture medium was aspirated, washed twice with PBS, and 100 μL of CCK8 solution (90 μL DMEM culture medium + 10 μL CCK8) was added. The plates were placed in the dark in an incubator and incubated for 30 minutes. The absorbance (OD) at 450 nm was then detected using a microplate reader. Six parallel samples were repeated at each time point, and the relative cell proliferation rate was calculated according to the following formula: relative growth rate (RGR) % = OD 实验组 / OD 空白组 *100%.

[0103] The cytotoxicity level was determined according to the cytotoxicity grade in Table 4, where the positive control should not be lower than grade 3. Cells with a grade of 0-1 were considered qualified.

[0104] Table 4 Cell proliferation response grading standards

[0105] We investigated the cytotoxicity of β-SF-EA and EA on L929 and LO2 cells using a CCK8 assay. As shown in Figure 17, the relative proliferation rate of all β-SF-EA groups exceeded 85%, indicating a cytotoxicity rating of 0-1 according to the ISO 10993-1 toxicity grading standard. Furthermore, the addition of silk fibroin significantly improved the biocompatibility of ellagic acid. In summary, β-SF-EA did not affect cell proliferation and demonstrated good cytocompatibility.

[0106] (2) Hemolysis test

[0107] Dilute 8mL of fresh rabbit blood with 10mL of normal saline. Put 20mg of β-SF-EA into a 50mL centrifuge tube, add 10mL of normal saline, and place in a 37℃ water bath for 10min. Then add 0.2mL of diluted rabbit blood, shake gently, and centrifuge at 1000rpm for 5min after 60min in a water bath. Take the supernatant and measure the absorbance at 540nm. The positive control group used 10mL of distilled water plus 0.2mL of rabbit blood, and the negative control group used 10mL of normal saline plus 0.2mL of rabbit blood. The operation method was the same. Each group was repeated three times. The hemolysis rate was calculated using the following formula: Hemolysis rate (%) = (absorbance of test sample - absorbance of negative control) / (absorbance of positive control - absorbance of negative control) * 100%.

[0108] Direct contact between various materials and blood can cause red blood cell rupture and hemolysis. Hemolysis testing assesses the hemolysis of a material by measuring hemoglobin concentration. The calculated hemolysis rate of β-SF-EA was 1.13 ± 0.02% (Table 5). According to GB / T 4233.2, a hemolysis rate below 5% is considered acceptable. Therefore, the prepared β-SF-EA meets international standards.

[0109] Table 5 Hemolysis rate (n=3)

[0110] Example 14 Degradation Performance Investigation

[0111] A certain amount of β-SF-EA was weighed and placed in a PBS solution containing 0.1 μg / mL protease XIV and incubated at 37°C. A PBS solution without protease XIV was used as a blank sample. Samples (n=3) were incubated in PBS solution and PBS solution containing 0.1 μg / mL protease XIV for 5, 10, 15, 20, 25, and 30 days. All degradation samples were replaced with fresh solutions at a fixed time every day. The degradation products were dried at 60°C and the samples were weighed to a constant weight. The residual mass retention rate R M The calculation formula is as follows: M / %=M dt / M i *100%, where M iis the initial mass, M dt is the mass preserved after t days, mg.

[0112] As a hemostatic agent for in vitro and in vivo use, it must exhibit good biodegradability. Ideally, during the initial wound healing phase, hemostatic powder should firmly bind tissues together to prevent cracking. Ideally, it should degrade slowly, gradually degrading the drug as the wound heals. The remaining drug components should be completely degraded after wound healing and absorbed by the body or excreted through metabolism. The degradation of β-SF-EA at 37°C is shown in Figure 18 . It can be seen that the degradation rate of β-SF-EA in PBS is slow, with a residual mass of 90.11 ± 3.51% at day 30. In PBS containing proteinase XIV, the degradation of β-SF-EA can be roughly divided into two stages: the first stage (0-15 days), during which the degradation rate is relatively slow, with a residual mass of 67.83 ± 2.25% at day 15; the second stage (15-30 days), during which the degradation rate begins to increase significantly, with a residual mass of 24.50 ± 0.70% at day 30, indicating that the drug has been largely degraded, demonstrating its good biodegradability.

