CN111944074A

CN111944074A - Laminarin strontium complex and its preparation method and application

Info

Publication number: CN111944074A
Application number: CN202010638844.4A
Authority: CN
Inventors: 唐斌; 马粉波; 张元俊; 葛永梅; 李慧丽
Original assignee: Southwest University of Science and Technology
Current assignee: Southwest University of Science and Technology; Southern University of Science and Technology
Priority date: 2020-07-06
Filing date: 2020-07-06
Publication date: 2020-11-17

Abstract

The invention relates to a laminarin strontium complex and a preparation method and application thereof. The preparation method of the laminarin strontium complex comprises the following steps: reacting laminarin with water-soluble strontium salt under the condition that the pH value is 9-11 to prepare reaction liquid; adding a precipitator into the reaction solution, standing for reaction, and performing solid-liquid separation to prepare the laminarin strontium complex. Experiments prove that the laminarin strontium complex with better bone repair effect can be prepared by adopting the preparation method of the laminarin strontium complex, and has better anti-inflammatory effect compared with laminarin.

Description

Laminarin strontium complex and its preparation method and application

Technical Field

The invention relates to the field of biomedicine, in particular to a laminarin strontium complex and a preparation method and application thereof.

Background

Bone regeneration and repair has become one of the major clinical challenges worldwide, involving multiple processes, including inflammatory responses, angiogenesis, and bone regeneration. Strontium is a trace element which has a beneficial effect on cartilage and bone and it promotes bone regeneration. However, too much free strontium ions are toxic in vivo, making strontium limited for use in the field of bone regeneration and repair.

Disclosure of Invention

Accordingly, there is a need for a method for preparing a strontium laminarin complex that is non-toxic and can promote bone regeneration.

In addition, a laminarin strontium complex and applications thereof are also provided.

A method for preparing laminarin strontium complex comprises the following steps:

reacting laminarin with water-soluble strontium salt under the condition that the pH value is 9-11 to prepare reaction liquid; and

adding a precipitator into the reaction solution, standing for reaction, and performing solid-liquid separation to prepare the laminarin strontium complex.

In one embodiment, the molar mass ratio of strontium element in laminarin and the water-soluble strontium salt is 60g:1mol to 250g:1 mol.

In one embodiment, in the step of reacting laminarin with the water-soluble strontium salt in the aqueous solution with the pH of 9-11, the reaction time is at least 1 hour, and preferably, the reaction time is 1 hour to 3 hours.

In one embodiment, the step of reacting laminarin with a water-soluble strontium salt in an aqueous solution having a pH of 9 to 11 comprises:

respectively dissolving the laminarin and the water-soluble strontium salt in water to prepare laminarin solution and strontium salt water solution;

adjusting the pH value of the laminarin solution to 9-11, adding the strontium salt aqueous solution into the laminarin solution, and continuously stirring and reacting for more than 1 hour under the condition that the pH value is 9-11.

In one embodiment, the laminarin solution has a concentration of 0.5g/mL to 5 g/mL; and/or the concentration of strontium element in the strontium salt aqueous solution is 1.5-2.0 mol/L; and/or in the step of adjusting the pH of the laminarin solution to 9-11, the used reagent is sodium hydroxide and/or potassium hydroxide.

In one embodiment, the precipitating agent is selected from at least one of ethanol and acetone.

In one embodiment, the volume ratio of the precipitant to the reaction liquid is 2: 1-5: 1.

In one embodiment, the standing reaction time is at least 12 hours, and preferably, the standing reaction time is 24 to 30 hours.

In one embodiment, after the step of solid-liquid separation, a step of purifying the precipitate after solid-liquid separation with an aqueous ethanol solution is further included.

In one embodiment, the step of purifying the precipitate after solid-liquid separation with an aqueous ethanol solution comprises: and sequentially washing the precipitate by using ethanol water solutions with different concentrations, and drying the precipitate.

