CN114306713B - Absorbable hemostatic wound-protecting dressing and preparation method thereof - Google Patents
Absorbable hemostatic wound-protecting dressing and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an absorbable hemostatic wound-protecting dressing and a preparation method thereof, wherein the method comprises the following steps: drying and crushing bletilla striata, mixing and leaching the bletilla striata with an extracting solution with the pH value of 11.5-13, performing ultrasonic treatment, performing deproteinization treatment on filtrate, performing precipitation by using ethanol, and performing freeze drying to obtain bletilla striata polysaccharide; the bletilla striata polysaccharide is processed to obtain bletilla striata polysaccharide sulfonated polymer, and then the bletilla striata polysaccharide sulfonated polymer is prepared into the spongy hemostatic wound-protecting dressing. The obtained rhizoma bletilla polysaccharide has high purity, and the dressing has good hemostatic effect and high tissue compatibility, and is porous and expected to be used as medicine carrying material.
Description
Technical Field
The invention relates to the technical field of medical instruments, in particular to an absorbable hemostatic wound-protecting dressing and a preparation method thereof.
Background
The absorbable hemostatic material is used for hemostasis in operations, and common absorbable hemostatic materials include chitosan gel, gelatin sponge, regenerated cellulose, potato starch and the like. However, the short plates of the existing hemostatic materials are: firstly, the histocompatibility of chitosan (crustacean organisms) and gelatin (fish/pig/cattle and the like) biological products of animal sources has a large risk; secondly, regenerated cellulose belongs to viscose cellulose, and clinical verification proves that the viscose cellulose is difficult to absorb in vivo, is easy to cause adverse events such as foreign matter wrapping, inflammatory reaction and the like, and has larger potential safety hazard; the sponge and the powder are in a gel-like and non-fixed state in the environment of physiological liquid in vivo, lack necessary mechanical strength, and easily wander and shift in the organism along with the physiological movement of human organs, thereby influencing the local hemostasis function; fourthly, the function of inducing the rapid repair of normal connective tissues (such as blood vessels, muscles and epithelia) is not achieved; fifthly, the medicine carrying structure is lacked, the medicine carrying structure cannot carry anti-tumor medicines, and the residual tumor cells cannot be subjected to target site clearing treatment.
Bletilla striata (in particular to the pseudobulb of plant bletilla striata) is a traditional Chinese medicine in China, has obvious medical effects of stopping bleeding, reducing swelling, promoting tissue regeneration and healing sore historically, and researches show that bletilla striata polysaccharide has various pharmacological activities of healing wound, enhancing immunity, resisting inflammation, resisting oxidation, resisting ulcer and the like. The extraction method of bletilla striata polysaccharide comprises hot water extraction, ultrasonic extraction, microwave extraction, enzyme-assisted extraction and the like, and bletilla striata polysaccharide extracted by different methods has different purity and yield.
The bletilla striata polysaccharide is excellent in the aspects of antivirus, antitumor and the like after being modified by sulfonation, and the anticoagulation effect can be enhanced to different degrees. In addition, the sulfonated product of the bletilla striata polysaccharide has a three-dimensional reticular porous structure, can be made into spongiform with various appearance forms through mould induction shaping, and is expected to meet the hemostasis requirement of clinical operation.
However, researches find that the properties of the spongy hemostatic materials prepared from bletilla striata polysaccharide obtained by different extraction methods are greatly different, especially in terms of hemostatic performance and tissue compatibility.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides an absorbable hemostatic wound-protecting dressing and a preparation method thereof, and the dressing has good histocompatibility and hemostatic effect by optimizing the extraction process of bletilla polysaccharide and controlling the sulfonation condition of the bletilla polysaccharide.
In order to achieve the above object, the technical solution of the present invention is as follows:
a method of making an absorbable hemostatic wound dressing comprising the steps of:
s1, drying and crushing bletilla striata, mixing the dried and crushed bletilla striata with the extracting solution, leaching for 0.5-1.5 h at the temperature of 40-60 ℃, performing ultrasonic treatment for 3-10 min, and filtering to remove precipitates; the pH value of the extracting solution is 11.5-13;
s2, deproteinizing the filtrate to remove precipitates;
s3, adding absolute ethyl alcohol into the filtrate obtained in the step S2 until precipitates are separated out, refrigerating and standing the precipitates, keeping the precipitates, and freeze-drying the precipitates to obtain bletilla striata polysaccharide;
s4, sulfonating and modifying bletilla striata polysaccharide to obtain a bletilla striata polysaccharide sulfonated polymer;
s5, preparing the bletilla polysaccharide sulfonated polymer into the sponge-shaped hemostatic wound-protecting dressing.
