CN111303461A

CN111303461A - High molecular polysaccharide hydrogel and preparation method thereof

Info

Publication number: CN111303461A
Application number: CN202010314743.1A
Authority: CN
Inventors: 肖扬; 陈万程
Original assignee: Guangzhou Dakang Gene Technology Co ltd
Current assignee: Guangzhou Dakang Gene Technology Co ltd
Priority date: 2019-11-05
Filing date: 2020-04-21
Publication date: 2020-06-19

Abstract

The invention discloses a macromolecular polysaccharide hydrogel and a preparation method thereof, the material takes cellulose and pectin as raw materials, the main process is heating, cooling and washing, in particular, ionic liquid is added for crosslinking at high temperature, and isopropanol aqueous solution is used for soaking until colloid is separated out. The hydrogel is mild in preparation conditions, simple in method, few in reagents and free of violent reaction. The result shows that the composite hydrogel material has a good adsorption effect on methylene blue and can be developed into a drug adsorption material. Cell compatibility experiments show that the cells are still fusiform on the hydrogel, do not crack, grow in a flaky and adherent manner, have good growth conditions, show that the material has no obvious cytotoxicity, is a material with good biocompatibility, has a hemostatic effect, and can be developed into a material for medical use.

Description

High molecular polysaccharide hydrogel and preparation method thereof

Technical Field

The invention relates to the field of medical materials, in particular to a high-molecular polysaccharide hydrogel and a preparation method thereof.

Background

The hydrogel is a special dispersion system with a space network structure by taking water as a dispersion medium, and is characterized in that colloidal particles or macromolecules are connected with each other under certain conditions, and the water medium is filled in gaps of the structure and is filled in the gaps. The structure mainly forms a net structure through chemical bonds, and also can form the net structure through intermolecular force, physical entanglement, hydrogen bonds or metal complexation. The hydrogel can absorb water and keep water due to the characteristics of high elasticity, non-linear plasticity and the like of a three-dimensional network structure. The hydrogel has wide application in various fields such as agriculture, food, medicine, cosmetics, environmental protection and the like at home and abroad. Such as drug carriers, medical materials, adsorption and separation, biosensors, water retention and moisture retention, immobilized enzymes, intelligent textiles and the like. At present, the application of hydrogel as a drug carrier material is the most important and mature at home and abroad. The traditional hydrogel synthesized by adopting small molecular monomers such as acrylamide, acrylic acid and the like is limited in practical utilization due to poor biocompatibility, difficult biodegradation and environmental friendliness. The prior art urgently needs a material and a method which have good synthetic biocompatibility, are environment-friendly, have lower cost and are simple to manufacture.

Skin injury and bleeding are common health hazards, the skin injury and heavy bleeding caused by trauma often evolve into large-area skin defects which are difficult to heal, the heavy bleeding is one of the main causes of death caused by natural disasters and traffic accidents, and the life quality of patients is seriously influenced. Currently, the methods for treating skin wounds and stopping bleeding which are commonly used clinically mainly include skin transplantation, medical gelatin sponge, calcium alginate fiber pads and the like, and all have the defects that the skin transplantation source is limited, and the gelatin sponge and the calcium alginate fiber pads belong to hemostatic materials with single raw materials, so that the hemostatic treatment effect on infection or deep wounds is poor. Therefore, the development of a novel effective multifunctional composite material for stopping bleeding and promoting injury repair has clinical significance.

Disclosure of Invention

The invention aims to provide a high-polymer polysaccharide hydrogel which has high water absorption, high drug adsorption and a hemostatic function, and a preparation method thereof.

The purpose of the invention is realized by the following technical scheme:

the first aspect of the technical scheme of the invention is a method for preparing high-molecular polysaccharide hydrogel, which comprises the following steps:

the method comprises the following steps: mixing cellulose and pectin, and adding a chemical cross-linking agent;

step two: heating the mixture in the step one, stirring and dissolving;

step three: cooling the mixture obtained in the second step to room temperature;

step four: adding isopropanol and water solution with the volume ratio of 1: 1 into the mixture in the third step, and soaking until colloid is separated out;

optionally replacing the isopropanol and aqueous solution in the test tube multiple times during the soaking;

optionally fully soaking and washing the colloid with deionized water;

and step five, freeze-drying the colloid to obtain the high-molecular polysaccharide hydrogel.

