CN110604742B - Eucommia polysaccharide strontium complex and preparation method and application thereof - Google Patents
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Abstract
本发明涉及一种杜仲多糖锶络合物及其制备方法和应用。一种杜仲多糖锶络合物的制备方法，包括如下步骤：在控制pH为9～11的条件下，将杜仲多糖和水溶性锶盐在水中反应，得到反应液；在反应液中加入有机沉淀剂，然后静置反应，再经固液分离，收集沉淀，得到杜仲多糖络合物。上述杜仲多糖锶络合物的毒性较小且具有较好的骨修复和关节病防治效果。
The invention relates to a polysaccharide strontium complex of Eucommia ulmoides and its preparation method and application. A preparation method of Eucommia polysaccharide strontium complex, comprising the following steps: reacting Eucommia polysaccharide and water-soluble strontium salt in water to obtain a reaction solution; adding organic precipitate to the reaction solution Then, the reaction is allowed to stand, and then the solid-liquid separation is carried out to collect the precipitate to obtain the Eucommia polysaccharide complex. The above-mentioned Eucommia polysaccharide strontium complex has less toxicity and better bone repair and joint disease prevention and treatment effects.
Description
Technical Field
The invention relates to the field of biological medicines, in particular to an eucommia polysaccharide strontium complex and a preparation method and application thereof.
Background
Strontium plays an important role in bone formation, and can promote the proliferation of osteoblasts and multifunctional stem cells, improve bone metabolism and the like, however, free strontium ions have toxicity in vivo and are difficult to develop for the application of strontium.
Disclosure of Invention
Therefore, a preparation method of eucommia polysaccharide strontium complex with low toxicity and good bone repair and joint disease prevention and treatment effects is needed.
In addition, an eucommia polysaccharide strontium complex and application thereof are also provided.
A preparation method of eucommia polysaccharide strontium complex comprises the following steps:
reacting eucommia ulmoides polysaccharide and water-soluble strontium salt in water under the condition that the pH value is controlled to be 9-11 to obtain a reaction solution; and
and adding an organic precipitator into the reaction solution, standing for reaction, performing solid-liquid separation, and collecting precipitate to obtain the eucommia ulmoides polysaccharide complex.
In one embodiment, the mass molar ratio of the eucommia ulmoides polysaccharide to the strontium element in the water-soluble strontium salt is 60g:1mol to 250g:1 mol.
In one embodiment, the step of reacting eucommia ulmoides polysaccharide and a water-soluble strontium salt in water under the condition of controlling the pH to be 9-11 comprises the following steps:
respectively dissolving the eucommia ulmoides polysaccharide and the water-soluble strontium salt in water to obtain an aqueous solution of the eucommia ulmoides polysaccharide and an aqueous solution of the water-soluble strontium salt; adding the water-soluble strontium salt water solution into the eucommia ulmoides polysaccharide water solution under the conditions of continuously stirring and controlling the pH value to be 9-11, and then continuously stirring and reacting for more than 1 hour under the condition of controlling the pH value to be 9-11.
In one embodiment, the concentration of the eucommia ulmoides polysaccharide aqueous solution is 0.5 g/mL-1.5 g/mL; and/or the concentration of strontium element in the water solution of the water-soluble strontium salt is 1.5-2 mol/L; and/or the volume ratio of the organic precipitator to the reaction liquid is 2: 1-5: 1.
In one embodiment, the water soluble strontium salt is strontium chloride; and/or the organic precipitator is anhydrous acetone or anhydrous ethanol.
In one embodiment, the standing reaction time is more than 24 hours.
In one embodiment, sodium hydroxide is used as a pH regulator in the step of reacting the eucommia ulmoides polysaccharide and the water-soluble strontium salt in water under the condition of controlling the pH to be 9-11.
In one embodiment, the step of reacting the eucommia ulmoides polysaccharide and the water-soluble strontium salt in water is performed at 10-40 ℃.
In one embodiment, the method further comprises the step of washing the precipitate with ethanol.
In one embodiment, the washing the precipitate with ethanol comprises: and sequentially washing the precipitate by using ethanol with different concentrations, and drying the precipitate.
The eucommia polysaccharide strontium complex prepared by the preparation method of the eucommia polysaccharide strontium complex.
The application of the eucommia ulmoides polysaccharide complex in preparing bone repair materials.
