Oxidized sanguisorba officinalis cross-linked collagen hemostatic sponge and preparation method thereof

Abstract

The invention provides oxidized sanguisorba officinalis cross-linked collagen hemostatic sponge and a preparation method thereof.The preparation method comprises the steps that firstly, sanguisorba officinalis extract is oxidized with sodium periodate, dialyzed and freeze-dried into powder, then the powder is dissolved with an acetic acid solution, a collagen solution is dropwise added, dialyzed and freeze-dried, and an oxidized sanguisorba officinalis cross-linked collagen sponge product is obtained; due to the introduction of the oxidized sanguisorba officinalis extract, the blood coagulation effect of collagen is effectively promoted, the endogenous blood coagulation effect of collagen can be remarkably improved, and the collagen has the capacity of inducing cell adhesion, proliferation and growth and meets the use requirements of medical materials.

Description

TECHNICAL FIELD

[0001] The present application relates to an oxidized Sanguisorba officinalis cross-linked collagen hemostatic sponge and a preparation method thereof BACKGROUND

[0002] Blood is an important substance for maintaining human life activities, accounting for about 7-8% of the human body. Blood undertakes many functions of the human body, such as oxygen and nutrient transport, maintenance of body temperature, etc. Bleeding occurs widely in various scenarios, such as accidental injuries in daily life, such as sharp instrument cuts, falls, car accidents, or medical emergencies during surgery. Therefore, effective pre-hospital treatment can play a crucial role in reducing blood loss rate and improving survival rate of wounded personnel. It is far from enough to rely solely on the body's own repair hemostasis function for hemostasis. Timely application of effective exogenous hemostatic materials can effectively promote wound hemostasis and reduce the loss caused by bleeding. Therefore, hemostatic materials have attracted widespread attention from researchers, especially lightweight and portable hemostatic materials with simple use methods.

[0003] Due to its unique biological activity triple helix structure, collagen has many biological properties that other biological materials do not have. In the field of hemostasis, the advantages of collagen mainly include good degradability, low immunogenicity, excellent biocompatibility, promotion of cell adhesion and cell proliferation, etc. More importantly, collagen-based hemostatic materials can activate the intrinsic pathway of the secondary hemostasis process. At present, a large number of collagen-based hemostatic materials have been developed, including hydrogels, fibers, sponges, microspheres, etc. Collagen hemostatic materials of various preparations use collagen as the main body, which is compounded with other hemostatic components, and plays a good effect in the field of wound hemostasis, attracting widespread attention from researchers. However, compared with some hemostatic materials (such as fibrinogen), the hemostatic speed of collagen materials still has a gap, and shortening the hemostatic time has become one of the main research directions for improving the hemostatic performance of collagen materials. SUMMARY

[0004] To solve the above technical problems, the technical scheme adopted by the present application is: an oxidized Sanguisorba officinalis cross-linked collagen hemostatic sponge and a preparation method thereof, characterized in that the preparation method comprises:

[0005] S1. Preparation of oxidized Sanguisorba officinalis (OSo)

[0006] S1. Preparation of oxidized Sanguisorba officinalis (OSo) S1. Preparation of oxidized Sanguisorba officinalis (OSo) S2. Preparation of oxidized Sanguisorba officinalis cross-linked collagen hemostatic sponge (OSo-Collagen)

[0007] S2. Preparation of oxidized Ulmus pumila cross-linked collagen (OC)

[0008] Dissolve collagen and the prepared oxidized Ulmus pumila powder in acetic acid solution respectively to form solutions. Add the acetic acid solution of the oxidized Ulmus pumila with different oxidation degrees to the collagen solution in a mass ratio of 10%. After the reaction, adjust the pH of the reaction system to neutral with NaOH solution, dialyze the collagen solution after the reaction with a dialysis bag with a molecular weight of 8-10 kDa, change the water every day to remove impurities such as salts, and then freeze and dry to obtain the oxidized Ulmus pumila cross-linked collagen sponge product, which is recorded as OC.

