CN114230688A - Simple and efficient heparin sodium recovery method for blood purification - Google Patents
Simple and efficient heparin sodium recovery method for blood purification Download PDFInfo
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- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
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Abstract
The invention provides a simple and efficient heparin sodium recovery method for blood purification, which comprises the following steps: s1, adjusting the pH value: recovering waste liquid generated by the coupling process with heparin sodium as ligand, and adjusting the pH value of the waste liquid to 6-7.5; s2, precipitation: adding ethanol into the waste liquid treated in the step S1 according to a preset volume ratio, centrifuging for a preset time, and collecting precipitates; s3, freeze-drying: dissolving the precipitate with water, and freeze-drying to obtain massive heparin sodium; s4, powder grinding: and (3) pulverizing the massive heparin sodium by using a pulverizer to obtain powdery heparin sodium. According to the invention, the pH value of the waste liquid is adjusted to be neutral, then heparin sodium is precipitated in ethanol with a certain proportion, the ethanol dosage is obviously reduced under the condition of ensuring that the heparin sodium is precipitated to the maximum extent, and finally, the powdery heparin sodium is obtained by freeze-drying and crushing, so that the method has important significance for improving the utilization rate of raw materials and reducing the preparation cost of the blood adsorbent.
Description
Technical Field
The invention relates to the technical field of heparin sodium purification and recovery, in particular to a simple and efficient heparin sodium recovery method for blood purification.
Background
Heparin sodium (heparin) is also called heparin, is a polymer formed by alternately connecting two polysaccharides, is a mucopolysaccharide containing sulfate groups, has the molecular weight of 1.5 ten thousand, has anticoagulation effect in vivo and in vitro, and has an anticoagulation mechanism that the anticoagulation mechanism is combined with anticoagulation enzyme II, can inhibit the effect between factors IX a, VIII and PF3 at low concentration, and can strengthen antithrombin III to inactivate serine protease, thereby preventing thrombin from forming; it also has effects in inhibiting self-catalysis of thrombin and inhibiting factor X. The preparation is mainly used for thromboembolic diseases, myocardial infarction, cardiovascular operations, cardiac catheter examination, extracorporeal circulation, hemodialysis and the like in clinic.
The heparin sodium is white or quasi-white powder, has no odor and odor, has hygroscopicity, is easily soluble in water, and is insoluble in organic solvents such as ethanol, acetone, dioxane, etc. Heparin sodium is a currently known biomolecule with the strongest electronegativity, is widely present in liver, lung and intestinal mucosa of mammals, and is combined with proteins to form a complex. The enzymolysis protein can separate heparin sodium, and the heparin sodium is mucopolysaccharide containing sulfuric acid, amino and aldonic acid. At pH8-9, it has negative charge, and can be ion exchanged with anion exchanger for crude separation, and polysaccharide solution can be purified by precipitating in high concentration ethanol.
Immunoadsorption therapy is a new technology that has developed over the last 20 years. It combines highly specific antigen, antibody or some substance (ligand) with specific physical and chemical affinity with adsorption material (carrier) to make adsorbent (column), and utilizes its specific adsorption property to selectively or relatively specifically remove endogenous pathogenic factor in patient's blood so as to attain the goal of relieving disease condition.
When heparin sodium is used as a ligand to prepare the adsorbent, the cost of the adsorbent is greatly increased due to the limitation of the preparation process of the adsorbent and the high cost of a ligand solution, and the ligand is easily inactivated due to the influence of various factors such as temperature, time and the like in the process of coupling reaction with a carrier, so that the adsorption efficiency of the adsorbent is influenced, and the utilization rate of the heparin sodium is reduced. However, additives such as catalysts and the like are usually required to be added in the process of the coupling reaction of heparin sodium and the adsorption carrier, so that the components of the reaction solution of the coupling reaction are complex, and the content of the heparin sodium is also obviously reduced, so that the heparin sodium in the coupling reaction waste liquid is rarely recovered by the prior art, the waste of the heparin sodium is caused, and the production cost is increased. In view of the above, there is a need to design an improved method for recovering heparin sodium for blood purification with simplicity and high efficiency to solve the above problems.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a simple and efficient method for recovering heparin sodium for blood purification.
In order to achieve the above object, the present invention provides a simple and efficient method for recovering heparin sodium for blood purification, comprising the following steps:
s1, adjusting the pH value: recovering waste liquid generated by the coupling process with heparin sodium as ligand, and adjusting the pH value of the waste liquid to 6-7.5;
s2, precipitation: adding ethanol into the waste liquid treated in the step S1 according to a preset volume ratio, centrifuging for a preset time, and collecting precipitates;
s3, freeze-drying: dissolving the precipitate with water, and freeze-drying to obtain massive heparin sodium;
s4, powder grinding: and (3) pulverizing the massive heparin sodium by using a pulverizer to obtain powdery heparin sodium.