Experiments prove that: according to the preparation method of the eucommia polysaccharide strontium complex, the eucommia polysaccharide strontium complex with good bone repair and joint disease prevention and treatment effects can be prepared according to the steps, and has low toxicity compared with free strontium ions.
Drawings
FIG. 1 is a flowchart of a method for preparing an eucommia ulmoides polysaccharide complex according to an embodiment;
FIG. 2 is an XRD pattern of the product of example 1 and the eucommia ulmoides polysaccharide of comparative example 3;
FIG. 3 is an infrared spectrum of the product obtained in example 1 and the eucommia ulmoides polysaccharide of comparative example 3;
FIG. 4 is a scanning electron micrograph of the product of example 1;
FIG. 5 is a scanning electron micrograph of a eucommia ulmoides polysaccharide of comparative example 3;
FIG. 6 is a bar graph of macrophages cultured on the culture solution prepared from the eucommia ulmoides polysaccharide strontium complex of example 1 and the control experiment at the third day, respectively.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the following description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
As shown in fig. 1, a method for preparing a eucommia ulmoides polysaccharide strontium complex according to an embodiment includes the steps of:
step S110: and reacting the eucommia ulmoides polysaccharide and the water-soluble strontium salt in water under the condition of controlling the pH value to be 9-11 to obtain a reaction solution.
The pH is controlled to 9 to 11 so that the reaction proceeds smoothly, thereby producing a eucommia polysaccharide strontium complex.
Specifically, the mass molar ratio of the eucommia ulmoides polysaccharide to the strontium element in the water-soluble strontium salt is 60g:1 mol-250 g:1 mol. Too much strontium can leave a large amount of strontium ions in the complex, which can cause the material to be harmful in vivo. If the strontium element is less, the eucommia polysaccharide is excessive, and the complex reaction is insufficient.
In one embodiment, step S110 includes: respectively dissolving eucommia ulmoides polysaccharide and water-soluble strontium salt in water to obtain an aqueous solution of eucommia ulmoides polysaccharide and an aqueous solution of water-soluble strontium salt; adding a water-soluble strontium salt aqueous solution into the eucommia ulmoides polysaccharide aqueous solution under the conditions of continuously stirring and controlling the pH value to be 9-11, and then continuously stirring and reacting for more than 1 hour under the condition of controlling the pH value to be 9-11. Preferably, the water used in the step of dissolving the eucommia ulmoides polysaccharide and the water-soluble strontium salt in water is deionized water, so as to reduce impurities as much as possible and improve the purity of the product.
It is understood that, in other embodiments, the step S110 is not limited to the above-mentioned step, and in other embodiments, an aqueous solution of eucommia ulmoides polysaccharide may be added to an aqueous solution of water-soluble strontium salt; or directly adding the eucommia ulmoides polysaccharide and the water-soluble strontium salt into water, and stirring for reaction.
In one embodiment, the concentration of the aqueous solution of the eucommia ulmoides polysaccharide is 0.5 g/mL-1.5 g/mL, the maximum concentration of the aqueous solution of the eucommia ulmoides polysaccharide is 1.5g/mL, and the concentration of the aqueous solution is too low, so that the content of the eucommia ulmoides polysaccharide is too low, and the complex reaction is difficult to occur. The concentration of strontium element in the water solution of the water-soluble strontium salt is 1.5-2 mol/L, the strontium ion concentration is too high, the hydrolysis degree of the salt is reduced, and hydrogen ions in the solution are increased, so that the pH is influenced, and the generated complex is unstable. If the concentration of strontium is too low, the formation of coordinate bonds is less and the reaction is incomplete.
The water-soluble strontium salt can be a strontium salt which is commonly used in the field and can be dissolved in water, such as strontium chloride or strontium nitrate, and preferably, the water-soluble strontium salt is strontium chloride which can enable the prepared eucommia polysaccharide strontium complex to have fewer byproducts, higher purity and almost no toxicity.
Specifically, in step S110, sodium hydroxide or potassium hydroxide is used as a pH adjuster. It is understood that the pH regulator is not limited to sodium hydroxide, and in other embodiments, the pH regulator may be ammonia water, etc., however, the eucommia ulmoides polysaccharide contains sodium and potassium, and the sodium hydroxide or potassium hydroxide is used for regulating the pH regulator, so that redundant impurities are not generated, and the ammonia water may introduce new impurities into the system. In one embodiment, the pH regulator is sodium hydroxide aqueous solution with the concentration of 1.5-2 mol/L or potassium hydroxide aqueous solution with the concentration of 1.5-2 mol/L. The sodium hydroxide and the potassium hydroxide are strong bases, the increase of the concentration can cause local oxidation of the eucommia polysaccharide, the excessively low concentration can cause the overlarge volume of the eucommia polysaccharide, so that the concentration of each substance in a reaction system is reduced too much, and the reaction is not easy to occur.