In a preferred embodiment of the invention, the cellulose is selected from pineapple pomace cellulose, bagasse cellulose, and/or tea leaf cellulose.

In a preferred embodiment of the present invention, the mass ratio of the cellulose to the pectin is 5:1 to 5: 4.

In a preferred embodiment of the invention, the chemical cross-linking agent is an ionic liquid, more preferably the ionic liquid is selected from [ AMIM ] Cl, [ Bdmim ] Cl, [ R1R2mim ] Cl, [ C2mim ] Br, [ Hemim ] Br, and/or [ bmim ] Cl.

In a preferred embodiment of the present invention, the chemical crosslinking agent is added in an amount of 90% to 95%.

In a preferred embodiment of the invention, the second step is carried out in a constant-temperature magnetic stirring oil bath, wherein the temperature of the constant-temperature magnetic stirring oil bath is 100 ℃, and the rotating speed is set to be 50-100 r/min.

The second aspect of the present invention is a polymer polysaccharide hydrogel prepared by the above method.

The third aspect of the technical scheme of the invention is the application of the prepared macromolecular polysaccharide hydrogel in the aspects of medicine adsorption materials and the like.

The fourth aspect of the technical scheme of the invention is the application of the prepared high molecular polysaccharide hydrogel in the aspect of preparing the hemostatic.

Compared with the prior art, the invention has the following advantages and beneficial effects:

firstly, the pectin/cellulose composite hydrogel prepared from pectin and cellulose has better drug adsorption, biocompatibility, biodegradability and other properties. Can be used as a drug adsorbent in the field of environmental protection. The result shows that the composite hydrogel material has a good adsorption effect on methylene blue and can be developed into a drug adsorption material. Cell compatibility experiments show that the cells are still fusiform on the hydrogel, do not crack, grow in a flaky and adherent manner, have good growth conditions, show that the material has no obvious cytotoxicity, is a material with good biocompatibility, and can be developed into a material for medical application.

Secondly, the solvent ionic liquid for preparing the hydrogel is stable to heat, high in chemical stability, non-volatile, colorless, tasteless and non-combustible, and is in a relatively stable liquid state within a certain temperature range; soluble cellulose having high polarity; and is easy to separate from other substances, can be cleaned by soaking in isopropanol-water (1: 1) solution, and has almost no residue in hydrogel.

Thirdly, the pineapple dreg cellulose, bagasse cellulose or tea leaf cellulose is used for preparing the hydrogel, the used reagents are few, the toxicity is low, and the preparation process is simple and operable. The hydrogel is mild in preparation conditions, simple in method and free of violent reaction.

Fourth, the pectin/cellulose hydrogel obtained by the invention can promote the repair of skin injury and accelerate hemostasis. The hydrogel has the advantages of rapid gelation, good biocompatibility and the like, so the hydrogel can be used as a gel type hemostatic and applied to trauma hemostasis and surgical hemostasis.

Drawings

FIG. 1 is a schematic illustration of the preparation and efficacy of the present invention, FIG. 1A is a process flow diagram, FIG. 1B is a schematic illustration of the promotion of wound repair, and FIG. 1C is a schematic illustration of the promotion of hemostasis;

FIG. 2 is a pictorial representation of the present invention, and FIG. 2A is an optical image of a pectin/cellulose composite hydrogel; FIG. 2B is a Scanning Electron Microscope (SEM) image of a pectin/cellulose composite hydrogel; fig. 2C is an SEM image of a cellulose hydrogel, and fig. 2D is an SEM image of a pectin hydrogel.

Fig. 3 shows the physicochemical parameters of pectin, pectin/cellulose composite hydrogel (CH-P40%) and cellulose gum, fig. 3A shows fourier transform infrared spectroscopy (FTIR), fig. 3B shows X-ray diffraction analysis (XRD), and fig. 3C shows Thermogravimetric (TG) and differential thermal weight loss (DTG) analyses.