[0009] The preparation method of the oxidized Ulmus pumila cross-linked collagen sponge described above, characterized in that the Ulmus pumila extract in step one includes one or more of ethanol extract, ethyl acetate extract, dichloromethane extract, n-butanol extract and water extract of Ulmus pumila.

[0010] The preparation method of the oxidized Ulmus pumila cross-linked collagen sponge described above, characterized in that the oxidation degree of the oxidized Ulmus pumila extract is 3%-75%.

[0011] The preparation method of the oxidized Ulmus pumila cross-linked collagen sponge described above, characterized in that the collagen includes one or more of bovine skin collagen, bovine tendon collagen, pig skin collagen and fish skin collagen.

[0012] The preparation method of the oxidized Ulmus pumila cross-linked collagen sponge described above, characterized in that the key performance indicators of the oxidized Ulmus pumila cross-linked collagen sponge are as follows:

[0013] (1) In vitro hemostatic performance: the activated partial thromboplastin time is 15±5s, the prothrombin time is 8±4s, and the thrombin time is 14±4s according to the detection of fresh rabbit blood; the in vitro blood clotting index is 5.44±5.34%, and the in vitro whole blood clotting time is 5.56±3.82s.

[0014] (2) Platelet adhesion promoting performance: the number of surface platelet adhesion significantly increases according to the detection of fresh rabbit blood.

[0015] (3) Hemolysis rate: the hemolysis rate is 1.24±0.34% according to the detection of fresh rabbit blood.

[0016] (4) Cell compatibility: the L929 cells in the 72h extract solution are uniform and complete in shape, and the density is relatively high; the relative proliferation rate of the cells on the 5th day is 157±40%, and the cytotoxicity is 0 level.

[0017] (5) In vivo hemostatic performance: In the rat liver scratch hemostasis model, the bleeding amount was 127.03±42.28mg and the hemostasis time was 30.99±2.47s; in the rat tail amputation hemostasis model, the hemostasis time was 18.50±0.63s;

[0018] Technical advantages of this invention:

[0019] Based on the excellent hemostatic properties and biocompatibility of collagen, and combining it with the active ingredient of Sanguisorba officinalis, a traditional Chinese medicine, we have successfully developed an oxidized Sanguisorba officinalis powder with different degrees of oxidation by selectively oxidizing the ortho-dihydroxy group in the active ingredient of Sanguisorba officinalis with sodium periodate, oxidizing the hydroxyl group to a reactive aldehyde structure. Then, we used the Schiff base reaction to cross-link the chemically activated active ingredient of Sanguisorba officinalis with collagen. This hemostatic sponge combines the good bioactivity of collagen with the excellent hemostatic properties of Sanguisorba officinalis as a traditional Chinese hemostatic drug. The product not only has good hemostatic properties but also excellent cell compatibility. Attached Figure Description

[0020] Appendix Figure 1 FT-IR spectra of So and OSo with different oxidation degrees obtained by this invention;

[0021] Appendix Figure 2 Oxidation degree detection results of different groups of oxidized Sanguisorba officinalis extracts prepared by this invention

[0022] Appendix Figure 3 The following are the PT evaluation results, TT evaluation results, and APTT evaluation results of OC samples with different oxidation degrees prepared by this invention: (a) PT evaluation results; (b) TT evaluation results; (c) APTT evaluation results.

[0023] Appendix Figure 4 The coagulation index (BCI) of OC samples with different oxidation degrees obtained by this invention.

[0024] Appendix Figure 5 (a) Whole blood coagulation test and (b) Whole blood coagulation time (CT) of OC samples with different oxidation degrees prepared by this invention.