Specifically, the step of reacting the eucommia ulmoides polysaccharide and the water-soluble strontium salt in water is performed under a room temperature condition. Herein, room temperature means 10 ℃ to 40 ℃.
Step S120: adding an organic precipitator into the reaction solution, standing for reaction, performing solid-liquid separation, and collecting precipitate to obtain the eucommia ulmoides polysaccharide complex.
Adding organic precipitant, and standing for reaction to precipitate the eucommia ulmoides polysaccharide complex in the reaction solution. The organic precipitant may be an organic precipitant commonly used in the art. Specifically, the organic precipitant is anhydrous acetone or anhydrous ethanol. Preferably, the organic precipitator is absolute ethyl alcohol, and the toxicity of the absolute ethyl alcohol is lower compared with that of absolute acetone, so that the prepared eucommia polysaccharide strontium complex has lower toxicity, and the absolute ethyl alcohol is cheap, and is beneficial to reducing the production cost.
Specifically, the reaction time is 24 hours or more so that the eucommia ulmoides polysaccharide complex in the reaction solution can be precipitated as much as possible.
Specifically, the volume ratio of the organic precipitator to the reaction solution is 2: 1-5: 1, so that the purpose of removing impurities by washing is achieved on the premise of not wasting more organic precipitator.
Further, after step S120, a step of washing the precipitate with ethanol is further included to perform a purification process on the eucommia ulmoides polysaccharide complex. Washing with ethanol to remove strontium chloride in the precipitate and improve the purity of the eucommia ulmoides polysaccharide complex.
Specifically, the step of washing the precipitate with ethanol comprises: washing the precipitate with ethanol of different concentrations, and drying the precipitate. In one embodiment, the drying temperature in the step of drying the precipitate is 37 ℃ to 45 ℃.
In one embodiment, the step of sequentially washing the precipitate with ethanol of different concentrations comprises: ethanol with a volume percentage of 80%, ethanol with a volume percentage of 60% and ethanol with a volume percentage of 40% were mixed with the precipitate in sequence, with stirring for at least 30 minutes each time. Specifically, the volume ratio of ethanol with each concentration to the reaction solution obtained in step S110 is 2.5:1 to 5:1, so as to achieve the purpose of effective washing.
Experiments prove that: the eucommia polysaccharide strontium complex prepared by the preparation method of the eucommia polysaccharide strontium complex can avoid the toxicity of free strontium ions through the complexation reaction of strontium and macromolecules of eucommia polysaccharide. Meanwhile, the prepared eucommia polysaccharide strontium complex also has the characteristics of the eucommia polysaccharide strontium complex and enables strontium to be effectively released in vivo, plays the roles of reducing inflammation and proliferating bone cells in strontium and can better assist in treating osteoarthritis. The eucommia polysaccharide strontium complex can change the immune environment of bones and promote the proliferation and differentiation of cells related to bone repair. Can play an important role in bone repair by regulating bone immunity.
The eucommia ulmoides polysaccharide strontium complex prepared by the preparation method of eucommia ulmoides polysaccharide strontium complex of one embodiment. The eucommia polysaccharide strontium complex has good bone repair and joint disease prevention and treatment effects, and has low toxicity.
The eucommia polysaccharide complex can be used for preparing bone repair materials, so that the bone repair materials have good bone repair and joint disease prevention and treatment effects and low toxicity.
The following are specific examples (the following examples, unless otherwise specified, contain no other components not specifically indicated except for unavoidable impurities):
example 1
The preparation process of the eucommia ulmoides polysaccharide strontium complex of the embodiment is as follows:
(1) dissolving eucommia ulmoides polysaccharide in deionized water to prepare 1g/mL eucommia ulmoides polysaccharide water solution; dissolving strontium chloride in deionized water to prepare a strontium chloride aqueous solution with the concentration of 1.8 mol/L; sodium hydroxide is dissolved in deionized water to prepare a sodium hydroxide aqueous solution with the concentration of 1.8 mol/L.