FIG. 4 is a graph showing the results of the biotoxicity test of the present invention, FIG. 4A is the biocompatibility of the present invention to cells, and FIG. 4B is the results of the toxicity test of the present invention to cells by CCK 8; FIG. 4C shows the results of a flow cytometry assay to detect toxicity to cells of the present invention;

FIG. 5 shows the experimental results of the present invention on the repair of skin damage in mice, wherein FIG. 5A is a control group and FIG. 5B is an experimental group;

fig. 6 is a result of a hemostasis test of the present invention, fig. 6A is a result of an in vitro hemostasis function test, fig. 6B is a result of an in vivo hemostasis test, fig. 6C is a schematic view of hemostasis promotion of the present invention, and fig. 6D is a schematic view of a hydrogel hemostasis process.

Detailed Description

Compared with the traditional high-molecular hydrogel material, the high-molecular polysaccharide hydrogel has the advantages of natural non-toxicity, biodegradability, better biocompatibility, special functionality and the like, so that the high-molecular polysaccharide hydrogel is widely concerned and applied, cellulose is high-molecular polysaccharide formed by connecting D-glucose by β -1, 4-glycosidic bonds, and the molecular general formula is (C)₆H₁₀O₅)_nN is determined by the degree of polymerization, and is generally 2000 to 15000 or more. Is a renewable biomass resource which widely exists in organisms, and the main sources are plants such as cotton and hemp, wheat straw, bamboo and the like. Has the advantages of wide source, low price, good biocompatibility, environmental protection and the like. The natural pectin substances are widely present in fruits, roots, stems and leaves of plants in the forms of protopectin, pectin and pectic acid, and are one of the components of cell walls. Pectin is essentially a linear polysaccharide polymer containing hundreds to about 1000 anhydrogalacturonic acid residues with a corresponding average relative molecular mass of 50000-150000.

The pectin/cellulose composite hydrogel prepared by taking cellulose as a matrix and simultaneously adding a certain proportion of pectin is a high-molecular polysaccharide material which has a loose and porous structure, high water absorption and high drug adsorption. It has wide material source, low cost and simple preparation process. Has stable structure and excellent performance, and is a hydrogel material with application prospect. Compared with the traditional high-molecular hydrogel material, the high-molecular polysaccharide hydrogel has the advantages of natural non-toxicity, biodegradability, better biocompatibility, multiple biological functions and the like, so that the high-molecular polysaccharide hydrogel is widely concerned and applied.

Ionic liquids (or ionic liquids) are liquids composed entirely of ions, such as KCI at high temperature, KOH in liquid state, and they are ionic liquids. Substances composed of ions which are liquid at or around room temperature are called room temperature ionic liquids, room temperature molten salts (room temperature ionic liquids are often accompanied by the presence of hydrogen bonds and are defined as room temperature molten salts to be somewhat rare), organic ionic liquids, and the like, and there is no unified name at present, but ionic liquids are apt to be abbreviated as ionic liquids. In the ionic compound, the acting force between the anions and cations is coulomb force, the magnitude of which is related to the charge quantity and radius of the anions and cations, and the larger the ionic radius is, the smaller the acting force between the anions and the cations is, and the lower the melting point of the ionic compound is. Certain ionic compounds have bulky anions and cations and loose structures, resulting in low forces between them, such that the melting point is close to room temperature.

Cellulose is a high molecular weight polysaccharide formed by connecting D-glucose (D-glucose) by β -1, 4-glycosidic bonds, and has a molecular formula of (C6H 10O5) n, wherein n is determined by the degree of polymerization and is generally 2000 to 15000. therefore, the cellulose of pineapple residue, bagasse or tea leaves is basically close in structure, and the degree of polymerization is slightly different.

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.

Example 1

A preparation method of a high-molecular polysaccharide hydrogel capable of adsorbing drugs comprises the following steps:

respectively putting 0.50g of pineapple dreg cellulose (PC) and 0.40g of pectin into a test tube, then adding 10g of [ AMIM ] Cl, placing the test tube into a constant-temperature magnetic stirring oil bath kettle, stirring and dissolving at 100 ℃, and setting the rotating speed to be 60 revolutions per minute. After the heating, the mixture was cooled to room temperature. Then adding isopropanol-water (1: 1) solution into the tube, and soaking until colloid is separated out. To wash the ionic liquid thoroughly, the isopropanol-water solution in the test tube was replaced several times during the soaking period. And finally, fully soaking and washing the gel with deionized water, and freeze-drying at-80 ℃ to obtain the pineapple residue cellulose-pectin composite hydrogel (as shown in figures 1 and 2).