As a further improvement of the invention, in step S1, the pH is adjusted by adding hydrochloric acid.
As a further improvement of the present invention, in step S1, the content of heparin sodium in the waste liquid is 50%.
As a further improvement of the invention, the coupling process is that aldehyde group or epoxy group on the carrier is coupled with amino group on the heparin sodium.
In a further improvement of the invention, in step S2, the volume ratio of the waste liquid to the ethanol is 1mL: 0.2-3 mL.
As a further improvement of the invention, the volume ratio of the waste liquid to the ethanol is 1mL: 0.5-1.5 mL.
As a further improvement of the invention, the volume ratio of the waste liquid to the ethanol is 1mL:0.9 mL.
As a further improvement of the present invention, in step S2, the purity of the ethanol is 95%.
In a further improvement of the present invention, in step S2, the centrifugation time is 5-30 min.
As a further improvement of the invention, the step S2 is carried out at 0-40 ℃.
The invention has the beneficial effects that:
1. the simple and efficient heparin sodium recovery method for blood purification provided by the invention has the advantages that the pH value of the waste liquid is adjusted to be neutral, then heparin sodium is precipitated in ethanol with a certain proportion, the ethanol dosage is obviously reduced under the condition of ensuring that the heparin sodium is precipitated to the maximum extent, and finally, the powdery heparin sodium is obtained by freeze-drying and crushing, so that the method has important significance for improving the utilization rate of raw materials and reducing the preparation cost of a blood adsorbent. The recovery rate of the invention can reach more than 85 percent, the purity of the invention can reach more than 99 percent, the influence of the physicochemical property of the recovered heparin sodium is small, the heparin sodium can still be used as a coupling agent to carry out coupling preparation of the adsorbent again, the reduction rate of the adsorption rate of the adsorbent is less than 3 percent compared with the original adsorbent, and the invention has obvious recycling value.
2. The method firstly adjusts the pH value to be near neutral, then adds a proper amount of ethanol, and simultaneously carries out centrifugation to precipitate and separate out the heparin sodium, and obviously reduces the ethanol dosage and shortens the precipitation time under the condition of ensuring the maximum separation of the heparin sodium, so the whole precipitation process is simple and easy to operate, the raw materials are cheap and easy to obtain, the usage amount is small, and the recovery cost is low.
3. The precipitated heparin sodium is dissolved in water, on one hand, water-insoluble impurities can be removed, on the other hand, ethanol which can be adsorbed in the precipitation process can be removed through freeze-drying, and finally, the obtained powdery heparin sodium is convenient to store and use.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in detail below with reference to specific embodiments.
It should be noted that, in order to avoid obscuring the present invention with unnecessary details, only the structures and/or processing steps closely related to the scheme of the present invention are shown in the specific embodiments, and other details not closely related to the present invention are omitted.
In addition, it is also to be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
The invention provides a simple and efficient heparin sodium recovery method for blood purification, which comprises the following steps:
s1, adjusting the pH value: recovering waste liquid generated by coupling process with heparin sodium as ligand, and adjusting pH value to 6-7.5, preferably 6.5-7; the pH is preferably adjusted by addition of hydrochloric acid.
In step S1, the content of heparin sodium in the waste liquid is generally less than 50%. The content of heparin sodium in the original waste liquid is low, after the heparin sodium is recovered by the recovery method, the recovery rate can reach more than 85 percent, the purity can reach more than 99 percent, the influence of the physicochemical property of the recovered heparin sodium is small, the recovered heparin sodium can still be used as a coupling agent to carry out coupling preparation of the adsorbent again, the reduction rate of the adsorption agent is less than 3 percent compared with that of the original adsorbent, and the recovery and reutilization value of the heparin sodium is obvious.
The coupling process is a coupling reaction between aldehyde group or epoxy group on the carrier and amino group on the heparin sodium. The components in the waste liquid obtained by the coupling reaction are complex, the pH value is adjusted to be nearly neutral, and the appropriate amount of ethanol is added to precipitate and separate out the heparin sodium, so that the ethanol dosage is obviously reduced under the condition of ensuring the maximum separation of the heparin sodium, the recovery purity and the recovery rate are higher, and the cost is obviously reduced.
S2, precipitation: adding ethanol into the waste liquid treated in the step S1 according to a preset volume ratio at the temperature of 0-40 ℃, centrifuging for a preset time, and collecting precipitates; in step S2, the volume ratio of the waste liquid to the ethanol is 1mL: 0.2-3 mL, preferably 1mL: 0.5-1.5 mL, and more preferably 1mL:0.9 mL.