(2) Under the condition of room temperature and under the condition of continuously stirring and controlling the pH value to be 10 by using an aqueous sodium hydroxide solution, adding an aqueous solution of strontium chloride into the aqueous solution of eucommia ulmoides polysaccharide according to the mass molar ratio of 150g to 1mol of the eucommia ulmoides polysaccharide to the strontium element, and after the addition of the aqueous solution of strontium chloride is finished, continuously stirring and reacting at the pH value of 10 for 1 hour at room temperature to obtain a reaction solution.
(3) Adding an organic precipitator into the reaction solution, standing for 24 hours, filtering, and collecting precipitate. Wherein the organic precipitator is absolute ethyl alcohol, and the volume ratio of the organic precipitator to the reaction liquid is 2: 1.
(4) Mixing the precipitate with 80% ethanol by volume, stirring for 30 min, and filtering to obtain filter residue; mixing the filter residue with ethanol with the volume percentage concentration of 60%, stirring for 30 minutes, filtering, mixing the filter residue with ethanol with the volume percentage concentration of 40%, stirring for 30 minutes, filtering, collecting solid, and drying the solid at 41 ℃ for 12 hours to obtain the eucommia polysaccharide strontium complex, wherein the volume mass ratio of ethanol with each concentration to the precipitate in the step (4) is 3.5: 1.
Example 2
The preparation process of the eucommia ulmoides polysaccharide strontium complex of the embodiment is as follows:
(1) dissolving eucommia ulmoides polysaccharide in deionized water to prepare an aqueous solution of the eucommia ulmoides polysaccharide with the concentration of 0.5 g/mL; dissolving strontium chloride in deionized water to prepare a strontium chloride aqueous solution with the concentration of 2 mol/L; sodium hydroxide is dissolved in deionized water to prepare a sodium hydroxide aqueous solution with the concentration of 1.5 mol/L.
(2) Under the condition of room temperature and under the condition of continuously stirring and controlling the pH value to be 9 by using an aqueous sodium hydroxide solution, adding an aqueous solution of strontium chloride into the aqueous solution of eucommia ulmoides polysaccharide according to the mass molar ratio of 60g to 1mol of the eucommia ulmoides polysaccharide to the strontium element, and after the addition of the aqueous solution of strontium chloride is finished, continuously stirring and reacting at the pH value of 9 for 2 hours at room temperature to obtain a reaction solution.
(3) Adding an organic precipitator into the reaction solution, standing for 25 hours, filtering, and collecting precipitate. Wherein the organic precipitator is absolute ethyl alcohol, and the volume ratio of the organic precipitator to the reaction liquid is 3: 1.
(4) Mixing the precipitate with 80% ethanol by volume, stirring for 40 min, and filtering to obtain filter residue; mixing the filter residue with ethanol with the volume percentage concentration of 60%, stirring for 30 minutes, filtering, mixing the filter residue with ethanol with the volume percentage concentration of 40%, stirring for 50 minutes, filtering, collecting solid, and drying the solid at 37 ℃ for 12 hours to obtain the eucommia polysaccharide strontium complex, wherein the volume mass ratio of ethanol with each concentration to the precipitate in the step (4) is 2.5: 1.
Example 3
The preparation process of the eucommia ulmoides polysaccharide strontium complex of the embodiment is as follows:
(1) dissolving eucommia ulmoides polysaccharide in deionized water to prepare an aqueous solution of the eucommia ulmoides polysaccharide with the concentration of 1.5 g/mL; dissolving strontium chloride in deionized water to prepare an aqueous solution of strontium chloride with the concentration of 1.5 mol/L; sodium hydroxide is dissolved in deionized water to prepare a sodium hydroxide aqueous solution with the concentration of 2 mol/L.
(2) Under the condition of room temperature and under the condition of continuously stirring and controlling the pH value to be 11 by using an aqueous sodium hydroxide solution, adding an aqueous solution of strontium chloride into the aqueous solution of eucommia ulmoides polysaccharide according to the mass molar ratio of 250g to 1mol of the eucommia ulmoides polysaccharide to the strontium element, and after the addition of the aqueous solution of strontium chloride is finished, continuously stirring and reacting at the pH value of 11 for 3 hours at room temperature to obtain a reaction solution.
(3) Adding an organic precipitator into the reaction solution, standing for 30 hours, filtering, and collecting precipitate. Wherein the organic precipitator is absolute ethyl alcohol, and the volume ratio of the organic precipitator to the reaction liquid is 3: 1.