The hydrogel prepared by the method of the present invention was subjected to the following performance measurement.

(1) Morphological observation

The ultrastructure of the invention is observed using a Scanning Electron Microscope (SEM) under conventional study conditions. The probiotic microcapsules after freeze drying were adhered to a sample stage with double-sided tape, and the surface structure of the microcapsules was observed with a scanning electron microscope after gold spraying, and the results are shown in fig. 2. The results show that the structure of the invention is loose and porous, the surface is uneven, and the structure has a fibrous cord structure.

(2) Determination of physicochemical parameters

The physical and chemical parameters of the pectin/cellulose composite hydrogel (CH-P40%) and the cellulose gel obtained in the invention were characterized by FTIR, XRD, TG and DTG. The results show that FTIR shows that the FTIR spectrum of CH-P40% presents characteristic peaks of chemical structures and functional groups of cellulose and pectin, and the characteristic peaks of the composite hydrogel are similar to those of the cellulose and the pectin, and the ionic liquid does not involve any derivatization reaction during the heating and dissolving processes of the cellulose and the pectin and only plays a role in dissolving. XRD patterns show that the characteristic peak property of the composite hydrogel is more similar to that of cellulose, and the addition of pectin does not destroy the crystal structure of the cellulose hydrogel. The two show that the cellulose-pectin composite hydrogel synthesized by the method better retains the chemical structures of cellulose and pectin and does not influence the respective functional characteristics. As can be seen from the TG and DTG plots, the initial decomposition temperature of CH-P40% increased from 337 ℃ to 345 ℃ compared to CH, indicating that pectin may slightly improve the thermal stability of the hydrogel prepared

(3) Drug adsorption and drug sustained release experiments

Drawing of a Methylene blue (Methylene blue) standard curve: accurately weighing 0.0100g of methylene blue, dissolving in deionized water, and diluting to 100mL to obtain a methylene blue standard solution with the concentration of 100 mg/L. Then, the solution was diluted to a standard solution having a concentration gradient of 10mg/L, 20mg/L, 40mg/L, 80mg/L, or 100 mg/L. And measuring the light absorption value of the standard curve at 665nm by using a microplate reader, taking the concentration of the methylene blue solution as an abscissa and the corresponding light absorption value as an ordinate, drawing a standard curve, and obtaining a regression equation of the standard curve, wherein y is 0.0117x + 0.1406.

Accurately weigh 0.10g of the lyophilized hydrogel sample into a 50mL serum bottle, and add 25mL of methylene blue solution at a concentration of 100.0 mg/L. Standing at room temperature, sampling at regular intervals, measuring the light absorption value of the solution at 665nm by using a microplate reader, and calculating the residual capacity of methylene blue in the solution by using a methylene blue standard curve. Adsorption capacity (Q) of sample to methylene blue_e) Margin adsorption ratio (R)_e) Calculated according to the following formula:

Qe＝(C₀-C_t)V/m------------------------------------------(1)

Re＝(1-C₀/C_t)*100％-------------------------------------(2)

in the formula, c₀Initial concentration of methylene blue solution (mg/L); c. C_tIs the concentration of the methylene blue solution at the time t (mg/L); v is the volume of methylene blue solution (mL); and m is the mass (g) of the hydrogel before drug loading. The calculation analysis shows that the adsorption capacity of the hydrogel added with the pectin is higher and can reach 13.51mg/g in a certain range. However, the loading rate of the hydrogel sample to methylene blue does not change obviously with the increase of the addition amount of the pectin.

(4) Biocompatibility experiment

Spreading the prepared hydrogel in a 6-well plate, soaking and sterilizing with 75% ethanol solution for 24 hr, and washing the residual ethanol solution with phosphate buffer solution for several times. Then, 3T3 cells were diluted with the culture medium to the desired number of cells and added to 6-well plates, and after 24 hours of culture, morphologically observed under a microscope. The growth states of the cells of the hydrogel Sample (Sample) and the Control group (Control) are similar, the cells are fusiform, the cells are not cracked, and grow in a large scale and adherent manner, so that the growth condition is good. As the above experiment, the hydrogel of the experiment has excellent biocompatibility, drug adsorption and drug sustained release properties (as shown in FIG. 3).