[0113] Example 15 Preparation of polyhydroxyphenol compound-loaded silk fibroin nanoparticles (β-SFN-polyhydroxyphenol compound)

[0114] Based on Example 1 or Example 2, the silk fibroin complex loaded with polyhydroxyphenol compounds is nanosized using chemical methods (desolvation, salting out, microemulsion method, etc.), instrumental methods (electrospray coating, electric field and supercritical fluid technology, etc.), and other methods (polymer blending method, pH modification method, nanoimprinting method), to prepare silk fibroin nanoparticles loaded with polyhydroxyphenol compounds (β-SFN-polyhydroxyphenol compounds). This can then be prepared into dosage forms with hemostatic efficacy, including gauze, sponges, spinning, sprays, powders, granules, gels, sealants, ointments, films, patches, and embolic agents.

[0115] Example 16 Preparation of Ellagic Acid-Loaded Silk Fibroin Nanoparticle Hemostatic Powder

[0116] The silk fibroin is diluted 5-10 times with water and then slowly poured into an excess of vigorously stirred organic solvent. The mixture is stirred for 2-4 hours to complete the transformation of the β-fold conformation. The liquid portion is removed by suction filtration, high-speed centrifugation (10,000 rpm for 15-20 minutes), dialysis, etc. The remaining portion is repeatedly washed and then freeze-dried to obtain the β-folded silk fibroin. The resulting β-SF is prepared into a suspension with water and placed in a magnetic stirrer at 500-2000 rpm. The polyphenol is suspended in a solvent such as water, ethanol, propylene glycol, or butylene glycol. The polyphenol suspension is added dropwise to the β-SF suspension and mixed and stirred for 12-48 hours. The resulting solution is mechanically separated by suction filtration, centrifugation, membrane filtration, etc. to remove the liquid portion. The remaining portion is freeze-dried using a freeze dryer. The freeze-dried solid is ground and crushed, and then sieved to obtain a hemostatic powder.

[0117] Example 17 Preparation of Ellagic Acid-Loaded Silk Fibroin Nanoparticle Hemostatic Spray

[0118] After diluting the silk fibroin 5-10 times with water, it is slowly poured into a vigorously stirred excess organic solvent and stirred for 2-4 hours to complete the transformation of the β-fold conformation. The liquid portion is removed by filtration, high-speed centrifugation (10,000 rpm for 15-20 minutes), dialysis, etc. The remaining portion is repeatedly washed and then freeze-dried to obtain the β-fold silk fibroin. The resulting β-SF is prepared into a suspension with water and placed in a magnetic stirrer at 500-2000 rpm. The polyphenol is suspended in a solvent such as water, ethanol, propylene glycol, and butylene glycol. The polyphenol suspension is added dropwise to the β-SF suspension and mixed and stirred for 12-48 hours. The resulting solution is mechanically separated by filtration, centrifugation, membrane filtration, etc. to remove the liquid portion. It is then dissolved in water to prepare a 30% mass fraction suspension and injected into a pressure-resistant lightweight handheld spray bottle. The bottle is shaken for 10 seconds to evenly mix to form a hemostatic spray.