In one embodiment, in the drying step, the drying temperature is-10 ℃ to 0 ℃; and/or the step of sequentially washing the precipitate with ethanol aqueous solutions with different concentrations comprises the following steps: and sequentially mixing and stirring 75-85% by volume of ethanol aqueous solution, 55-65% by volume of ethanol aqueous solution and 35-45% by volume of ethanol aqueous solution with the precipitate for at least 30 minutes.

A laminarin strontium complex is prepared by the above preparation method.

The application of the laminarin strontium complex in preparing bone repair materials or anti-inflammatory materials.

Experiments prove that: the preparation method of the laminarin strontium complex can avoid toxicity caused by excessive free strontium ions through the complexation reaction of strontium and laminarin macromolecules, and the prepared laminarin strontium complex can promote bone repair and regeneration through promoting angiogenesis and osteoblast proliferation, and can be applied to the field of bone repair.

Drawings

FIG. 1 is a process flow diagram of one embodiment of a method for preparing a strontium laminarin complex;

FIG. 2 is an XRD pattern of the product of example 1, comparative example 3;

FIG. 3 is an infrared spectrum of the product obtained in example 1 and laminarin of comparative example 3;

FIG. 4 is a graph showing the results of the proliferation test of human umbilical vein endothelial cells of example 1, comparative example 3 and control group;

FIG. 5 is a graph showing the results of experiments on proliferation of preosteoblasts of mice in example 1, comparative example 3 and control group;

FIG. 6 is an alkaline phosphatase staining pattern of preosteoblasts of mice of example 1, comparative example 3, induction group and control group;

FIG. 7 is a graph showing the results of gene expression analysis of vascular endothelial growth factor VEGF by human umbilical vein endothelial cells of example 1, comparative example 3 and control group;

FIG. 8 is a graph showing the results of gene expression analysis of the inflammatory factor IL-6 in preosteoblasts of mice in example 1, comparative example 3 and control groups.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description taken in conjunction with the accompanying drawings. The detailed description sets forth the preferred embodiments of the invention. 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.

Strontium is a trace element which has a beneficial effect on cartilage and bone and it promotes bone regeneration. Laminarin has a protective effect on blood vessels. The laminarin strontium complex prepared by complexing laminarin and strontium is found to have the advantages of good bone regeneration and repair promoting effect, no toxicity and good anti-inflammatory effect compared with laminarin.

Specifically, referring to fig. 1, the method for preparing a laminarin strontium complex according to an embodiment of the present invention includes the steps of:

step S110: reacting laminarin with water-soluble strontium salt under the condition that the pH value is 9-11 to prepare reaction liquid.

Wherein the laminarin refers to macromolecular polysaccharide substance in thallus laminariae. The laminarin can reduce urine protein, purify uric acid in blood, and improve blood lipid concentration, and has good prevention and treatment effects on gout, hyperlipemia, nephropathy, and early and middle stage renal failure. Laminarin, unlike general polysaccharides, contains sulfate groups at the molecular chain ends, and sulfate groups are linked to polysaccharides via ester bonds. In this embodiment, laminarin is complexed with strontium ions, so that the laminarin strontium complex has the effect of promoting angiogenesis and osteoblast proliferation, thereby promoting bone regeneration and repair.

Specifically, laminarin is reacted with a water soluble strontium salt in water.

By controlling the pH to 9 to 11, the reaction proceeds smoothly, and a laminarin strontium complex is produced. In one embodiment, the pH is 9, 9.5, 10, 10.5, or 11. Experiments have shown that too high or too low a pH, strontium cannot be incorporated into laminarin and therefore cannot form laminarin strontium complex.

Specifically, the mass molar ratio of the laminarin to the strontium element in the water-soluble strontium salt is 60g:1 mol-250 g:1 mol. In one embodiment, the molar mass ratio of laminarin to strontium element in the water-soluble strontium salt is 60g:1mol, 100g:1mol, 150g:1mol, 200g:1mol, or 250g:1 mol. Preferably, the mass molar ratio of the laminarin to the strontium element in the water-soluble strontium salt is 80g:1mol to 160g:1 mol. Too much strontium leaves a large amount of strontium ions in the complex, and the residual strontium ions are toxic in vivo, resulting in harmful effects of the material in vivo. If the amount of strontium element added is too small, the laminarin is excessive, so that the complexation reaction is insufficient or part of laminarin is not complexed with strontium.