Example 2

0.50g of bagasse cellulose (SC) and 0.30g of pectin are respectively put into a test tube, 10g of [ Bdmim ] Cl is added into the test tube, the test tube is placed into a constant-temperature magnetic stirring oil bath kettle, and is stirred and dissolved at 100 ℃, and the rotating speed is set as 50 revolutions per minute. After the heating, the mixture was cooled to room temperature. Then adding isopropanol-water (1: 1) solution into the tube, and soaking until colloid is separated out. To wash the ionic liquid thoroughly, the isopropanol-water solution in the test tube was replaced several times during the soaking period. And finally, fully soaking and washing the gel with deionized water, and freeze-drying at-80 ℃ to obtain the bagasse cellulose-pectin composite hydrogel. As the experiment, the hydrogel of the experiment has excellent biocompatibility, drug adsorption and drug slow release performance.

Example 3

A preparation method of a high molecular polysaccharide hydrogel capable of adsorbing drugs comprises the following steps as shown in figure 1:

0.50g of Tea Cellulose (TC) and 0.10g of pectin are respectively put in a test tube, 10g of [ bmim ] Cl is added, the test tube is placed in a constant-temperature magnetic stirring oil bath kettle and is stirred and dissolved at 100 ℃, and the rotating speed is set as 60 revolutions per minute. After the heating, the mixture was cooled to room temperature. Then adding isopropanol-water (1: 1) solution into the tube, and soaking until colloid is separated out. To wash the ionic liquid thoroughly, the isopropanol-water solution in the test tube was replaced several times during the soaking period. And finally, fully soaking and washing the colloid with deionized water, and freeze-drying at-80 ℃ to obtain the tea cellulose-pectin composite hydrogel.

Example 4

The hydrogel prepared in the above example is subjected to compatibility detection, and the selected study objects are mouse macrophage cell line RAW263.7 and breast cancer cell 3T 3. The results are shown in FIG. 4, the cells are still fusiform on the hydrogel, do not crack, grow in sheets and adherently, and grow well. Meanwhile, cell activity detection shows that the material has no obvious cytotoxicity, is a material with good biocompatibility and can be developed into a material for medical use.

Example 5

Selecting a plurality of C57 mice with the weight of about 25g in 4 weeks, after anesthesia, constructing a skin injury model by using a puncher, randomly dividing the mice into 2 groups, covering the mice by using the hydrogel obtained by the invention in an experimental group, smearing the mice in a control group by using PBS, and observing the wound repair process of the two groups of mice. As a result, as shown in FIG. 5, the hydrogel obtained in the present invention can promote the healing process of the damaged tissue. Meanwhile, HE staining is carried out on the damaged tissue, and the result shows that the damaged tissue treated by the hydrogel provided by the invention has obvious hyperplasia of new dense fibrous tissue structure and small angiogenesis compared with the control group. The result shows that the hydrogel obtained by the invention can accelerate damage repair.

Example 6

Firstly, the hemostatic function of the hydrogel obtained by the invention is detected by performing a blood clot index experiment in vitro, and the experimental result shows that the hydrogel can obviously promote the formation of a blood clot. Meanwhile, a plurality of C57 mice with the weight of about 25g in 4 weeks are selected, after anesthesia, a liver bleeding model is constructed, the mice are randomly divided into 2 groups, the experimental group is covered by the hydrogel obtained by the invention, the control group is smeared by PBS, and the bleeding conditions of the two groups of mice are observed (as shown in figure 6). The experimental result shows that the hydrogel obtained by the invention can complete the hemostasis process after 3 minutes after rapidly covering the wound. Fig. 6D is a schematic illustration of a hydrogel hemostasis process.

In vitro experiment results show that the pectin/cellulose hydrogel obtained by the invention does not influence the cell activity, and the results are shown in FIGS. 3A-B; meanwhile, the influence of the pectin/cellulose hydrogel on the apoptosis level is detected by flow cytometry, and the result is shown in fig. 4C, and the pectin/cellulose hydrogel obtained by the method does not influence the apoptosis of cells. The result shows that the invention has good biocompatibility and no toxicity to cells. In vivo experiments show that the pectin/cellulose hydrogel prepared by the invention can promote skin injury repair and accelerate hemostasis. The hydrogel has the advantages of rapid gelation, good biocompatibility and the like, so the hydrogel can be used as a gel type hemostatic and applied to trauma hemostasis and surgical hemostasis.