Further, in the above technical scheme, the weight ratio of the bletilla striata to the extracting solution is 1: 30 to 50.
Further, in the above technical solution, the extracting solution is a sodium hydroxide solution, a potassium hydroxide solution or a mixed solution of the two.
Further, in the above technical solution, the deproteinization treatment process is: concentrating the filtrate, adding sevage reagent with the same volume, shaking for reaction, standing for layering, and filtering to remove precipitate.
Further, in the above technical scheme, the precipitate filtered in step S3 is redissolved, and then purified twice with absolute ethanol; furthermore, the precipitate is redissolved and then decolorized by activated carbon.
Further, in the above technical solution, the sulfonation modification method comprises: dissolving bletilla striata polysaccharide in a formamide or dimethyl sulfoxide solution to obtain a suspension, then dropwise and slowly adding the suspension into a chlorosulfonic acid-pyridine esterification reagent, reacting for 3-5 hours under the condition of ice-water bath, adjusting the pH value to be neutral, centrifuging, and freeze-drying.
Further, in the technical scheme, the bletilla polysaccharide sulfonated polymer is prepared into the spongy hemostatic wound-protecting dressing by a low-temperature freeze-drying method.
The invention has the beneficial effects that: the hemostatic dressing prepared by the invention can be used for hemostasis in vivo and can be absorbed (verified by animal experiments), thereby widening the use forms and ways of the traditional Chinese medicine (namely oral administration and external application); secondly, the hemostatic dressing prepared by the invention only takes bletilla striata polysaccharide as a raw material, and can be slowly degraded into low molecular weight saccharides in an organism so as to be absorbed and metabolized by the organism; the hemostatic dressing has a loose and porous three-dimensional structure, has a physical space structure with good adsorption and carrying performance, and can be used as a strong liquid absorbing material and a carrier for carrying medicaments; fourthly, the bletilla striata polysaccharide has high purity, and the prepared hemostatic dressing has good tissue compatibility and hemostatic effect; in the preparation process, less organic reagent is used, no toxic or harmful reagent is used, and the safety degree is high.
Drawings
FIG. 1 is an enlarged view of the outer surface of the absorbable hemostatic wound dressing prepared in examples 1-3;
FIG. 2 is a scanning electron micrograph (a is a longitudinal plane, and b is a cross-sectional plane) of the absorbable hemostatic and wound-protecting dressing prepared in example 1;
FIG. 3 is an optical micrograph of a pathological section of the muscle tissue of the subject (a is a negative control, b is a positive control, and c is a sample).
Detailed Description
The present invention is further illustrated by the following specific examples, which should not be construed as limiting the scope of the invention.
Example 1
An absorbable hemostatic wound-protecting dressing, which is prepared by the following specific steps:
(1) drying and crushing bletilla striata, sieving with a 200-mesh sieve, adding a sodium hydroxide solution with the pH value of 13, wherein the solid-liquid weight ratio is 1: 40. Soaking at 60 deg.C for 60min under magnetic stirring, ultrasonic extracting for 8min, and vacuum filtering. Concentrating the filtrate at 60 ℃ by using a rotary evaporator, cooling, adding an isovolumetric sevage reagent (chloroform and n-butanol in a volume ratio of 5: 1), shaking at normal temperature for 15min, standing for layering, and filtering out precipitates (namely impurity proteins). Adding anhydrous ethanol into the clear liquid until precipitation is achieved, refrigerating and standing at 4 ℃ for 12h, performing suction filtration under negative pressure, taking the filter cake, adding ultrapure water for redissolving (properly heating at the temperature of not more than 60 ℃ if necessary), decoloring with active carbon, filtering out insoluble substances, adding anhydrous ethanol into the filtrate until precipitation is achieved, refrigerating and standing at 4 ℃ for 12h, performing suction filtration under negative pressure, and freeze-drying the filter cake. Weighing and calculating the yield, and freezing and storing at-18 ℃ for later use.