In step S2, the purity of ethanol is preferably 95%. The preset time of centrifugation is 5-30 min, preferably 10-20min, and more preferably 15 min.
As can be seen from the precipitation conditions, the precipitation conditions of the invention are mild, the requirement on the purity of ethanol is not high, so the usage amount is also low, and the precipitation time can be obviously shortened by adding ethanol and then centrifuging, so the whole precipitation process is simple and easy to operate, the raw materials are cheap and easy to obtain, the usage amount is small, and the recovery cost is low.
S3, freeze-drying: dissolving the precipitate with water of the same volume, and freeze-drying to obtain massive heparin sodium;
s4, powder grinding: and (3) pulverizing the massive heparin sodium by using a pulverizer to obtain the powdery heparin sodium.
Dissolving the precipitated heparin sodium in water, on one hand, removing water-insoluble impurities, on the other hand, removing ethanol which can be absorbed in the precipitation process through freeze-drying, and finally obtaining the powdery heparin sodium which is convenient to store and use.
Example 1
A simple and efficient method for recovering heparin sodium for blood purification comprises the following steps:
first, an adsorbent for blood purification is prepared to obtain a waste liquid to be subjected to recovery treatment: adding an alkaline activating reagent into a certain amount of agarose gel resin, carrying out an activation reaction, cleaning to obtain activated agarose microspheres, and coupling with a ligand solution to obtain the adsorbent for blood purification and waste liquid. The following examples use the waste liquid generated by the coupling process, and the reaction conditions for preparing the adsorbent for blood purification are kept consistent.
(1) Removing impurities: adding hydrochloric acid into the heparin sodium waste liquid generated by the coupling process, and adjusting the pH value to be neutral;
(2) and (3) precipitation: adding 95% ethanol in water bath at 40 ℃, centrifuging for 30min, and collecting precipitate, wherein the ratio of the heparin sodium waste liquid to the 95% ethanol is 1mL: 3 mL; wherein 95% ethanol is obtained by purchasing.
(3) Freeze-drying: dissolving the precipitate with water of the same volume, and lyophilizing to obtain block-shaped heparin sodium;
(4) powdering: and (3) pulverizing the massive heparin sodium by using a pulverizer to obtain the powdery heparin sodium.
For the recovery rate of heparin sodium, the recovery rate of heparin sodium is calculated according to the following formula:
the yield is 100% multiplied by M/M0
In the formula:
m is the mass of the powdered heparin sodium after being powdered, and the unit is g;
M0-the mass of heparin sodium in the ligand solution used for coupling, in g.
Example 2
A simple and efficient method for recovering heparin sodium for blood purification adopts coupling reaction waste liquid obtained by the same method as in example 1, and is different from example 1 in that 95% ethanol is added into a water bath at 25 ℃ in step (2), and precipitates are collected by centrifugation for 20min, wherein the ratio of the heparin sodium waste liquid to the 95% ethanol is 1mL: 1.5 mL.
Example 3
A simple and efficient method for recovering heparin sodium for blood purification adopts coupling reaction waste liquid obtained by the same method as in example 1, and is different from example 1 in that 95% ethanol is added at room temperature in step (2), the mixture is centrifuged for 10min, and precipitates are collected, wherein the ratio of the heparin sodium waste liquid to the 95% ethanol is 1mL: 1 mL.
Example 4
A simple and efficient method for recovering heparin sodium for blood purification adopts coupling reaction waste liquid obtained by the same method as in example 1, and is different from example 1 in that 95% ethanol is added at room temperature in step (2), the mixture is centrifuged for 5min, and precipitates are collected, wherein the ratio of the heparin sodium waste liquid to the 95% ethanol is 1mL: 1 mL.
Example 5
A simple and efficient method for recovering heparin sodium for blood purification adopts coupling reaction waste liquid obtained by the same method as in example 1, and is different from example 1 in that 95% ethanol is added in ice bath in step (2), the mixture is centrifuged for 15min, and precipitates are collected, wherein the ratio of the heparin sodium waste liquid to the 95% ethanol is 1mL:0.9 mL.
Example 6
A simple and efficient method for recovering heparin sodium for blood purification adopts coupling reaction waste liquid obtained by the same method as in example 1, and is different from example 1 in that 95% ethanol is added at room temperature in step (2), the mixture is centrifuged for 10min, and precipitates are collected, wherein the ratio of the heparin sodium waste liquid to the 95% ethanol is 1mL:0.5 mL.