[0119] Example 18 Preparation of Ellagic Acid-Loaded Silk Fibroin Nanoparticle Hemostatic Granules

[0120] The silk fibroin is diluted 5-10 times with water and slowly poured into an excess of vigorously stirred organic solvent. The mixture is stirred for 2-4 hours to complete the transformation of the β-sheet conformation. The liquid portion is removed by filtration, high-speed centrifugation (10,000 rpm for 15-20 minutes), dialysis, and the remaining portion is repeatedly washed and then freeze-dried to obtain the β-sheet silk fibroin. The resulting β-SF is prepared into a suspension with water and placed in a magnetic stirrer at 500-2000 rpm. The polyphenol is suspended in a solvent such as water, ethanol, propylene glycol, or butylene glycol. The polyphenol suspension is added dropwise to the β-SF suspension and mixed and stirred for 12-48 hours. The resulting solution is mechanically separated by filtration, centrifugation, or membrane filtration to remove the liquid portion. The remaining portion is freeze-dried using a freeze dryer to obtain silk fibroin accumulation particles (β-SF-polyphenol). Finally, the freeze-dried particles are ground, sieved, and other treatments as needed to obtain hemostatic granules of the desired particle size.

[0121] In Examples 19-22, EA was used as a model drug, and β-SFN-EA powder was prepared using the method of Example 16: a silk fibroin protein complex (β-SF-EA) was prepared under an optimized process, and β-SFN-EA was prepared by adding a nano-process. The optimized process was EA:β-SF = 1:4 (w / w). The ellagic acid in step (2) was suspended in propylene glycol as a solvent. After incubation with SF, the final propylene glycol concentration in the resulting solution was 10%. β-SFN without EA was also used as a control.

[0122] Example 19 Characterization of Ellagic Acid-Loaded Silk Fibroin Nanoparticle Hemostatic Powder (β-SFN-EA)

[0123] Figures 19A-C show the macromorphology of silkworm cocoons, β-SFN, and β-SFN-EA. Morphological examination was performed using a TESCAN MAIA 3GMU field emission scanning electron microscope. Scanning electron microscope images (SEM) in Figures 19D-F show that the morphology of β-SF and β-SF-EA is spherical, and the volume and particle size of β-SF-EA are observed to increase. Particle size was measured by dynamic light scattering (DLS) using a NANO ZS90. The results in Table 6 show that the average particle size of β-SFN-EA (201.18±2.17nm) is larger than that of β-SFN (170.33±0.15nm). The zeta potential of both samples is large (<-20mV), indicating that β-SFN-EA tends to remain stable in aqueous solution due to electrostatic repulsion. The polydispersity index (PdI) for the β-SFN group was <0.1, indicating uniform particle size distribution. However, the PdI for the β-SFN-EA group increased to 0.39±0.05, indicating a decrease in particle size uniformity after preparation. Furthermore, it was observed that the β-SFN group had a light, thin texture and tended to float in solution, while the prepared β-SFN-EA group had a heavier texture and tended to settle in solution.

[0124] Table 6 Particle size, PdI, zeta potential (n=3)

[0125] Example 20 Investigation of hemostatic effect

[0126] New Zealand white rabbits (female, 2.5-3.0 kg) were fixed on an iron plate for surgery and anesthetized with an intraperitoneal injection of 3% sodium pentobarbital. The positive control was a commercial chitosan hemostatic powder purchased from Saikesaisi Biotechnology Co., Ltd., which is an instantaneous composite microporous polysaccharide hemostatic powder with chitosan as its main active ingredient.

[0127] Auricular artery hemostasis model: The rabbit hair on the back of the rabbit's ear was shaved, and the auricular artery was cut 7 cm from the tip of the ear. Blood immediately gushed out from the wound. After the blood bleeds freely for 5 seconds, the ear wound is gently wiped with a sterile cotton ball. 150 mg of β-SFN-EA is immediately sprinkled on the bleeding site. A pre-weighed cotton ball is gently placed vertically on it. The time is immediately started. The cotton ball is gently removed every 30 seconds to observe whether the bleeding continues. If the wound continues to bleed, the cotton ball is continued to hang down and observed every 30 seconds until the bleeding stops. The bleeding time is recorded and the gushing blood is absorbed with a cotton ball. The control group is treated with 150 mg of commercially available chitosan hemostatic powder, and the blank group is treated with only cotton balls. Other steps are the same as above. After hemostasis is completed, the cotton ball is weighed and the blood loss is calculated by differential weight method. Surviving rabbits are euthanized. The experiment is repeated 6 times for each sample, and the average value is taken.