[0025] Appendix Figure 6 Evaluation results of hemolysis rate of OC samples with different oxidation degrees obtained by this invention

[0026] Appendix Figure 7 Cell compatibility of OCs with different oxidation degrees obtained by this invention

[0027] Appendix Figure 8(a1) intact rat liver; (a2) rat liver bleeding of the blank group; (a3) rat liver bleeding of the application OC-1.3; (b1) intact rat tail; (b2) rat tail bleeding of the blank group; (b3) rat tail bleeding of the application OC-1.3

[0028] Figure 2 shows the hemostatic effect of the application OC-1.3 in the rat liver hemostatic model and the rat tail hemostatic model. Figure 9 (a) hemostatic time of the rat liver hemostatic model; (b) hemostatic time of the rat tail hemostatic model DETAILED DESCRIPTION

[0029] For those skilled in the art to understand the features and effects of the present application, the following is a general description and definition of the terms and phrases mentioned in the specification and claims. Unless otherwise specified, all technical and scientific words used herein have their usual meanings to those skilled in the art of the present application, and in case of conflict, the definition in the specification shall prevail.

[0030] Theories or mechanisms described and disclosed herein, whether correct or not, should not be used to limit the scope of the present application, i.e. the present application can be practiced without being limited by any particular theory or mechanism.

[0031] Herein, all features defined in the form of numerical ranges or percentage ranges, such as numerical values, quantities, contents and concentrations, are for the sake of brevity and convenience. Therefore, the description of numerical ranges or percentage ranges should be considered to have encompassed and specifically disclosed all possible sub-ranges and individual numerical values (including integers and fractions) within the range.

[0032] Herein, for the sake of brevity, all possible combinations of the technical features in each embodiment or example are not described. Therefore, as long as the combinations of the technical features do not conflict, the technical features in each embodiment or example can be combined arbitrarily, and all possible combinations should be considered as the scope of the present specification.

[0033] The present application provides a preparation method of oxidized Sanguisorba officinalis cross-linked collagen hemostatic sponge, specifically comprising:

[0034] S1. Preparation of oxidized Sanguisorba officinalis (OSo)

[0035] S1. Preparation of oxidized Sanguisorba officinalis (OSo)

[0036] S2. Preparation of oxidized sumac cross-linked collagen (OC)

[0037] The collagen and the oxidized sumac powder prepared in the previous step were dissolved in acetic acid solution respectively to form solutions. The collagen solution was added with the oxidized sumac acetic acid solution with different oxidation degrees in a mass ratio of 10%. After the reaction, the pH of the reaction system was adjusted to neutral with NaOH solution. The collagen solution after the reaction was dialyzed with a dialysis bag with a molecular weight cut-off of 8-10 kDa. The water was changed every day to remove impurities such as salts. After pre-freezing, the collagen solution was freeze-dried to obtain the oxidized sumac cross-linked collagen sponge product, which was recorded as OC.

[0038] The application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the application and not used to limit the scope of the application. Furthermore, it should be understood that after reading the content of the application, those skilled in the art can make various modifications or changes to the application, and these equivalent forms also fall within the scope of the appended claims.

[0039] The following examples use the conventional instruments and equipment in the art. The experimental methods in the following examples not specified in the specific conditions are usually carried out according to the conventional conditions or the conditions recommended by the manufacturers. The various raw materials used in the following examples are commercially available unless otherwise specified, and the specifications are conventional specifications in the art. In the specification of the application and the following examples, unless otherwise specified, “%” means weight percent, “parts” means weight parts, and the ratio means weight ratio.

[0040] Example 1

[0041] S1. Preparation of oxidized sumac (OSo)

[0042] 10 parts by weight of sumac extract (So) was dissolved in 200-800 parts by weight of pure water at room temperature with magnetic stirring to accelerate dissolution. After complete dissolution, 4 parts by weight of sodium periodate was added, and the reaction was carried out at room temperature with magnetic stirring for 24-72 h in the dark with tin foil. 5-15 parts by weight of ethylene glycol was added to terminate the oxidation reaction for 0.5 h. The reaction product was dialyzed in pure water with a dialysis bag with a molecular weight cut-off of 500-2000 Da for 1-3 days, and the water was changed 4-6 times a day to remove unreacted reactants and other impurities. Then the product was pre-frozen and freeze-dried to obtain oxidized sumac powder (OSo) with a certain oxidation degree.