(2) Modifying by adopting a chlorosulfonic acid-pyridine method to obtain the bletilla polysaccharide sulfonated polymer. The method specifically comprises the following steps: dissolving 200mg of polysaccharide powder in 15mL of formamide solution, adding 25mL of chlorosulfonic acid-pyridine esterification reagent into another container, placing the container in an ice water bath, dropwise and slowly adding the polysaccharide suspension into the esterification reagent, stirring for reaction for 4 hours, adjusting the pH to be neutral, centrifuging (without dialysis), and freeze-drying to obtain the polysaccharide powder. The preparation method of the chlorosulfonic acid-pyridine esterification reagent comprises the following steps: pyridine is added into a three-neck flask with a condensing device and a stirring device under the condition of ice-water bath, and then chlorosulfonic acid (in the embodiment, the volume ratio of the pyridine to the chlorosulfonic acid is 6: 1) is slowly added dropwise to react to generate an esterification reagent.
(3) Redissolving the bletilla striata polysaccharide sulfonated polymer in pure water, pouring the solution into a mould, inducing the polymer to freeze and crystallize in a fixed direction in an environment of-25 ℃ to ensure that the fiber is oriented in the stacking process, and then freeze-drying in a vacuum environment to prepare a spongy object (the process is a physical process, and no other chemical substances except the pure water are introduced).
Example 2
An absorbable hemostatic wound-protecting dressing, which is prepared by the following specific steps:
(1) different from the example 1, the extracting solution adopts sodium hydroxide solution with pH of 11.7, and the solid-to-liquid ratio is 1: 45, the soaking time of the extracting solution is 90min, the ultrasonic extraction time is 5min, and the rest is the same as the example 1.
(2) And (3) in accordance with example 1.
Example 3
An absorbable hemostatic wound-protecting dressing is prepared by the following specific preparation processes:
(1) different from the example 1, the extracting solution adopts a potassium hydroxide solution with the pH value of 12.5, and the solid-to-liquid ratio is 1: 35 soaking the extractive solution for 45min, and ultrasonic extracting for 3min, which are the same as in example 1.
(2) And (3) in accordance with example 1.
Comparative example 1
An absorbable hemostatic wound-protecting dressing, which is prepared by the following specific steps:
(1) the same procedure as in example 1 was repeated except that the extract solution used in example 1 was a sodium hydroxide solution having a pH of 14.
(2) And (3) in accordance with example 1.
Comparative example 2
An absorbable hemostatic wound-protecting dressing, which is prepared by the following specific steps:
(1) the same procedure as in example 1 was repeated except that a sodium hydroxide solution having a pH of 9 was used as the extract solution in example 1.
(2) And (3) in accordance with example 1.
Comparative example 3
An absorbable hemostatic wound-protecting dressing, which is prepared by the following specific steps:
(1) the method is different from the method of example 1 in that pure water is used as an extracting agent, and extraction is carried out at 90 ℃ for 3h, and the method is otherwise the same as the method of example 1.
(2) And (3) in accordance with example 1.
Comparative example 4
An absorbable hemostatic wound-protecting dressing, which is prepared by the following specific steps:
(1) the method is different from the method of the embodiment 1 in that pure water is used as an extracting agent, and ultrasonic treatment is directly carried out in hot water at 60 ℃ for 50min, and the method is the same as the method of the embodiment 1.
(2) And (3) in accordance with example 1.
Comparative example 5
Unlike example 1, the procedure of step (2) is as follows:
modifying by adopting a chlorosulfonic acid-pyridine method to obtain a bletilla polysaccharide sulfonated polymer, which specifically comprises the following steps: dissolving 200mg of polysaccharide powder in 15mL of formamide solution to obtain a suspension, adding 25mL of chlorosulfonic acid-pyridine esterification reagent (same as in example 1) into the suspension at one time, stirring at room temperature (30 ℃) for reaction for 4 hours, adjusting the pH to be neutral, centrifuging, and freeze-drying to obtain the polysaccharide powder.