[0128] Liver hemostasis model: After anesthetizing the rabbit, the abdomen was shaved and placed with the abdomen facing upward. The abdomen was disinfected with 75% medical alcohol, and the peritoneal cavity was opened. The left medial lobe of the liver was removed from the peritoneal cavity and placed on sterile gauze. A 3.0 cm long and 0.5 cm deep incision was made in the liver. Free bleeding was allowed for 5 seconds. Immediately, 50 mg of β-SFN-EA was sprinkled over the bleeding site. A pre-weighed cotton ball was gently placed vertically on the wound. Time was immediately measured, and the cotton ball was gently removed every 30 seconds to observe whether bleeding continued. If bleeding continued, the above procedure was continued until bleeding stopped. Bleeding time was recorded, and the gushing blood was absorbed with a cotton ball. A control group was treated with 50 mg of commercially available chitosan hemostatic powder, while a blank group was treated with only a cotton ball. After hemostasis was achieved, the cotton ball was weighed, and blood loss was calculated by differential weight. Surviving rabbits were euthanized. The experiment was repeated six times for each sample, and the average value was calculated.

[0129] Femoral Artery Hemostasis Model: After anesthesia, rabbits were placed with their abdomen facing upwards. The hind legs were shaved, and the hind legs were disinfected with 75% medical alcohol. The skin and soft tissue were dissected with a scalpel to expose the femoral artery. The femoral artery was directly transected. After 5 seconds of free bleeding, the wound was gently wiped clean with a sterile cotton ball. Immediately, 150 mg of β-SFN-EA was sprinkled on the bleeding site. A pre-weighed cotton ball was gently placed vertically on the wound. The timer was immediately started, and the cotton ball was gently removed every 30 seconds to observe whether bleeding continued. If bleeding continued, the cotton ball was left hanging for 30 seconds until bleeding stopped. The bleeding time was recorded, and the gushing blood was absorbed with a cotton ball. A control group was treated with 150 mg of commercially available chitosan hemostatic powder, while a blank group was treated with only the cotton ball. After hemostasis was achieved, the cotton ball was weighed, and blood loss was calculated by differential weight. Surviving rabbits were euthanized. The experiment was repeated six times for each sample, and the average value was calculated.

[0130] Figures 20 and 21 are comparisons of the bleeding time and bleeding volume of rabbits, respectively.

[0131] The dorsal artery of the rabbit ear is comparable in size to the aorta in an adult's arm. Conventional hemostatic materials are difficult to stop, making it an ideal model for testing the hemostatic properties of biomaterials. In the auricular artery experiment, the β-SFN-EA group showed significant improvements over the chitosan group in both bleeding time and bleeding volume (P < 0.05). The chitosan group experienced an average hemostatic time of 209.0 seconds, while the β-SFN-EA group experienced an average hemostatic time of 111.5 seconds, a reduction of 87.44%. In terms of bleeding volume, the chitosan group experienced an average of 6.69 g, while the β-SFN-EA group experienced an average of 2.24 g, a reduction of 198.66%. This demonstrates the superior hemostatic effect of β-SFN-EA.

[0132] The human liver is the most vascular of all internal organs, and hepatic bleeding is a particularly challenging problem during surgery. Therefore, the rabbit liver bleeding model is representative of human visceral bleeding. A 3.0 cm long and 0.5 cm deep wound was created in the rabbit liver to induce profuse bleeding. β-SFN-EA demonstrated significant absorption of the gushing blood, while a commercial chitosan hemostatic powder group failed to do so, with a significant difference (P < 0.05). Interestingly, β-SFN-EA not only adhered to the wound to prevent secondary bleeding but also acted as a barrier, isolating the injured organ from surrounding tissues and preventing postoperative adhesions. Bleeding time averaged 123.2 seconds in the chitosan group, compared to 76.0 seconds in the β-SFN-EA group, a 61.84% reduction. Blood loss averaged 0.35 g in the chitosan group, compared to 0.07 g in the β-SFN-EA group, a 400% reduction.