[0043] S2. Preparation of oxidized sumac cross-linked collagen (OC)

[0044] The collagen and the oxidized Sanguisorba officinalis powder prepared in the preceding step were dissolved in acetic acid solution respectively to form solutions. The collagen solution was added with the acetic acid solution of the oxidized Sanguisorba officinalis with different oxidation degrees at a mass ratio of 10%. After the reaction, the pH of the reaction system was adjusted to neutral with NaOH solution. The collagen solution after the reaction was dialyzed with a dialysis bag with a molecular weight cut-off of 8-10 kDa. The water was changed every day to remove impurities such as salts. After pre-freezing, the collagen solution was freeze-dried to obtain the oxidized Sanguisorba officinalis cross-linked collagen sponge product, which was recorded as OC.

[0045] Example 2

[0046] The difference between the present embodiment and Example 1 is that the amount of sodium periodate added in step S1 is 6 parts by weight. The other steps and parameters are the same as those in Example 1.

[0047] Example 3

[0048] The difference between the present embodiment and Example 1 is that the amount of sodium periodate added in step S1 is 8 parts by weight. The other steps and parameters are the same as those in Example 1.

[0049] Example 4

[0050] The difference between the present embodiment and Example 1 is that the amount of sodium periodate added in step S1 is 13 parts by weight. The other steps and parameters are the same as those in Example 1.

[0051] Example 5

[0052] The difference between the present embodiment and Example 1 is that the amount of sodium periodate added in step S1 is 15 parts by weight. The other steps and parameters are the same as those in Example 1.

[0053] In the present application, the experimental process for detection is as follows:

[0054] 1. Characterization of the oxidized Sanguisorba officinalis (OSo) prepared in the present application:

[0055] (1) Detection of the infrared structure of the oxidized Sanguisorba officinalis (OSo)

[0056] 2-3 mg of the oxidized Sanguisorba officinalis powder with different oxidation degrees was mixed with KBr at a ratio of 1:100, dried in an infrared drying oven, and then ground in a jade mortar until no reflective particles were present. The mixture was pressed into a semi-transparent sheet in a tablet press for 30 s. The sheet was placed in a Fourier infrared spectrometer (FT-IR) for analysis and detection. The wavelength range was 4000-500 cm -1 , the resolution was 4 cm -1 , and the test conditions were 25℃ dry environment. The mixture of the oxidized Sanguisorba officinalis powder with different oxidation degrees and KBr was ground and pressed into a sheet for measurement. The Fourier infrared characterization of the So and OSo samples before and after oxidation was performed, and the results are shown in Figure 1as shown.

[0057] The characteristic absorption peak of aldehyde group appeared at 1732 cm -1 after oxidation, which confirmed the existence of aldehyde group in OSo. The structure of oxidized Ulmus pumila is similar to Ulmus pumila, and the absorption peak of hydroxyl group in So and OSo samples slightly shifted to high wavenumber at 3424 cm -1 , and the absorption peak of hydroxyl group in OSo-0.6 sample group was slightly weakened, and the absorption peak of hydroxyl group in OSo-0.4 and OSo-0.8 was slightly enhanced, but the overall change was not large, and the absorption peak of carbon hydrogen on saturated carbon at 2925 cm -1 , and the absorption peak of carbon oxygen double bond of carboxyl group at 1624 cm -1 , and the absorption peak of oxygen sulfur bond at 1240 cm -1 , and the absorption peak at 1080 cm -1 was the stretching vibration of C-O-C and C-O bond in Ulmus pumila monomer [5] , and the absorption peak in OSo with high oxidation degree was slightly weakened, which proved that the sodium periodate oxidation process basically retained the original structure of Ulmus pumila and did not destroy the effective components of Ulmus pumila itself.