In particular, the water-soluble strontium salt may be a strontium salt commonly used in the art that is capable of being dissolved in water, for example, the water-soluble strontium salt is strontium chloride or strontium nitrate. Preferably, the water soluble strontium salt is strontium chloride. Strontium chloride can make the prepared laminarin strontium complex have less by-products, higher purity and almost no toxicity.

Specifically, in step S110, sodium hydroxide and/or potassium hydroxide is used as a pH adjuster. It is understood that in other embodiments, the pH adjusting agent is not limited to sodium hydroxide and/or potassium hydroxide, but may be other alkaline agents commonly used in the art, such as ammonia, etc. However, in this embodiment, since laminarin itself contains sodium and potassium, the use of sodium hydroxide or potassium hydroxide as a pH adjuster does not produce excessive impurities, and ammonia may introduce new impurities into the system.

In one embodiment, the pH regulator is sodium hydroxide aqueous solution with the concentration of 1.5-2.0 mol/L or potassium hydroxide aqueous solution with the concentration of 1.5-2.0 mol/L. The sodium hydroxide and the potassium hydroxide are strong bases, local oxidation of laminarin can be caused by increasing concentration, the volume of laminarin is too large due to too low concentration and pH value is controlled to be 9-11, so that the concentrations of laminarin and water-soluble strontium salt in the reaction liquid are reduced too much, and the reaction is not easy to occur.

Specifically, the step of reacting laminarin with a water-soluble strontium salt in water is performed at room temperature. In the present embodiment, the room temperature is 10 to 40 ℃. The step of reacting laminarin with water-soluble strontium salt in water is carried out under stirring for at least 1h to allow the laminarin and the water-soluble strontium salt to react in a fully complex manner. Specifically, the reaction time is 1-3 h. In one embodiment, the reaction time is 1h, 2h, or 3 h.

In one embodiment, step S110 includes:

respectively dissolving laminarin and water-soluble strontium salt in water to prepare laminarin solution and strontium salt water solution;

adjusting the pH value of the laminarin solution to 9-11, adding a strontium salt aqueous solution into the laminarin solution, and continuously stirring and reacting for more than 1 hour under the condition that the pH value is 9-11.

Preferably, the water used in the step of separately dissolving laminarin and the water-soluble strontium salt in water is deionized water to reduce impurities as much as possible and improve the purity of the product.

In one embodiment, the laminarin solution has a concentration of 0.5g/mL to 1.5 g/mL. In one embodiment, the laminarin solution has a concentration of 0.5g/mL, 0.8g/mL, 1.0g/mL, 1.2g/mL, or 1.5 g/mL. The laminarin solution has a concentration of up to 1.5g/ml, and too low a concentration of the solution results in too low a laminarin content, resulting in a difficult complexation reaction.

The concentration of strontium element in the strontium salt water solution is 1.5 mol/L-2.0 mol/L. In one embodiment, the concentration of strontium element in the strontium salt aqueous solution is 1.5mol/L, 1.6mol/L, 1.7mol/L, 1.8mol/L or 2.0 mol/L. If the concentration of strontium ions is too high, the hydrolysis degree of strontium salt is reduced, hydrogen ions in the solution are increased, and the pH is affected, so that the generated complex is unstable. If the concentration of strontium ions is too low, the formation of coordinate bonds is less, and the reaction is incomplete.

It is understood that in other embodiments, step S110 is not limited to the above-described step, and the laminarin solution may be added to the strontium salt aqueous solution; or directly adding laminarin and water-soluble strontium salt into water to carry out stirring reaction.

Step S120: adding precipitant into the reaction solution, standing for reaction, and performing solid-liquid separation to obtain laminarin strontium complex.