Evaluation of results
1. When the bletilla striata polysaccharide prepared in the examples 1-3 is measured, the average yield of the polysaccharide processed by a plurality of batches is 1.16%, the extraction rate is low, but the extraction purity is as high as 99%. The heavy metal and ethanol residues of the bletilla striata polysaccharide prepared in example 1 are shown in the following table:
2. the absorbable hemostatic and wound-protecting dressing prepared in the examples 1 to 3 is light yellow white or grey white flat sponge-shaped, soft to touch, and has fibrous stripes in appearance as shown in fig. 2; the longitudinal surface and the transverse surface of the drug carrier are scanned by an electron microscope and are shown in figure 3, and the drug carrier has a more regular layered three-dimensional structure, is loose and porous and is expected to be used as a drug carrier.
3. Evaluation of tissue compatibility of the dressings prepared in examples
Whether the dressing has good histocompatibility is determined by a general acute toxicity test of New Zealand Long-ear rabbits and a routine blood test and a local reaction test after muscle implantation. The specific process is as follows:
1) taking 6 New Zealand Long-ear rabbits, randomly dividing the New Zealand Long-ear rabbits into A, B, C three groups according to sex, wherein each group comprises 2 animals, and the male and female animals are 1 respectively; wherein group a is a negative control group, group B is a positive control group (the positive control is sugricel, a fast spun regenerated oxidized fiber, purchased from shanghai medical devices, ltd.), and group C is a sample group (the dressing prepared in example 1).
2) The long-ear rabbits were anesthetized one by one and tested, and the material was embedded under the right leg ventral muscle of each group of new zealand long-ear rabbits by a planting method (the negative group formed only the same wound and did not implant the material). After continuous observation for 72h, no animal in A, B, C groups died, no adverse reaction, normal body temperature, insignificant weight change and normal animal activity.
3) After 3 weeks of the experiment, the animals were anesthetized, the skin of the pre-implantation material site was cut open, and the morphology of the muscle tissue at the implantation site was observed. Visible to the naked eye: A. b, C groups had normal muscle tissue, well-healed wounds, no bleeding, no nodules, complete absorption of Surgicel and test samples, no residue to the naked eye; scar tissues are slightly visible at the wound part and are scars generated by self healing.
Taking out the muscle tissue of the operation part, preparing tissue sections and observing by a microscope. Experimental site HE section fiber observations (fig. 3) show: A. b, C groups of muscle fibers have good continuity and no obvious inflammatory reaction, only a small amount of inflammatory cells infiltrate into interstitial tissues, a small amount of fibrous tissues proliferate at the edges of embedded tissues, and the tissue structure is normal, which are judged as the inflammatory reaction grade 0.
The three groups of test results are similar, so the biocompatibility of the test product is equivalent to that of the similar products sold at present, and the test product has good biocompatibility.
4. The hemostatic properties of the dressings prepared in the examples were verified
The evaluation of the hemostatic performance is an in vitro coagulation test and the establishment of a living body hemostatic model is a conventional means for comprehensively evaluating the hemostatic performance of the hemostatic material. The in vitro coagulation test evaluates procoagulant properties of whole blood by measuring the coagulation time, Activated Partial Thromboplastin Time (APTT), Prothrombin Time (PT), and Thrombin Time (TT), and preliminarily predicts the hemostatic mechanism thereof.
1) Whole blood clotting time assay
Let test, control and blank groups, n-9, use disposable sterile plastic test tubes. The absorbable hemostatic and wound-protecting dressing and instant yarn prepared in example 1 were prepared into 5% strength solutions with physiological saline, and the solutions were recorded as a sample solution and a control solution, respectively, and were ready for use. The test group contained 20. mu.l of sample solution per tube, the control group contained 20. mu.l of control solution per tube, and the blank group contained 20. mu.l of physiological saline. The three sets of tubes were sealed and pre-warmed at 37 ℃ for use.