[0058] (2) Detection of oxidation degree of oxidized Ulmus pumila

[0059] 0.100 g of oxidized Ulmus pumila product with different sodium periodate addition amounts was taken, and 25 mL of 0.25 mol / L hydroxylamine hydrochloride-methyl orange solution was added, and the mixture was stirred to form a uniform solution, and the reaction was carried out for 2 h. 0.100 mol / L standard NaOH aqueous solution was used as titrant, and the amount of HCl released was determined by potentiometric titration method, and then the concentration of aldehyde group on the oxidized polyaldehyde Ulmus pumila was calculated by the amount of NaOH solution consumed after neutralization reaction, and then the oxidation degree of oxidized Ulmus pumila with different oxidation degrees was calculated, and the formula was as shown in the formula.

[0060]

[0061] , M is the molecular weight of the sample monomer, the molecular weight of Ulmus pumila monomer is calculated based on the molecular weight of Ulmus pumila saponin I766.95, the unit is g / moL; C is the concentration of standard titrant, the unit is mol / L; ΔV is the volume of standard titrant added at the end of titration reaction; m is the dry weight of sample, and 6 is the number of hydroxyl groups on Ulmus pumila monomer that can be oxidized.

[0062] The aldehyde group concentration of OSo sample prepared by oxidation with different amounts of sodium periodate was determined by hydroxylamine hydrochloride-potentiometric titration method, and the oxidation degree of OSo was calculated by the volume of NaOH consumed in the titration process. As Figure 2As shown, the actual oxidation degrees of OSo-0.4, OSo-0.6, OSo-0.8, OSo-1.3, OSo-1.5 were 6.40%, 19.14%, 25.44%, 31.99%, 51.13% respectively calculated by hydroxylamine hydrochloride-potentiometric titration curve. The determination of the oxidation degrees of OSo confirmed that sodium periodate had successfully oxidized part of the hydroxyl groups in the willow extract to aldehyde groups, and each group of OSo contained aldehyde groups with good chemical reactivity, which could be used to crosslink collagen. The type I collagen was crosslinked with the above OSo crosslinking agent to prepare a new type of hemostatic material.

[0063] 2. Characterization of the prepared oxidized willow crosslinked collagen (OC) of the application:

[0064] (3) In vitro prothrombin time (PT), partial activated thromboplastin time (APTT), thrombin time (TT) detection

[0065] Fresh healthy New Zealand rabbit ear venous blood was collected, and the upper plasma was taken by centrifugation at 1500 rpm for 10 min. 2 mg of hemostatic material containing different amounts of crosslinking agent was dispersed in 10 mL of 0.9% NaCl solution to prepare a 0.2 mg / mL sample suspension, which was mixed with plasma at a ratio of 1:2 for determination. At the same time, the saline and plasma were mixed at a ratio of 1:2 to serve as a blank group. The PT, APTT and TT of each sample were detected in the coagulation detection system, and three parallel samples were set for each group. The detection results are shown in Figure 3 As shown, compared with the pure collagen protein OC-0 group, the PT and TT of the OC hemostatic material prepared from the crosslinked collagen with different oxidation degrees OSo changed little, and some groups showed a small increase, but the APTT of each group after crosslinking was significantly shortened and had a significant difference compared with the OC-0 group. This confirmed that OC could improve the expression level of coagulation factors V, VIII, IX, X and XI to some extent, and the willow extract could promote the formation of prothrombin and fibrinogen, i.e. achieve hemostatic efficacy by activating the endogenous coagulation system.