[0133] Among human extremity hemorrhages, the femoral artery bleeds most rapidly and carries the highest risk. The femoral artery at the base of the rabbit thigh is similar in size to an adult's calf artery, making the rabbit femoral artery bleeding model a representative model for simulating massive extremity hemorrhage. We established a lethal femoral artery injury model during surgical intervention in rabbits. The control group failed to achieve visible hemostasis, requiring compression for true hemostasis. However, the β-SFN-EA group achieved spontaneous hemostasis without the need for external force, significantly outperforming the chitosan group (P < 0.05). Bleeding time decreased by 167.39% in the chitosan group (mean 246.0 s) and 92.0 s in the β-SFN-EA group. Blood loss decreased by 245.73% to 5.67 g in the chitosan group and 1.64 g in the β-SFN-EA group. In conclusion, this rabbit model of massive hemorrhage demonstrates the remarkable and rapid hemostatic effect of β-SFN-EA.

[0134] At the same time, it was observed in three rabbit bleeding models that β-SFN-EA has a faster blood sedimentation rate than other drugs and can easily settle quickly to the bleeding site. The commercially available chitosan powder has a lighter texture and is more likely to float on the surface of whole blood and be washed away by the blood flow, thus affecting its hemostatic effect.

[0135] Example 21 In vivo biocompatibility study

[0136] In vivo biocompatibility was assessed by subcutaneous implantation in the back of SD rats for 7, 15, and 30 days, followed by histopathological examination (Figure 22). H&E staining showed acute inflammation at the particle / tissue interface after 7 days, decreased inflammation and collagen deposition after 15 days, and no residual material or obvious tissue reaction was observed by 30 days, indicating initial inflammation, followed by fibroblast proliferation and accelerated healing. After 30 days, β-SFN-EA was completely absorbed.

[0137] Example 22 Investigation of tissue healing effects

[0138] (1) Mouse full-thickness skin defect experiment

[0139] A full-thickness skin defect model was used to evaluate the effect of a single use of β-SF-EA on the subsequent skin wound healing effect. C57B mice were divided into a blank group, a β-SFN-EA group, a chitosan group, and a Johnson & Johnson Speed ​​Gauze group. The mice were anesthetized using an air anesthesia machine, the back hair was removed, and the back area was disinfected with iodine tincture and alcohol. Subsequently, the healing pad was sutured to the back of the mouse, and a circular skin defect (d = 8 mm) was established in the back pad of each mouse using a skin punch. On day 0, each group was administered 10 mg of the drug, and the blank group was not treated. All wounds were fixed with breathable medical dressings to prevent the movement of the drug. The wounds were photographed and the dressings were changed with a digital camera on days 0, 3, 7, and 14, and the wound size was measured using Image J software. Wound healing rate (%) = ((A o -A t ) / A o )*100% of which A o is the initial wound area, A t Wound area at different time points (days 3, 7, and 14). To evaluate the healing process, four mice per group were randomly euthanized on days 3, 7, and 14. Skin and muscle tissue from the wound site were sampled and fixed in 4% paraformaldehyde for 24 hours. The harvested skin was then embedded in paraffin and stained with H&E and Masson's trichrome staining. Healing was evaluated by analyzing epithelial thickness, integrity, scab formation, neovascularization, inflammatory cells, and collagen fiber deposition. Collagen deposition density was quantified using Image J software. Immunofluorescence detection of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) was used to investigate the anti-inflammatory and angiogenic effects of β-SFN-EA on the wound site. Immunohistochemical staining was used to detect the expression of IL-6, tumor necrosis factor-α (TNF-α), and platelet endothelial cell adhesion molecule-1 (CD31) in the skin wound. Positive markers were analyzed using Image J software.