After blood is taken from the heart of the New Zealand Long-ear rabbit, the blood is uniformly mixed with sodium citrate according to the ratio of 9:1, and the anticoagulated whole blood is obtained. Adding anticoagulated whole blood into test tubes of test group, control group and blank group1 mL/tube, incubating at 37 deg.C for 3min, adding 25mmol/L CaCl 2 Immediately after the solution, a stopwatch was started, and the tube was tilted every 30s to see if blood flowed until blood coagulated (tube inverted with no blood flow). Recording from the addition of CaCl 2 The time that the solution is solidified to the liquid surface and does not flow is the whole blood coagulation time.
2) Determination of in vitro coagulation indices APTT, PT and TT
The anticoagulated whole blood obtained in the above step is centrifuged at 3000rpm for 15 minutes at low temperature, and the upper plasma is collected for standby.
Three in vitro coagulation indexes APTT, PT and TT are measured by setting a test group, a control group and a blank group, wherein n is 10, each test tube of the test group is placed with a small piece of 2mg of multifunctional absorbable hemostatic wound-protecting dressing, each test tube of the control group is placed with a small piece of 20mg of Surgicel, the blank group is not placed with substances, and all test tubes are preheated for 5min at 37 ℃ for standby.
And (3) APTT determination: respectively placing 0.5 mL/branch of blood plasma into test group, control group and blank group, keeping the temperature at 37 deg.C for 5min, adding 0.5 mL/branch of APTT reagent with pre-temperature of 37 deg.C, mixing, incubating at 37 deg.C for 5min, adding 25mmol/L CaCl with pre-temperature of 37 deg.C 2 The solution was 0.5 mL/arm and a stopwatch was started immediately until a stop watch of plasma clotting occurred and plasma clotting time (in seconds) was recorded.
And (3) PT and TT determination: PT and TT kits are respectively adopted, and the test method is the same as above.
3) Test results
The results of the measurement of the whole blood coagulation time, Activated Partial Thromboplastin Time (APTT), Prothrombin Time (PT) and Thrombin Time (TT) are shown in the following table.
| Coagulation time of whole blood | APTT | PT | TT | |
| Blank group | 226.7±29.14 | 23.8±5.3 | 29.6±3.39 | 7.9±0.22 |
| Control group | 186.1±20.63** | 21.8±2.81* | 26.5±3.09* | 7.4±0.27** |
| Test group | 170.6±32.40** | 20.9±2.31** | 20.2±3.04** | 7.1±0.29** |
Note: the data in this table were analyzed for one-way anova using the blank as a control.
As can be seen from the above table, the whole blood coagulation time of the sample group of the multifunctional absorbable hemostatic and wound-protecting dressing is about 170s, the whole blood coagulation time of the control group of the commercially available Surgicel whole blood is about 186s, and the whole blood coagulation time of the blank group is about 226 s. Through SPSS-19.0 software ANOVA analysis, the test group can absorb three groups of the hemostatic dressing group, the contrast group commercially available absorbable gelatin sponge and the blank group are compared pairwise, P between the test group, the contrast group and the blank group is less than 0.001, and the difference is significant; p is more than 0.05 between the test group and the control group, and the difference has no significance. The whole blood coagulation time measurement result shows that the absorbable hemostatic dressing has good hemostatic effect, and the effect is equivalent to that of a commercially available hemostatic material.
In the whole blood coagulation time test, compared with a blank group, the p of the sample group is less than 0.001, and the difference is significant; compared with the commercial absorbable Surgicel hemostatic material, the difference is not significant, and p is greater than 0.05. The multifunctional absorbable hemostatic wound-protecting dressing sample has good hemostatic effect, and has the same effect as a sold hemostatic material.
The result of the APTT, PT and TT kit shows that the significant difference p is less than 0.001 when the sample group is compared with the blank group, and the kit has the performance of obviously shortening the blood coagulation time. Indicates p <0.001 in statistical analysis, significant differences were evident; indicates p <0.01 in statistical analysis, significant differences were evident.
The dressings prepared in the embodiments 2 to 3 are evaluated by the same method, and the results show that the dressings have the similar properties to those of the embodiment 1, and have good compatibility, hemostatic effect and mechanical strength.