[0066] (4) In vitro blood clotting test (BCI)

[0067] 20 mg of sample was pre-incubated in a colorimetric tube at 37℃ for 5 min, and 100 μL of fresh New Zealand rabbit ear venous blood was added. 20 μL of 0.2 mol / L CaCl2 solution was added to the surface of the sample, and it was incubated at 37℃ for 5 min. 25 mL of deionized water was added, and it was incubated at 37℃ for another 5 min. The supernatant was taken, and the absorbance at 545 nm was measured. The above blood 100 μL and deionized water 25 mL were used as a control group, and the absorbance at 415 nm was measured. The BCI calculation formula is shown in the formula.

[0068]

[0069] Wherein, Abs1 is the absorbance of the supernatant of the sample group at 545 nm, and Abs0 is the absorbance of the supernatant of the control group at 415 nm.

[0070] The test results are shown in Table 2. Figure 4 As shown in Table 2, each group of OC materials showed a low BCI, and the collagen added with OSo for crosslinking showed a lower BCI, and all had a significant difference with the pure collagen protein group (14.25±0.28%) without adding OSo, among which the OC-1.3 group showed the lowest BCI (5.44±0.34%), confirming that the crosslinking of the oxidized Sanguisorba officinalis extract can improve the hemostatic ability of collagen. With the increase of oxidation degree, the blood clotting index of the OC sponge first showed a downward trend and then an upward trend, and the OC-1.3 group with an oxidation degree of 31.99% showed the best blood clotting performance, but with the further increase of the oxidation degree, the blood clotting index showed an upward trend. This should be because in the process of gradually increasing the oxidation degree, the binding degree of Sanguisorba officinalis extract and collagen is improved, thereby improving the hemostatic performance of OC, but with the further increase of the oxidation degree, the sugar ring and other effective components in Sanguisorba officinalis extract are damaged to a certain extent, thereby weakening the improvement of the hemostatic performance of OC. Combined with the results of platelet adhesion and PT, APTT, and TT analysis, this should be because after the blood contacts the OC hemostatic material, the expression of Ⅴ, Ⅷ, Ⅸ, Ⅹ, and XI is activated through the endogenous coagulation system, and at the same time, a large amount of thrombus can be effectively and rapidly formed through the good adsorption of the material to blood cells, thereby achieving the effect of rapid hemostasis.

[0071] (5) Whole blood clotting test (CT)

[0072] Take 1×1 cm 2 The hemostatic sponges of different sample groups were incubated at 37°C for 20 min, 0.025 mol / L CaCl2 20 μl was added to each sample, and then 200 μl of freshly collected New Zealand rabbit ear vein blood was added. The blood was timed in a 37°C water bath until it completely coagulated, and the coagulation time was recorded. Three parallel controls were set for each sample. The test results are shown in Table 2. Figure 5 Figure 5 (b) As can be clearly seen, except for the OC-0.4 group and the uncrosslinked control group OC-0 (26.03±0.78s), the rest of the groups can significantly shorten the in vitro whole blood clotting time, especially the OC-1.3 group (5.56±0.82s) which shows extremely excellent in vitro whole blood clotting time, which indicates that crosslinking OSo can combine the hemostatic effect of Sanguisorba officinalis as a traditional hemostatic Chinese medicine, significantly improve the hemostatic performance of collagen, and promote collagen to achieve hemostasis through the "coagulation cascade" effect. ​

[0073] (6) Hemolysis test

[0074] 2 mg samples were sterilized by ultraviolet light in advance and placed in sterile test tubes. 10 mL of 0.9% saline and 0.2 mL of freshly collected New Zealand rabbit ear marginal vein blood were added to each tube. After the samples were thoroughly mixed, they were incubated at 37°C for 1 h and centrifuged at 1500 rpm for 10 min. Meanwhile, 10 mL of ultrapure water and 0.2 mL of venous blood were used as a positive control, and 10 mL of saline and 0.2 mL of venous blood were used as a negative control. Each group was repeated in triplicate. The hemolysis rate was calculated as shown in the formula.