(4) Mixing the precipitate with 80% ethanol by volume, stirring for 35 min, and filtering to obtain filter residue; mixing the filter residue with ethanol with the volume percentage concentration of 60%, stirring for 50 minutes, filtering, mixing the filter residue with ethanol with the volume percentage concentration of 40%, stirring for 30 minutes, filtering, collecting solid, and drying the solid at 45 ℃ for 12 hours to obtain the eucommia polysaccharide strontium complex, wherein the volume mass ratio of ethanol with each concentration to the precipitate in the step (4) is 5: 1.
Example 4
The preparation process of the eucommia ulmoides polysaccharide strontium complex of the embodiment is as follows:
(1) dissolving eucommia ulmoides polysaccharide in deionized water to prepare an aqueous solution of the eucommia ulmoides polysaccharide with the concentration of 0.8 g/mL; dissolving strontium chloride in deionized water to prepare an aqueous solution of strontium chloride with the concentration of 1.6 mol/L; sodium hydroxide is dissolved in deionized water to prepare a sodium hydroxide aqueous solution with the concentration of 1.6 mol/L.
(2) Under the condition of room temperature and under the condition of continuously stirring and controlling the pH value to be 10 by using an aqueous sodium hydroxide solution, adding an aqueous solution of strontium chloride into the aqueous solution of eucommia ulmoides polysaccharide according to the mass molar ratio of 100g to 1mol of the eucommia ulmoides polysaccharide to the strontium element, and after the addition of the aqueous solution of strontium chloride is finished, continuously stirring and reacting at the pH value of 10 for 3 hours at room temperature to obtain a reaction solution.
(3) Adding an organic precipitator into the reaction solution, standing for 26 hours, filtering, and collecting precipitate. Wherein the organic precipitator is absolute ethyl alcohol, and the volume ratio of the organic precipitator to the reaction liquid is 4: 1.
(4) Mixing the precipitate with 80% ethanol by volume, stirring for 30 min, and filtering to obtain filter residue; mixing the filter residue with ethanol with the volume percentage concentration of 60%, stirring for 30 minutes, filtering, mixing the filter residue with ethanol with the volume percentage concentration of 40%, stirring for 30 minutes, filtering, collecting solid, and drying the solid at 38 ℃ for 12 hours to obtain the eucommia polysaccharide strontium complex, wherein the volume mass ratio of ethanol with each concentration to the precipitate in the step (4) is 4: 1.
Example 5
The preparation process of the eucommia ulmoides polysaccharide strontium complex of the embodiment is as follows:
(1) dissolving eucommia ulmoides polysaccharide in deionized water to prepare an aqueous solution of the eucommia ulmoides polysaccharide with the concentration of 1.2 g/mL; dissolving strontium chloride in deionized water to prepare an aqueous solution of strontium chloride with the concentration of 1.7 mol/L; sodium hydroxide is dissolved in deionized water to prepare a sodium hydroxide aqueous solution with the concentration of 1.6 mol/L.
(2) Under the condition of room temperature and under the condition of continuously stirring and controlling the pH value to be 9 by using an aqueous sodium hydroxide solution, adding an aqueous solution of strontium chloride into the aqueous solution of eucommia ulmoides polysaccharide according to the mass molar ratio of 200g to 1mol of the eucommia ulmoides polysaccharide to the strontium element, and after the addition of the aqueous solution of strontium chloride is finished, continuously stirring and reacting at the pH value of 9 for 2 hours at room temperature to obtain a reaction solution.
(3) Adding an organic precipitator into the reaction solution, standing for 28 hours, filtering, and collecting precipitate. Wherein the organic precipitator is absolute ethyl alcohol, and the volume ratio of the organic precipitator to the reaction liquid is 5: 1.
(4) Mixing the precipitate with 80% ethanol by volume, stirring for 30 min, and filtering to obtain filter residue; mixing the filter residue with ethanol with the volume percentage concentration of 60%, stirring for 30 minutes, filtering, mixing the filter residue with ethanol with the volume percentage concentration of 40%, stirring for 30 minutes, filtering, collecting solid, and drying the solid at 38 ℃ for 12 hours to obtain the eucommia polysaccharide strontium complex, wherein the volume mass ratio of ethanol with each concentration to the precipitate in the step (4) is 3: 1.
Example 6
The preparation process of the eucommia ulmoides polysaccharide complex of this example is similar to that of the eucommia ulmoides polysaccharide complex of example 1, except that the step (4) is not performed in this example.