Adding precipitant, and standing for reaction to precipitate the strontium laminarin complex in the reaction solution. Specifically, the precipitant is an organic precipitant. The precipitating agent may be an organic precipitating agent commonly used in the art. Specifically, the precipitant is at least one selected from acetone and ethanol. Further, the precipitant is absolute ethyl alcohol. Compared with anhydrous acetone, the toxicity of the anhydrous ethanol is low, so that the prepared laminarin strontium complex has low toxicity, and the anhydrous ethanol is low in price, and is beneficial to reducing the production cost.

Specifically, the time for the standing reaction is 12 hours or more so that the laminarin strontium complex in the reaction solution can be precipitated as much as possible. Further, the standing reaction time is 24-30 h. In one embodiment, the standing reaction time is 24h, 25h, 26h, 28h or 30 h.

Specifically, the volume ratio of the precipitant to the reaction solution is 2: 1-5: 1, so that the laminarin strontium complex is precipitated sufficiently on the premise of not wasting more precipitant. In one embodiment, the volume ratio of the precipitant to the reaction solution is 2:1, 3:1, 4:1, or 5: 1.

Further, after step S120, a purification process is also included. Specifically, the precipitate after solid-liquid separation is washed with an ethanol aqueous solution to purify the laminarin strontium complex. Washing with an aqueous ethanol solution to wash out the strontium chloride in the precipitate improves the purity of the laminarin strontium complex without re-dissolving the laminarin strontium complex.

Specifically, the step of washing the precipitate with an aqueous ethanol solution comprises: washing the precipitate with ethanol water solution of different concentrations, and drying the precipitate. In one embodiment, the drying temperature in the step of drying the precipitate is from-10 ℃ to 0 ℃. The drying time was 12 h. In one embodiment, the drying temperature is-10 ℃, -5 ℃, or 0 ℃. Experiments prove that the freeze drying mode does not damage the structure of the prepared laminarin strontium complex, thereby maintaining the performance of the laminarin strontium complex. It is understood that in other embodiments, the drying step may be performed under heating.

In one embodiment, the step of sequentially washing the precipitate with ethanol aqueous solutions of different concentrations comprises: and mixing 75-85% by volume of ethanol aqueous solution, 55-65% by volume of ethanol aqueous solution and 35-45% by volume of ethanol aqueous solution with the precipitate in sequence, wherein the mixing and stirring time is at least 30 minutes respectively. Specifically, the volume ratio of the ethanol aqueous solution with each concentration to the reaction solution obtained in step S110 is 2.5: 1-5: 1, so as to achieve the purpose of effective washing and purification. It is understood that in other embodiments, the ethanol aqueous solution with different concentrations is not limited to the concentrations of 75-85%, 55-65% and 35-45% by volume percentage, and may have other concentrations.

Experiments prove that: the laminarin strontium complex prepared by the preparation method of the laminarin strontium complex can avoid the toxicity of excessive free strontium ions when used for bone repair through the complexation reaction of strontium and laminarin macromolecules. Meanwhile, the prepared laminarin strontium complex also has the characteristics of the laminarin strontium complex and can effectively release strontium in vivo, play the role of relieving inflammation of strontium and better assist bone repair by promoting angiogenesis and bone cell proliferation.

In addition, laminarin is adopted to be complexed with strontium, and laminarin has better protection effect on blood vessels than other polysaccharide macromolecules are adopted to be complexed with strontium.

The present invention also provides an embodiment of the strontium laminarin complex, which is prepared by the method of preparing the strontium laminarin complex of the above embodiment. The laminarin strontium complex has good bone repairing effect and anti-inflammatory effect. In addition, the laminarin strontium complex can avoid toxicity of excessive free strontium ions when applied in vivo.

In addition, the present invention also provides an application of the laminarin strontium complex of an embodiment in the preparation of a bone repair material or an anti-inflammatory material. The bone repair material prepared from the laminarin strontium complex has good bone repair effect and blood vessel regeneration effect, and can effectively inhibit inflammation.