[0075]

[0076] wherein Abs sample is the absorbance of the sample, Abs negative is the absorbance of the negative control group, and Abs positive is the absorbance of the positive control group. As Figure 6 It is shown that the hemocompatibility of the hemostatic sponge was evaluated by fresh rabbit blood. When tested at 20 mg per sample, the hemolysis rates of the hemostatic sponges prepared from collagen cross-linked with different oxidation degrees OSo were slightly different, but all were less than 5%, meeting the standard of hemocompatibility of medical biological materials, confirming that OSo-Col materials can be used for wound hemostasis.

[0077] (7) Cell compatibility study

[0078] Logarithmic growth phase fibroblast L929 cells were used as model cells, and CCK-8 method was used to determine the cell compatibility of EC-0, EC-4, EC-8, EC-12, and EC-16. After being sterilized by ultraviolet light in advance, 10 mg of each sample was immersed in cell culture solution to prepare a 2 mg / mL extract, which was extracted at 37°C for 24 h. 100 μL of 1 x 10 4 / mL cell suspension was inoculated on a 96-well plate, and the L929 cells adhered after 4 h. After removing the original culture medium, the extract of different sample groups was inoculated on the plate at 100 μL per well, with four parallel holes in each group. At the same time, a control group inoculated only with cells and culture medium and a negative blank group inoculated only with culture medium were established, which were cultured in a 37°C CO2 incubator for 1 d, 3 d, and 5 d, with liquid change every other day. Before measurement, the extract was added to 100 μL of culture medium. 10 μL of CCK-8 reagent was added to each well, and incubated for 2 h. The absorbance was measured at 450 nm. As Figure 7 It is shown that each group of samples showed good cell compatibility, and the cell proliferation rate of the cells co-cultured with OC material extract was greater than 100% at 1 day, 3 days, and 5 days, and the cytotoxicity was 0 grade. Compared with the pure collagen protein group OC-0 without modification by cross-linking agent, it showed a certain degree of effect on promoting cell growth.

[0079] (9) Evaluation of in vivo hemostatic performance

[0080] The animal experiments described in this paper were conducted at the Animal Experiment Center of Sichuan University. All animal experimental procedures were approved by the Animal Protection and Use Committee of Sichuan University.

[0081] Liver hemorrhage model: Medical gauze and EC-12 were pre-cut into 10×10mm pieces. 2 A square, after sealing 60 Sterilization by Co irradiation. SD rats (300-320g) were divided into 3 groups and fed for one week before the experiment. Anesthesia was performed by intraperitoneal injection of 3% sodium pentobarbital (1mL / kg). The abdominal hair of the rats was shaved off, and the rats were placed supine on the operating table. The abdomen below the thoracic vertebrae and ribs was incised layer by layer using surgical scissors. The left lobe of the liver was carefully removed with hemostatic forceps, and a pre-weighed sterile filter paper was placed at the bottom. Immediately after bleeding, the wound was covered with EC-12, and the outflowing blood was absorbed with sterile filter paper. The time to hemostasis was recorded, and the blood loss was calculated by the change in the weight and mass of the sterile filter paper before and after the experiment. Wounds treated with medical gauze served as positive controls, and wounds not treated with medical gauze served as negative controls. The abdominal cavity was closed postoperatively. Figure 8 (a) Figure 9 As shown in (a), in the liver hemorrhage model, OC-1.3 exhibited superior hemostatic performance, with a bleeding volume of 127.03±42.28 mg and a hemostasis time of 30.99±2.47 s, which were significantly better than the bleeding volume of 229.63±30.63 mg and the hemostasis time of 42.38±1.74 s in the medical gauze group, respectively. This demonstrates that OC-1.3 sponge has superior hemostatic performance compared to commercially available medical gauze.