Example 7
The preparation process of the eucommia ulmoides polysaccharide complex of this example is similar to that of the eucommia ulmoides polysaccharide complex of example 1, except that the organic precipitant is anhydrous acetone in step (3).
Example 8
The procedure for the preparation of the eucommia ulmoides polysaccharide complex of this example was similar to that of example 1, except that strontium chloride was replaced with strontium nitrate in this example.
Example 9
The preparation process of the eucommia ulmoides polysaccharide complex of this example is similar to that of the eucommia ulmoides polysaccharide complex of example 2, except that the mass molar ratio of the eucommia ulmoides polysaccharide to the strontium element in step (2) of this example is 55g:1 mol.
Example 10
The preparation process of the eucommia ulmoides polysaccharide complex of this example is similar to that of the eucommia ulmoides polysaccharide complex of example 3, except that the mass molar ratio of the eucommia ulmoides polysaccharide to the strontium element in step (2) of this example is 255g:1 mol.
Example 11
The preparation process of the eucommia ulmoides polysaccharide complex of this example is similar to that of the eucommia ulmoides polysaccharide complex of example 1, except that potassium hydroxide is used instead of sodium hydroxide in this example.
Comparative example 1
The preparation process of the eucommia ulmoides polysaccharide strontium complex of comparative example 1 was similar to that of example 2, except that the pH was controlled to 8.5 in step (2) of comparative example 1.
Comparative example 2
The preparation process of the eucommia ulmoides polysaccharide strontium complex of comparative example 2 is similar to that of example 3, except that the pH is controlled to 11.5 in step (2) of comparative example 1.
Comparative example 3
Comparative example 3 is eucommia ulmoides polysaccharide.
(1) XRD test:
fig. 2 is an XRD pattern of the product of example 1 and the eucommia ulmoides polysaccharide of comparative example 3, which can be seen from the figure. As can be seen from fig. 2, the XRD result of the eucommia ulmoides polysaccharide of example 1 showed different peak positions compared to that of the eucommia ulmoides polysaccharide of comparative example 3, indicating that the crystallinity of the eucommia ulmoides polysaccharide was changed after the strontium was doped. XRD characterization proves that strontium is successfully introduced into the eucommia ulmoides polysaccharide and forms a new compound, namely a eucommia ulmoides polysaccharide strontium complex.
The products obtained in examples 2 to 11 have XRD patterns similar to those of the product in example 1, and the patterns are not provided repeatedly, namely, the eucommia ulmoides polysaccharide strontium complex is also obtained in examples 2 to 11. The products obtained in comparative examples 1 and 2 were similar to XRD of the eucommia ulmoides polysaccharide of comparative example 3, and the products of comparative examples 1 and 3 were also still eucommia ulmoides polysaccharide.
(2) Infrared spectrum test:
FIG. 3 is an infrared spectrum of the product obtained in example 1 and the eucommia ulmoides polysaccharide of comparative example 3. from FIG. 3, it can be seen that the basic skeleton of the strontium complex of eucommia ulmoides polysaccharide obtained in example 1 is not substantially changed with respect to the eucommia ulmoides polysaccharide of comparative example 3.
The products obtained in examples 2-11 have infrared spectrograms similar to those of the product in example 1, and the infrared spectrograms of the products obtained in comparative example 1 and comparative example 2 are similar to those of the eucommia ulmoides polysaccharide in comparative example 3, and are not described again.
(3) And (3) testing a scanning electron microscope:
scanning electron microscopy was used to scan the product of example 1 and the eucommia ulmoides polysaccharide of comparative example 3, fig. 4 and 5 are scanning electron microscopy images of the strontium complex of eucommia ulmoides polysaccharide of example 1 and the eucommia ulmoides polysaccharide of comparative example 3, respectively, and it can be seen from fig. 4 and 5 that the strontium complex of eucommia ulmoides polysaccharide of example 1 has a smaller particle size and is more uniformly distributed than the eucommia ulmoides polysaccharide of comparative example 3, probably because the incorporation of strontium facilitates optimization of the disordered structure of the eucommia ulmoides polysaccharide.