Comparative example 1

0.50g of Tea Cellulose (TC) and 0.08g of pectin are respectively put into a test tube, 10g of [ AMIm ] Cl is added into the test tube, the test tube is placed into a constant-temperature magnetic stirring oil bath kettle, and is stirred and dissolved at 100 ℃, and the rotating speed is set as 60 revolutions per minute. After the heating, the mixture was cooled to room temperature. Then adding isopropanol-water (1: 1) solution into the tube, and soaking until colloid is separated out. To wash the ionic liquid thoroughly, the isopropanol-water solution in the test tube was replaced several times during the soaking period. And finally, fully soaking and washing the colloid with deionized water, and freeze-drying at-80 ℃ to obtain the tea cellulose-pectin composite hydrogel.

Comparative example 2

0.50g of bagasse cellulose (SC) and 4.20g of pectin are respectively put into a test tube, 10g of [ AMIm ] Cl is added into the test tube, the test tube is placed into a constant-temperature magnetic stirring oil bath kettle, and is stirred and dissolved at 100 ℃, and the rotating speed is set as 60 revolutions per minute. After the heating, the mixture was cooled to room temperature. Then adding isopropanol-water (1: 1) solution into the tube, and soaking until colloid is separated out. To wash the ionic liquid thoroughly, the isopropanol-water solution in the test tube was replaced several times during the soaking period. And finally, fully soaking and washing the gel with deionized water, and freeze-drying at-80 ℃ to obtain the bagasse cellulose-pectin composite hydrogel.

The tea leaf cellulose-pectin composite hydrogel in the comparative example 1 and the bagasse cellulose-pectin composite hydrogel in the comparative example 2 are subjected to a drug adsorption experiment and a hemostasis experiment similar to those described above, and surprisingly, the effect of the hydrogel is remarkably inferior to that of the hydrogel prepared in the examples, wherein the mass ratio of cellulose to pectin is 5: 1-5: 4, and the hydrogel has an unexpected technical effect when the mass ratio of cellulose to pectin is 5: 1-5: 4, and has performances remarkably superior to those of hydrogels with other components and proportions in drug adsorption and hemostasis.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A method of preparing a high molecular polysaccharide hydrogel comprising the steps of:

step two: heating the mixture in the step one, stirring and dissolving;

optionally, fully washing the colloid with deionized water;

2. The method for preparing a polymeric polysaccharide hydrogel according to claim 1, wherein the cellulose is selected from pineapple residue cellulose, bagasse cellulose, and/or tea leaf cellulose.

3. The method for preparing the high-molecular polysaccharide hydrogel according to claim 1, wherein the mass ratio of the cellulose to the pectin is 5:1 to 5: 4.

4. The method for preparing a polymeric polysaccharide hydrogel of claim 1 wherein said chemical cross-linking agent is an ionic liquid.

5. The method for preparing a polymeric polysaccharide hydrogel of claim 4 wherein the ionic liquid is selected from the group consisting of [ AMIM ™ ]]Cl、[Bdmim]Cl、[R¹R²mim]Cl、[C₂mim]Br、[Hemim]Br, and/or [ bmim]Cl。

6. The method for preparing a polymeric polysaccharide hydrogel according to claim 1, wherein the chemical crosslinking agent is added in an amount of 90 to 95% by mass.

7. The method for preparing the polymeric polysaccharide hydrogel according to claim 1, wherein the second step is carried out in a constant temperature magnetic stirring oil bath, wherein the temperature of the constant temperature magnetic stirring oil bath is 100 ℃ and the rotation speed is 50-100 r/min.

8. A polymeric polysaccharide hydrogel prepared by the method of any one of claims 1 to 7.

9. Use of the polymeric polysaccharide hydrogel of claim 8 for the preparation of a drug-adsorbing material.

10. Use of the polymeric polysaccharide hydrogel of claim 8 in the preparation of a hemostatic agent.