[0082] Rat tail amputation hemostasis model: Anesthetized rats were fixed on an operating table, and the tail was cut off approximately 5 cm from the base using surgical scissors. Pre-weighed filter paper was placed underneath, and then an EC-12 hemostatic sponge was immediately placed on the severed tail. Hemostasis time was recorded, the weight of the filter paper before and after hemostasis was measured, and the amount of bleeding was calculated. Medical gauze served as a positive control, and untreated gauze as a negative control. Figure 8 (b) Figure 9 (b) shows that in the rat tail amputation hemostasis model, OC-1.3 hemostatic sponge exhibited superior hemostatic performance and significantly shortened the hemostasis time. The tail amputation hemostasis time of rats treated with OC-1.3 sponge was 18.50±0.63s, which was significantly better than the 38.20±1.11s of the medical gauze group and significantly better than the 135.21±12.43s of the untreated control group, demonstrating the good potential of OSo-Col hemostatic sponge for in vivo hemostasis.

Claims

1. An oxidized red vine leaf cross-linked collagen hemostatic sponge and a method for preparing the same, characterized in that, Comprising the following steps: S1. Preparation of oxidized Sanguisorba officinalis (OSo) Sanguisorba officinalis extract (So) was dissolved in pure water, and after complete dissolution, sodium periodate was added. The reaction was stirred magnetically at room temperature for 24-72 h, and ethylene glycol was added to terminate the oxidation reaction. The reaction product was dialyzed in pure water using a 500-2000 Da dialysis bag, and the water was changed every day to remove unreacted reactants and other impurities. Then the product was pre-frozen and freeze-dried to obtain oxidized Sanguisorba officinalis powder (OSo) with a certain degree of oxidation. S2. Preparation of oxidized Sanguisorba officinalis cross-linked collagen (OC) Collagen and the oxidized Sanguisorba officinalis powder prepared in the preceding step were dissolved in acetic acid solution, respectively, to form solutions. Different degrees of oxidized Sanguisorba officinalis acetic acid solution were added dropwise to the collagen solution at a mass ratio of 10%. After the reaction, the pH of the reaction system was adjusted to neutral with NaOH solution. The collagen solution after reaction was dialyzed using a dialysis bag with a molecular weight cut-off of 8-10 kDa, and the water was changed every day to remove impurities such as salts. The product was pre-frozen and freeze-dried to obtain oxidized Sanguisorba officinalis cross-linked collagen sponge, which was denoted as OC.

2. The oxidized Hamelbarn hickory cross-linked collagen hemostatic sponge according to claim 1, and a preparation method thereof, characterized in that The Sanguisorba officinalis extract includes one or more of ethanol extract, ethyl acetate extract, dichloromethane extract, n-butanol extract, and water extract of Sanguisorba officinalis.

3. An anticoagulant acellular dermal matrix according to claim 1, wherein the anticoagulant acellular dermal matrix is prepared by the method according to claim 2. The oxidized Sanguisorba officinalis extract has an oxidation degree of 3%-75%.

4. The oxidized collaeanthonyl hemostatic sponge according to claim 1, wherein The key performance indicators of the oxidized Sanguisorba officinalis cross-linked collagen hemostatic sponge are as follows: (1) In vitro hemostatic performance: The activated partial thromboplastin time was 15±5 s, the prothrombin time was 8±4 s, and the thrombin time was 14±4 s, as detected by fresh rabbit blood. The in vitro coagulation index was 5.44±5.34%, and the in vitro whole blood clotting time was 5.56±3.82 s. (2) Promoting platelet adhesion performance: The number of surface platelet adhesion significantly increased, as detected by fresh rabbit blood. (3) Hemolysis rate: The hemolysis rate was 1.24±0.34%, as detected by fresh rabbit blood. (4) Cell compatibility: The L929 cells in the 72 h extract solution were uniform and complete in shape, and the density was relatively high. The relative proliferation rate of the cells on the 5th day was 157±40%, and the cytotoxicity was grade 0. (5) In vivo hemostatic performance: In the rat liver scratch hemostasis model, the bleeding amount was 127.03±42.28 mg, and the hemostatic time was 30.99±2.47 s. In the rat tail transection hemostasis model, the hemostatic time was 18.50±0.63 s.

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