(4) And (3) purity testing: the mass percent of strontium in the eucommia polysaccharide strontium complexes obtained in examples 1-11 was tested by EDS (energy Dispersive spectrometer) method and is denoted as W1; and calculating the mass percentage content W2 of the strontium element in the reactant, wherein W2 is the mass of the strontium element in the water-soluble strontium salt 100%/(the mass of the water-soluble strontium salt + the mass of the eucommia polysaccharide), and the purity of the eucommia polysaccharide strontium complex is W1 is 100%/W2. The purity of the eucommia ulmoides polysaccharide strontium complexes of examples 1 to 11 is shown in table 1:
TABLE 1
| Purity (%) | |
| Example 1 | 96.2％ |
| Example 2 | 95.5％ |
| Example 3 | 94％ |
| Example 4 | 97.6％ |
| Example 5 | 95.3％ |
| Example 6 | 83.9％ |
| Example 7 | 96％ |
| Example 8 | 95％ |
| Example 9 | 92.3％ |
| Example 10 | 92％ |
| Example 11 | 96％ |
As can be seen from table 1, the purity of the eucommia ulmoides polysaccharide strontium complex prepared in example 1 is 96.2%, while the purity of the eucommia ulmoides polysaccharide complex prepared in example 6 is only 83.9%, which indicates that the eucommia ulmoides polysaccharide strontium complex obtained after ethanol purification has higher purity. The purity of the eucommia ulmoides polysaccharide strontium complex compound of example 11 was 96%, which is equivalent to that of example 1, and this shows that sodium hydroxide and potassium hydroxide as pH regulators do not greatly affect the purity of the eucommia ulmoides polysaccharide strontium complex compound. The purity of the eucommia ulmoides polysaccharide strontium complex prepared in example 2 is 95.5%, the purity of the eucommia ulmoides polysaccharide strontium complex prepared in example 9 is only 92.3%, the purity of the eucommia ulmoides polysaccharide strontium complex prepared in example 3 is 94%, and the purity of the eucommia ulmoides polysaccharide strontium complex prepared in example 10 is only 92%, which indicates that the mass molar ratio of the eucommia ulmoides polysaccharide to the strontium element is too large or too small, which can adversely affect the purity of the eucommia ulmoides polysaccharide strontium complex.
(5) Macrophage experiments:
the eucommia ulmoides polysaccharide strontium complexes of examples 1 to 11, the product of comparative example 1, the product of comparative example 2, and the eucommia ulmoides polysaccharide of comparative example 3 were used as samples, and DMEM medium containing FBS at a mass percentage concentration of 5% was used to prepare sample solutions at concentrations of 10 μ g/mL, 50 μ g/mL, 100 μ g/mL, and 500 μ g/mL, respectively, and DMEM medium containing FBS at a mass percentage concentration of 5% without adding the sample was used as a control solution.
Taking mouse macrophage RAW264.7 (because macrophage RAW264.7 plays an important role in regulating the immune environment of bones and the differentiation and proliferation of the macrophage RAW264.7 play a regulating role in bone cells, the macrophage RAW264.7 is realized by adopting the cells), scraping the cells by using a cell scraper, and blowing and beating the cells to prepare single cell suspension. After counting on counting plates, the cells were diluted with DMEM medium containing FBS (fetal bovine serum) at a concentration of 5% by volume and seeded into 96-well plates, approximately 5000 cells per well of which were added per well in 100 μ L of cell suspension. Then, the 96-well plate was placed in a carbon dioxide incubator and incubated for 24 hours, and then the culture medium in the 96-well plate was aspirated, and then 200. mu.L of the above sample solution was added to the wells of the 96-well plate as an experimental group, and 200. mu.L of a control solution was added as a control experiment, and four wells were arranged in parallel for each concentration of sample solution, for example, the eucommia ulmoides polysaccharide strontium complex of example 1 was formulated into four sample solutions having concentrations of 10. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL, and 500. mu.g/mL, four wells for each concentration of sample solution, and 200. mu.L of sample solution in each well.
Detecting the cell proliferation rate of the macrophage on the third day by using a cell counting kit-8, wherein the cell counting kit-8 has the following detection principle: the component WST-8 is bioreduced by cellular dehydrogenases into a medium-soluble orange formazan product, and the amount of formazan produced is directly proportional to the number of viable cells. Measuring the absorbance of each hole by using a microplate reader to obtain an absorbance value, and reflecting the cell proliferation rate by the absorbance value, wherein the absorbance value of the macrophage of a control experiment on the third day is shown in table 2, and the absorbance value of the macrophage of a control experiment group on the third day is shown in table 3:
TABLE 2
| The third day | |
| Control experiment | 0.30 |
TABLE 3
As can be seen from tables 2 to 3, the eucommia polysaccharide strontium complex can obviously promote the proliferation of macrophages on the third day, and the promotion effect is most obvious when the concentration is 50ug/ml to 100ug/ml compared with the eucommia polysaccharide strontium complex. However, at high concentrations, the eucommia polysaccharide strontium complex has an inhibitory effect on cell proliferation. Among them, the low absorbance values of examples 7 and 8 are because anhydrous acetone and strontium nitrate have toxicity, resulting in low absorbance. The low absorbance value of example 6 was because the product of example 6 was not subjected to step (4), resulting in a product containing strontium chloride, which has a certain inhibitory effect on cell proliferation.