The following are examples of the following (the following examples, unless otherwise specified, contain no other components not specifically indicated except for unavoidable impurities):

example 1

The preparation process of the laminarin strontium complex of this example is detailed below:

(1) dissolving laminarin in deionized water to prepare laminarin solution with the concentration of 1 g/mL; 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, adding a strontium chloride aqueous solution into the laminarin aqueous solution which is continuously stirred and has the pH value adjusted to 10 by using a sodium hydroxide aqueous solution according to the mass molar ratio of 150g to 1mol of laminarin to strontium element, and continuously stirring and reacting for 1 hour at the room temperature and the pH value of 10 after the strontium chloride aqueous solution is added 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 water solution, stirring for 30 min, and filtering to obtain filter residue; mixing the filter residue with an ethanol water solution with the volume percentage concentration of 60%, stirring for 30 minutes, filtering, mixing the filter residue with an ethanol water solution with the volume percentage concentration of 40%, stirring for 30 minutes, filtering again, collecting solids, and drying the solids at the temperature of minus 5 ℃ for 12 hours to obtain the laminarin strontium complex, wherein the volume mass ratio of the ethanol water solution with each concentration to the precipitate in the step (4) is 3.5: 1.

Example 2

(1) dissolving laminarin in deionized water to prepare laminarin solution 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, adding a strontium chloride aqueous solution into the laminarin aqueous solution which is continuously stirred and has the pH value adjusted to 9 by using a sodium hydroxide aqueous solution according to the mass molar ratio of 60g to 1mol of laminarin to strontium element, and continuously stirring and reacting for 2 hours at the room temperature and the pH value of 9 after the strontium chloride aqueous solution is added 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 water solution, stirring for 40 min, and filtering to obtain filter residue; mixing the filter residue with an ethanol water solution with the volume percentage concentration of 60%, stirring for 30 minutes, filtering, mixing the filter residue with an ethanol water solution with the volume percentage concentration of 40%, stirring for 50 minutes, filtering again, collecting solids, and drying the solids at 0 ℃ for 12 hours to obtain the laminarin strontium complex, wherein the volume mass ratio of the ethanol water solution with each concentration to the precipitate in the step (4) is 2.5: 1.

Example 3

(1) dissolving laminarin in deionized water to prepare laminarin solution 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) Adding a strontium chloride aqueous solution into the laminarin solution which is continuously stirred and has the pH value adjusted to 11 by using a sodium hydroxide aqueous solution according to the mass molar ratio of 250g to 1mol of laminarin to strontium element at room temperature, and continuously stirring and reacting for 3 hours at the room temperature and the pH value of 11 after the strontium chloride aqueous solution is added 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 water solution, stirring for 35 min, and filtering to obtain filter residue; mixing the filter residue with an ethanol water solution with the volume percentage concentration of 60%, stirring for 50 minutes, filtering, mixing the filter residue with an ethanol water solution with the volume percentage concentration of 40%, stirring for 30 minutes, filtering again, collecting solids, and drying the solids at-10 ℃ for 12 hours to obtain the laminarin strontium complex, wherein the volume mass ratio of the ethanol water solution with each concentration in the step (4) to the precipitate is 5: 1.

Example 4

(1) dissolving laminarin in deionized water to prepare laminarin solution 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, adding a strontium chloride aqueous solution into the laminarin aqueous solution which is continuously stirred and has the pH value adjusted to 10 by using a sodium hydroxide aqueous solution according to the mass molar ratio of laminarin to strontium element of 100g:1mol, and continuously stirring and reacting for 3 hours at the room temperature and the pH value of 10 after the strontium chloride aqueous solution is added 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 water solution, stirring for 30 min, and filtering to obtain filter residue; mixing the filter residue with an ethanol water solution with the volume percentage concentration of 60%, stirring for 30 minutes, filtering, mixing the filter residue with an ethanol water solution with the volume percentage concentration of 40%, stirring for 30 minutes, filtering again, collecting solids, and drying the solids at 0 ℃ for 12 hours to obtain the laminarin strontium complex, wherein the volume mass ratio of the ethanol water solution with each concentration to the precipitate in the step (4) is 4: 1.