[0140] As shown in Figure 23, the calculated wound closure rate on days 0, 3, 7, and 14 intuitively reflects the treatment effect. After 3 days of treatment, the wound area of ​​each group decreased to a certain extent. Compared with the β-SF-EA group, the blank group had the lowest wound closure rate, indicating the worst healing effect. Compared with the chitosan group and the Johnson & Johnson Speed ​​Yarn group, the β-SF-EA group had the highest wound closure rate. On day 7, the healing rate of the β-SF-EA group was still the highest among all groups, indicating the best treatment effect. H&E-stained sections after 7 days of healing (Figure 24) show that compared with the blank group, the chitosan group produced a large number of fibroblasts, but the defect was not completely filled. New capillaries sporadically appeared in the granulation tissue, indicating a certain repair effect. In addition, a large number of inflammatory cells appeared around the existing blood vessels, and the overall section morphology was irregular. Compared with the blank group, the Johnson & Johnson Speed ​​Yarn group showed obvious tissue damage in the sections, accompanied by inflammatory cell infiltration, but no large number of fibroblasts or granulation tissue formation was observed, indicating no significant repair ability. Compared with the blank group, the β-SF-EA group not only produced a large number of fibroblasts and granulation tissue, but also had dense new capillaries, which is conducive to wound repair. Among the three groups, Johnson & Johnson Speed ​​Yarn had the weakest repair effect. Chitosan had a slightly stronger effect, but severe inflammatory cell infiltration and incomplete tissue repair. The β-SF-EA group had the best repair effect, with a mild inflammatory reaction and a large number of fibroblasts and granulation tissue, which is conducive to repair. On the 15th day, the number of fibroblasts and granulation tissue in the chitosan group further increased, and the increase in new capillaries was beneficial to the process of wound repair. However, the overall section was still not regular enough, and the inflammation was alleviated compared with the 7th day. In the Johnson & Johnson Speedy Gauze group, obvious defects could be seen, indicating that the Johnson & Johnson Speedy Gauze had no repair effect. The fibroblast group and granulation tissue group in the β-SF-EA group decreased compared with the 7th day, the original tissue recovered, and the inflammation weakened.

[0141] (2) Rabbit femoral artery tissue healing experiment

[0142] New Zealand white rabbits were weighed and anesthetized, fixed with the abdomen facing up, the hind legs shaved, the hind legs disinfected with 75% medical alcohol, and the skin and tissue peeled with a scalpel to reveal the femoral artery. A non-penetrating arterial incision was made in the femoral artery using a (0.7*25mm) medical blood-drawing needle. Sterile medical cotton balls were used to absorb the gushing blood, and β-SF-EA was quickly administered. After the bleeding stopped, the intramuscular drug was removed and the muscle tissue and skin tissue were sutured. In the blank group, no cotton balls were used to absorb blood. The rabbits were placed in a rabbit cage and naturally lost blood to wake up. After death, the blood was cleaned, the weight was weighed, the blood loss was calculated, and the blood vessel specimens were collected and stained with H&E to observe the defects in the arterial tissue. After 30 days, the rabbits were killed and the femoral artery tissue was taken for H&E staining to observe the recovery of the arterial layer.

[0143] As shown in the results in Figure 25, the blood vessels in the β-SF-EA group were almost completely repaired on the 30th day. Although the blood vessels were contracted and narrowed, there was still blood flow, indicating that the blood supply was normal. Judging from the animals' performance, the lower limb activities were normal. It can be judged that β-SF-EA has a significant effect in promoting vascular tissue repair while being used to stop bleeding in non-penetrating wounds of the rabbit femoral artery.