In fig. 6, it can be seen from fig. 6 that the cell growth effect of macrophages cultured in the cell culture medium containing the solution of strontium eucommia ulmoides polysaccharide of example 1 at a concentration of 10 to 100 μ g/mL was significant and significantly different from the control group, and thus the prepared strontium eucommia ulmoides polysaccharide complex has a promoting effect on cell growth.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (11)
1. The preparation method of the eucommia polysaccharide strontium complex is characterized by comprising the following steps:
reacting eucommia ulmoides polysaccharide and water-soluble strontium salt in water under the condition that the pH value is controlled to be 9-11 to obtain a reaction solution; and
adding an organic precipitator into the reaction solution, standing for reaction, performing solid-liquid separation, collecting precipitate, and cleaning the precipitate with ethanol to obtain an eucommia polysaccharide complex; the mass molar ratio of the eucommia ulmoides polysaccharide to the strontium element in the water-soluble strontium salt is 60g:1 mol-250 g:1mol, the water-soluble strontium salt is strontium chloride, and the organic precipitator is anhydrous acetone or anhydrous ethanol.
2. The method for preparing eucommia ulmoides polysaccharide strontium complex according to claim 1, wherein the step of reacting eucommia ulmoides polysaccharide with water-soluble strontium salt in water under the condition of controlling pH to be 9-11 comprises the following steps:
respectively dissolving the eucommia ulmoides polysaccharide and the water-soluble strontium salt in water to obtain an aqueous solution of the eucommia ulmoides polysaccharide and an aqueous solution of the water-soluble strontium salt; adding the water-soluble strontium salt water solution into the eucommia ulmoides polysaccharide water solution under the conditions of continuously stirring and controlling the pH value to be 9-11, and then continuously stirring and reacting for more than 1 hour under the condition of controlling the pH value to be 9-11.
3. The method for preparing the eucommia ulmoides polysaccharide strontium complex according to claim 2, wherein the concentration of the eucommia ulmoides polysaccharide aqueous solution is 0.5 g/mL-1.5 g/mL; and/or the concentration of strontium element in the water solution of the water-soluble strontium salt is 1.5-2 mol/L; and/or the volume ratio of the organic precipitator to the reaction liquid is 2: 1-5: 1.
4. The method for producing a eucommia ulmoides polysaccharide strontium complex according to claim 1, wherein the time for the standing reaction is 24 hours or more.
5. The method for preparing eucommia ulmoides polysaccharide strontium complex according to claim 1, wherein sodium hydroxide is used as a pH regulator in the step of reacting eucommia ulmoides polysaccharide with a water-soluble strontium salt in water under the condition of controlling pH to 9-11.
6. The method for preparing eucommia ulmoides polysaccharide strontium complex according to claim 1, wherein the step of reacting eucommia ulmoides polysaccharide with water-soluble strontium salt in water is performed at 10-40 ℃.
7. The method for preparing eucommia ulmoides polysaccharide strontium complex according to claim 1, wherein the step of washing the precipitate with ethanol comprises: and sequentially washing the precipitate by using ethanol with different concentrations, and drying the precipitate.
8. The method for preparing eucommia ulmoides polysaccharide strontium complex according to claim 7, wherein the step of sequentially washing the precipitate with ethanol of different concentrations comprises: ethanol with a volume percentage of 80%, ethanol with a volume percentage of 60% and ethanol with a volume percentage of 40% are mixed with the sediment in turn, and are mixed for at least 30 minutes with each stirring.
9. The method for preparing eucommia ulmoides polysaccharide strontium complex according to claim 7, wherein the drying temperature in the step of drying the precipitate is 37 to 45 ℃.
10. The eucommia ulmoides polysaccharide strontium complex prepared by the method for preparing eucommia ulmoides polysaccharide strontium complex according to any one of claims 1 to 9.
11. Use of the eucommia ulmoides polysaccharide strontium complex of claim 10 in the preparation of a bone repair material.
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