Example 5

(1) dissolving laminarin in deionized water to prepare laminarin solution 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, adding a strontium chloride aqueous solution into the laminarin aqueous solution which is continuously stirred and has the pH value adjusted to 9 by using a sodium hydroxide aqueous solution according to the mass molar ratio of laminarin to strontium element of 200g:1mol, and continuously stirring and reacting for 2 hours at the room temperature and the pH value of 9 after the strontium chloride aqueous solution is added, thus obtaining 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 water solution, stirring for 30 min, and filtering to obtain filter residue; mixing the filter residue with an ethanol water solution with the volume percentage concentration of 60%, stirring for 30 minutes, filtering, mixing the filter residue with an ethanol water solution with the volume percentage concentration of 40%, stirring for 30 minutes, filtering again, collecting solids, and drying the solids at the temperature of minus 5 ℃ for 12 hours to obtain the laminarin strontium complex, wherein the volume mass ratio of the ethanol water solution with each concentration to the precipitate in the step (4) is 3: 1.

Example 6

The procedure for the preparation of the strontium laminarin complex of this example was similar to that of example 1, except that: step (4) was not performed in this example.

Example 7

The procedure for the preparation of the strontium laminarin complex of this example was similar to that of example 1, except that: in step (3) of this example, the organic precipitant is anhydrous acetone.

Example 8

The procedure for the preparation of the strontium laminarin complex of this example was similar to that of example 1, except that: strontium chloride was replaced with strontium nitrate in step (1) and step (2) of this example.

Example 9

The procedure for the preparation of the strontium laminarin complex of this example was similar to that of example 2, except that: in this example, the molar mass ratio of laminarin to strontium element in step (2) was 55g:1 mol.

Example 10

The procedure for the preparation of the strontium laminarin complex of this example was similar to that of example 3, except that: in this example, the molar mass ratio of laminarin to strontium element in step (2) was 255g:1 mol.

Example 11

The procedure for the preparation of the strontium laminarin complex of this example was similar to that of example 1, except that: in step (1) and step (2) of this example, potassium hydroxide was used instead of sodium hydroxide.

Comparative example 1

The preparation process of the strontium laminarin complex of comparative example 1 was similar to the preparation process of the strontium laminarin complex of example 2, except that: the pH in step (2) of comparative example 1 was controlled to 8.5.

Comparative example 2

The procedure for preparing the strontium laminarin complex of comparative example 2 was similar to that of example 3, except that: the pH in step (2) of comparative example 2 was controlled to 11.5.

Comparative example 3

Comparative example 3 laminarin used in example 1 was used without complexing.

The following are test sections:

1. XRD test:

figure 2 is an XRD pattern of the products of example 1 and comparative example 3. As can be seen from fig. 2, the strontium complex laminarin of example 1 had almost no XRD diffraction peak compared to the XRD result of laminarin of comparative example 3, indicating that the crystallinity of laminarin was changed after the strontium was incorporated, unlike the XRD pattern of comparative example 3. XRD characterization confirmed the successful introduction of strontium into laminarin and the formation of a new complex-laminarin strontium complex.

Wherein, the products prepared in examples 2 to 11 have similar XRD patterns to those of the product of example 1, and the pattern is not repeated here, that is, the laminarin strontium complex is also obtained in examples 2 to 11. The XRD of the products obtained in comparative examples 1 and 2 were similar to that of comparative example 3, indicating that the products of comparative examples 1 and 2 were also still laminarin, without the synthesized laminarin strontium complex.

2. Infrared spectrum test:

FIG. 3 is an infrared spectrum of the product obtained in example 1 and laminarin of comparative example 3. from FIG. 3, it can be seen that the basic skeleton of the laminarin strontium complex obtained in example 1 is not substantially changed with respect to the laminarin of comparative example 3.