[0144] (3) Liver tissue healing experiment

[0145] Sprague-Dawley rats were anesthetized with an intraperitoneal injection of 1% sodium pentobarbital. The anesthetized rats were placed supine on a dissecting table. A longitudinal incision was made along the midline of the abdomen to access the peritoneal cavity and expose the right lobe of the liver. Peritoneal fluid was blotted with clean gauze. A parallel wound approximately 2.0 cm long and 0.5 cm deep was made in the right lobe of the liver using a razor blade. Any bleeding from the wound was immediately blotted with pre-weighed cotton. 20 mg of each sample (β-SF-EA, chitosan, and Johnson & Johnson Super Gauze) was then applied to the wound surface. The wound was initially compressed with cotton for 30 seconds and observed for bleeding. If bleeding continued, cotton was applied again for 30 seconds and observed again. This cycle was repeated until bleeding stopped. The rats' peritoneal cavities were then sutured, collars were fitted to prevent biting, and the rats were housed individually. To assess the healing process, four rats from each group were randomly euthanized on days 3, 7, and 14. Liver tissue samples were collected from the wound site and fixed in 4% paraformaldehyde for 24 hours. The harvested skin was then embedded in paraffin and stained with H&E and Masson's trichrome staining to evaluate healing, with particular attention paid to liver fibrosis changes.

[0146] As shown in Figure 26, the liver wound healing rates at days 0, 3, 7, and 14 directly reflect the healing efficacy of each group. After 3 days of treatment, the wound area in each group decreased to some extent. Compared with the chitosan and Johnson & Johnson Speedy Gauze groups, the β-SF-EA group had the highest wound closure rate and the best healing effect. On days 7 and 14, the β-SF-EA group maintained the highest healing rate of all groups, demonstrating the best treatment efficacy. Repair was essentially complete by day 30.

[0147] Example 23 Investigation of Anti-adhesion Performance in Vivo

[0148] The in vivo anti-adhesion effect of β-SF-EA was evaluated in a rat sidewall defect-cecal abrasion model. SD rats were anesthetized with sodium pentobarbital (50 mg / kg) and their abdominal hair was shaved. Then a 5 cm long incision was made along the midline of the abdominal wall with surgical scissors. The cecum was separated and its serosal surface was gently rubbed with sterile surgical gauze until punctate bleeding appeared. A 1 cm × 2 cm peritoneal defect was formed on the corresponding outer side of the abdominal wall with a scalpel. In the β-SF-EA group, 1 mL of β-SF-EA sample was placed on the injured abdominal wall and cecum, the negative control group sprayed 1 mL of sterile saline on the wound surface, and the positive control group covered the abdominal wall defect with a commercially available polylactic acid anti-adhesion film. Four rats in each group were euthanized after 7 and 14 days, and the peritoneum was opened and examined for adhesions. Adhesions formed between the cecum and the abdominal wall were scored using a standard scoring system (Table 7).

[0149] The negative control group exhibited severe, unremovable adhesions on days 7 and 14, with weakened adhesion observed between the abdominal wall and cecum. This may be due to the commercially available polylactic acid anti-adhesion membrane being applied as a solid sheet, which failed to completely cover the injured surface, a drawback of this type of conventional anti-adhesion barrier. The β-SF-EA group showed no signs of adhesion on days 7 and 14. Furthermore, on day 14, the injured abdominal wall and cecum in the β-SF-EA group had returned to normal. In Table 7, the β-SF-EA group had the lowest score of the three groups, demonstrating its superior anti-adhesion effect.

[0150] Table 7 Scores of each group in the standard adhesion score table Note: Control is the blank group, Film is the commercially available polylactic acid anti-adhesive film, compared with the blank group, *P<0.05, **P<0.05

Claims

1. A silk fibroin complex loaded with polyhydroxy phenol compounds, characterized in that: It is prepared by self-assembly of polyhydroxyphenolic compounds and silk fibroin; the silk fibroin is treated by a β-sheet conformational transition process, and the mass ratio of the polyhydroxyphenolic compound to the silk fibroin is <1:

1.