The dressings prepared in comparative examples 1 to 3 were evaluated by the same method, and the results showed that the hemostatic effect and the tissue compatibility of the dressings prepared in comparative examples 1 to 3 were significantly inferior to those of the examples. Analysis of the bletilla striata polysaccharides prepared in each example shows that the polysaccharide prepared in the comparative example has higher yield but slightly lower purity, and the purities of comparative examples 1-3 are 94%, 89% and 92% respectively. Further analysis on the components of the bletilla striata polysaccharide shows that the saponin content in the comparative example is higher than that in the examples, and the saponin has a hemolytic function due to the special physiological activity, so that the saponin is presumed to be a main factor influencing the hemostatic effect. The above examples show that saponins in bletilla striata are sensitive to the pH of the extracting solution, the separation of saponins and polysaccharide is not facilitated when the pH is too high or too low, saponin sugar chains can be hydrolyzed in the early step of the extraction process by controlling the pH of the extracting solution, the subsequent dialysis and purification of bletilla striata polysaccharide are avoided, the time consumption is short, and the cost is low.
The dressing prepared in comparative example 4 is not only inferior in hemostatic effect, but also the worst in mechanical strength after the dressing is molded; the reason for this may be: the molecular chain is broken by long-time ultrasound, so that the molecular weight of the obtained bletilla striata polysaccharide is small.
The dressing prepared in comparative example 5 was dissolved in the histocompatibility assessment test before the surgical site recovered, resulting in a more pronounced inflammatory response at the surgical site. The reason for this is presumed to be that the polysaccharide is esterified to an excessively high degree, resulting in excessively high water solubility, which is not favorable for maintaining the mechanical structural strength of the dressing and the sustained-release effect of the drug over a certain period of time.
The present invention may be better understood and appreciated by those skilled in the art with reference to the following examples. However, the protection of the invention and the scope of the claims are not limited to the examples provided. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (8)
1. A method of making an absorbable hemostatic wound dressing comprising the steps of:
s1, drying and crushing bletilla striata, mixing the dried and crushed bletilla striata with the extracting solution, leaching for 0.5-1.5 h at the temperature of 40-60 ℃, performing ultrasonic treatment for 3-10 min, and filtering to remove precipitates; the pH value of the extracting solution is 11.5-13;
s2, deproteinizing the filtrate to remove precipitates;
s3, adding absolute ethyl alcohol into the filtrate obtained in the step S2 until precipitates are separated out, refrigerating and standing the precipitates, keeping the precipitates, and freeze-drying the precipitates to obtain bletilla striata polysaccharide;
s4, sulfonating splendid achnatherum polysaccharide to obtain a sulfonated bletilla striata polysaccharide polymer;
the sulfonation modification method comprises the following steps: dissolving bletilla striata polysaccharide in a formamide or dimethyl sulfoxide solution to obtain a suspension, gradually and slowly adding the suspension into a chlorosulfonic acid-pyridine esterification reagent, reacting in an ice-water bath for 3-5 hours, adjusting the pH value to be neutral, centrifuging, and freeze-drying;
s5, preparing the bletilla striata polysaccharide sulfonated polymer into the spongy hemostatic and wound-protecting dressing.
2. The method for preparing an absorbable hemostatic wound dressing according to claim 1, wherein the weight ratio of bletilla striata to the extracting solution is 1: 30 to 50.
3. The method for preparing an absorbable hemostatic and wound-protecting dressing according to claim 1, wherein the extraction solution is sodium hydroxide solution or potassium hydroxide solution.
4. The method for preparing an absorbable hemostatic and wound-protecting dressing according to claim 1, wherein the deproteinizing process of step S2 is: concentrating the filtrate, adding sevage reagent with the same volume, shaking for reaction, standing for layering, and filtering to remove precipitate.
5. The method for preparing the absorbable hemostatic and wound-protecting dressing according to claim 1, wherein the precipitate obtained after filtration in step S3 is re-dissolved and then subjected to a secondary precipitation treatment with absolute ethanol.
6. The method for preparing an absorbable hemostatic and wound-protecting dressing according to claim 5, wherein the precipitation is redissolved and then decolorized with activated carbon.
7. The method for preparing an absorbable hemostatic and wound-protecting dressing according to claim 1, wherein the bletilla polysaccharide sulfonated polymer is prepared into a spongy hemostatic and wound-protecting dressing by a low-temperature freeze-drying method.
8. An absorbable hemostatic wound dressing prepared by the method of any one of claims 1 to 7.
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