Wherein, the products obtained in examples 2-11 have similar infrared spectrogram as the product of example 1, and the infrared spectrogram of the products obtained in comparative example 1 and comparative example 2 is similar to that of laminarin in comparative example 3, and the description is omitted.

3. Proliferation experiment of human umbilical vein endothelial cells and mouse preosteoblasts:

the strontium laminarin complex of laminarin of example 1 and the laminarin of comparative example 3 were used as samples, and DMEM medium containing FBS (fetal bovine serum) at a concentration of 5% by volume was prepared as sample solutions at a concentration of 10 μ g/ml, respectively, and DMEM medium containing FBS at a concentration of 5% by volume without adding the sample was used as a control solution.

Human umbilical vein endothelial cells HUVECs are taken, digested by pancreatin and blown into a single cell suspension. After counting on counting plates, the cells were diluted with DMEM medium containing 5% FBS by volume and seeded into 96-well plates, approximately 5000 cells per well of which were added per well of 100 μ L single cell suspension. Then, the 96-well plate was placed in a carbon dioxide incubator and incubated for 24 hours, and then the medium in the 96-well plate was aspirated, and then 200. mu.L of the above sample solution was added to each well of the 96-well plate as an experimental group and 200. mu.L of a control solution as a control experiment, each sample solution being set in four wells in parallel, for example, a sample solution prepared from the strontium laminaran complex of example 1 being set in four wells, and the sample solution in each well being 200. mu.L.

The cell proliferation rate of the human umbilical vein endothelial cells on the first day and the fifth day is detected by adopting a cell counting kit-8, and the detection principle of the cell counting kit-8 is as follows: the component WST-8 is bioreduced by cellular dehydrogenases into an orange formazan product that is soluble in the culture medium and produces an amount of formazan product that is directly proportional to the number of viable cells. And measuring the absorbance of each hole by using a microplate reader to obtain an absorbance value, and reflecting the cell proliferation rate by using the absorbance value. The absorbance value of each concentration of sample solution is the average of the absorbance values measured in the four wells. And taking the mouse preosteoblasts, and detecting the cell proliferation rate of the mouse preosteoblasts on the first day and the fifth day by using the same method.

The bar graphs of the absorbance of human umbilical vein endothelial cells and pre-osteogenesis of mice of example 1, comparative example 3 and control are shown in fig. 4 and 5, respectively. Fig. 4 is a graph showing the results of the proliferation test of human umbilical vein endothelial cells in example 1, comparative example 3 and the control group, and fig. 5 is a graph showing the results of the proliferation test of mouse preosteoblasts in example 1, comparative example 3 and the control group. As can be seen from FIGS. 4 and 5, the stimulation of proliferation of human umbilical vein endothelial cells and mouse preosteoblasts was clearly observed on the fifth day with laminarin strontium complex.

In addition, 7 days after the culture, the mouse preosteoblasts MC3T3-E1 of example 1, comparative example 3 and control group were collected and tested for ALP activity, and the results shown in FIG. 6 were obtained. Briefly, after trypsinizing the collected cells, cell lysis buffer was added to lyse the cells on ice for 1 hour. The lysate was then transferred to centrifuge tubes and centrifuged at 4 ℃ (centrifugal force 12000g) for 5 minutes. The supernatant was collected and analyzed using alkaline phosphatase assay kit.

Alkaline phosphatase (ALP) is a marker for the osteogenic phenotype. As shown in FIG. 6, the higher level of ALP activity of example 1 compared to the control group and comparative example 3 indicates that strontium laminarin is more effective in accelerating the differentiation of MC3T3-E1 cells into osteoblasts, thereby facilitating bone regeneration.

In addition, it can be seen from the above experimental results that the strontium laminarin complex prepared in example 1 can promote the proliferation of human umbilical vein endothelial cells and mouse preosteoblasts, which also indicates that the strontium laminarin complex is non-toxic.