2. The silk fibroin complex according to claim 1, wherein: The mass ratio of the polyhydroxyphenolic compound to the silk fibroin is 1:2 to 1:

16.

3. The silk fibroin complex according to claim 2, characterized in that: The mass ratio of the polyhydroxyphenolic compound to the silk fibroin is 1:

4.

4. The silk fibroin complex according to any one of claims 1 to 3, wherein: The polyhydroxyphenolic 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.

5. The silk fibroin complex according to any one of claims 1 to 4, characterized in that: The polyhydroxyphenolic compound is a poorly soluble polyhydroxyphenolic compound.

6. The silk fibroin complex as described in claim 5, wherein: The poorly soluble polyhydroxyphenolic 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.

7. The silk fibroin complex according to claim 6, wherein: The poorly soluble polyhydroxyphenolic compound is ellagic acid.

8. The silk fibroin complex according to claim 7, characterized in that: The mass ratio of the ellagic acid to the silk fibroin is 1:2 to 1:

16.

9. The silk fibroin complex according to claim 8, characterized in that: The mass ratio of the ellagic acid to the silk fibroin is 1:

4.

10. The method for preparing the silk fibroin complex according to any one of claims 1 to 9, comprising two steps of silk fibroin conformational transition and self-assembly with polyhydroxyphenolic compounds: Step (1): The silk fibroin is subjected to a conformational transition process from an α-helix to a β-sheet to obtain a silk fibroin solution. 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, and 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, wherein the feeding mass ratio of the polyhydroxyphenolic compound to the silk fibroin is <1:1, and the silk fibroin complex loaded with the polyhydroxyphenolic compound is obtained.

11. The preparation method according to claim 10, wherein: In step (2), the feeding mass ratio of the polyhydroxyphenolic compound to the silk fibroin is 1:2 to 1:

16.

12. The silk fibroin complex according to claim 11, wherein: In step (2), the feeding mass ratio of the polyhydroxyphenolic compound to the silk fibroin is 1:

4.

13. The preparation method according to any one of claims 10 to 12, characterized in that: In step (1), polyols are used to promote the conformational transition of silk fibroin to β-sheet.

14. The preparation method according to claim 13, characterized in that: The polyol is ethanol.

15. The preparation method according to any one of claims 10 to 14, characterized in that: The polyhydroxyphenolic compound is a poorly soluble polyhydroxyphenolic compound, and 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.

16. The preparation method according to claim 15, characterized in that: The poorly soluble polyhydroxyphenolic compound is ellagic acid, and the organic solvent is propylene glycol. After the ellagic acid suspension is co-incubated with silk fibroin in step (2), the final concentration of propylene glycol in the obtained solution is 5-20%.

17. The preparation method according to claim 16, characterized in that: The final concentration of propylene glycol is 10%.

18. A silk fibroin complex loaded with a polyhydroxyphenolic compound prepared by the preparation method according to any one of claims 10 to 17.

19. Use of the silk fibroin complex according to any one of claims 1 to 9 and 18 in the preparation of a preparation with hemostatic efficacy, characterized in that: Comprising the silk fibroin complex according to any one of claims 1 to 9, 18 and a pharmaceutically acceptable excipient.

20. The application according to claim 19, characterized in that: The preparation with a hemostatic effect comprises gauze, sponge, spinning, spray, powder, granule, gel, sealant, ointment, film, patch, embolization agent.

21. Use of the fibroin complex according to any one of claims 1 to 9 and 18 in the preparation of a preparation having the efficacy of wound healing, and / or anti-infection, and / or anti-adhesion, characterized in that: Comprising the silk fibroin complex according to any one of claims 1 to 9, 16 and a pharmaceutically acceptable excipient.

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