4. Analysis of Gene expression

mRNA of the human umbilical vein endothelial cells of example 1 and comparative example 3 was extracted, and gene expression analysis was performed on vascular endothelial growth factor VEGF thereof, to obtain the experimental result of FIG. 7. VEGF is a key factor in inducing angiogenesis to accelerate bone healing, and as shown in fig. 7, VEGF mRNA expression of the human umbilical vein endothelial cells of example 1 was significantly up-regulated compared to comparative example 3 and the control experimental group. This is mainly due to the release of Sr ions from laminarin strontium, which can stimulate VEGF expression. Therefore, the laminarin strontium complex should be beneficial for angiogenesis.

mRNA of mouse preosteoblasts of example 1 and comparative example 3 was extracted, and gene expression analysis of IL-6, an inflammatory factor, was performed, and the results shown in FIG. 8 were obtained. As can be seen from FIG. 8, the expression of the inflammatory factors of comparative example 3 and example 1 was significantly decreased compared to the blank control, while the expression of the inflammatory factors of example 1 was more decreased than that of comparative example 3, thereby indicating that the laminarin strontium complex had a better anti-inflammatory effect.

As can be seen from the above experimental results, the laminarin strontium complex prepared in example 1 can promote bone repair and regeneration by promoting angiogenesis and osteoblast proliferation, and can be applied to the field of bone repair.

It should be noted that the above experimental tests were performed using the strontium laminarin complex prepared in example 1, and the effects of the strontium laminarin complexes prepared in examples 2 to 11 were equivalent to those of example 1, and thus the description thereof is omitted.

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

1. A preparation method of laminarin strontium complex is characterized by comprising the following steps:

2. The method of claim 1, wherein the molar mass ratio of strontium element in laminarin to water-soluble strontium salt is 60g:1mol to 250g:1 mol.

3. The method of claim 1, wherein the step of reacting laminarin with a water-soluble strontium salt in an aqueous solution having a pH of 9 to 11 is performed for a reaction time of at least 1 hour.

4. The method of preparing the laminarin strontium complex according to any one of claims 1 to 3, wherein the step of reacting laminarin with a water-soluble strontium salt at a pH of 9 to 11 comprises:

5. The method of claim 4, wherein the concentration of the laminarin solution is 0.5g/mL to 1.5 g/mL; and/or the concentration of strontium element in the strontium salt aqueous solution is 1.5-2.0 mol/L; and/or in the step of adjusting the pH of the laminarin solution to 9-11, the used reagent is sodium hydroxide and/or potassium hydroxide.

6. The method of claim 1, wherein the precipitating agent is at least one selected from the group consisting of ethanol and acetone.

7. The method for producing a laminarin strontium complex according to claim 1, wherein the volume ratio of the precipitant to the reaction solution is 2:1 to 5: 1.

8. The process of claim 1, wherein the standing reaction time is at least 12 hours.

9. The method for producing a laminarin strontium complex according to any one of claims 1 to 3 and 5 to 8, further comprising a step of purifying the precipitate after the solid-liquid separation with an aqueous ethanol solution after the solid-liquid separation step.

10. The method of claim 9, wherein the step of purifying the precipitate after the solid-liquid separation using an aqueous solution of ethanol comprises: and sequentially washing the precipitate by using ethanol water solutions with different concentrations, and drying the precipitate.

11. The method of claim 10, wherein the drying step is carried out at a temperature of-10 ℃ to 0 ℃; and/or the step of sequentially washing the precipitate with ethanol aqueous solutions with different concentrations comprises the following steps: and sequentially mixing and stirring 75-85% by volume of ethanol aqueous solution, 55-65% by volume of ethanol aqueous solution and 35-45% by volume of ethanol aqueous solution with the precipitate for at least 30 minutes.

12. A strontium laminarin complex, characterized by being produced by the process for producing a strontium laminarin complex according to any one of claims 1 to 11.

13. Use of the strontium laminarin complex of claim 12 for the preparation of a bone repair material or for the preparation of an anti-inflammatory material.