Example 7
A simple and efficient method for recovering heparin sodium for blood purification adopts coupling reaction waste liquid obtained by the same method as in example 1, and is different from example 1 in that 95% ethanol is added at room temperature in step (2), the mixture is centrifuged for 10min, and precipitates are collected, wherein the ratio of the heparin sodium waste liquid to the 95% ethanol is 1mL:0.2 mL.
Application example 1
The powdery heparin sodium obtained in examples 1 to 7 was weighed, and the results of the yield calculation are shown in Table 1.
TABLE 1 heparin sodium yield recovered from different groups
In the implementation process, the temperature has no influence on the recovery of the heparin sodium basically, so the heparin sodium can be recovered at room temperature.
The comparison between the examples 1 to 7 shows that the ratio of the heparin sodium waste liquid to the 95% ethanol is 1mL: when the concentration is 0.5-1.5 mL, the recovery rate of the heparin sodium is higher; the ratio of the heparin sodium waste liquid to the 95% ethanol is 1mL: when the concentration is 3mL, the recovery rate of the heparin sodium is higher, but considering that a proper amount of 95% ethanol can recover more heparin sodium, the preferable range is 1mL: 0.5-1.5 mL, and the optimal proportion is 1mL:0.9 mL.
The comparison between the examples 2 to 4 shows that the purpose of complete precipitation can be achieved when the centrifugation time is 10min or more, and when the proportion of the heparin sodium waste liquid to the 95% ethanol is 1mL: when the concentration is 1mL, the yield of 20min of centrifugation time is lower than that of 10min, which indicates that the centrifugation time is too long, but the centrifugation time is not beneficial to the efficient precipitation of heparin sodium, so the centrifugation time is selected to be 10 min.
From examples 4 and 5, it can be seen that the centrifugation time and the volume ratio of the heparin sodium waste solution to 95% ethanol together affect the yield, and the yield is higher as the non-ethanol content is higher. The ethanol content of the embodiment 5 is lower than that of the embodiment 4, but the centrifugation time is properly increased, the yield is obviously higher than that of the embodiment 4, and is equivalent to that of the embodiment 3, the centrifugation time of the embodiment 3 is slightly lower than that of the embodiment 5, but the ethanol content is slightly higher than that of the embodiment 5, so that an optimal collocation interval exists between the centrifugation time and the ethanol content.
Application example 2
Preparing a ligand solution from the powdery heparin sodium recovered in the embodiments 1 to 7, preparing 7 batches of adsorbents again according to the preparation method for preparing the adsorbent for purifying blood, and detecting the adsorption rate of the adsorbents, wherein the batches are recorded as the embodiments 1-2 to 7-2.
TABLE 2 adsorption Rate results for adsorbents prepared from recovered heparin sodium ligand solution
When the adsorbent is prepared by adopting the recovered heparin sodium, compared with the adsorption rate of the adsorbent prepared by original heparin sodium, the change rate of the adsorption rate is smaller and is within the detection error range, and as can be seen from the table 2, the adsorption rate of the adsorbent prepared by recovering the heparin sodium is not influenced by the recovered heparin sodium, and the recovered heparin sodium can be reused and can be subsequently recycled.
In conclusion, the simple and efficient heparin sodium recovery method for blood purification provided by the invention has the advantages that under the condition of ensuring that heparin sodium is separated out to the maximum extent, the ethanol dosage is obviously reduced, and finally, powdery heparin sodium is obtained by freeze-drying and crushing, so that the method has important significance for improving the utilization rate of raw materials and reducing the preparation cost of blood adsorbents. The recovery rate of the invention can reach more than 85 percent, the purity of the invention can reach more than 99 percent, the influence of the physicochemical property of the recovered heparin sodium is small, the heparin sodium can still be used as a coupling agent to carry out coupling preparation of the adsorbent again, the reduction rate of the adsorption rate of the adsorbent is less than 3 percent compared with the original adsorbent, and the invention has obvious recycling value.
Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the spirit and scope of the present invention.
Claims (10)
1. A simple and efficient method for recovering heparin sodium for blood purification is characterized by comprising the following steps:
s1, adjusting the pH value: recovering waste liquid generated by the coupling process with heparin sodium as ligand, and adjusting the pH value of the waste liquid to 6-7.5;
s2, precipitation: adding ethanol into the waste liquid treated in the step S1 according to a preset volume ratio, centrifuging for a preset time, and collecting precipitates;
s3, freeze-drying: dissolving the precipitate with water, and freeze-drying to obtain massive heparin sodium;
s4, powder grinding: and (3) pulverizing the massive heparin sodium by using a pulverizer to obtain powdery heparin sodium.
2. The simple and efficient method for recovering heparin sodium for blood purification according to claim 1, wherein the pH is adjusted by adding hydrochloric acid in step S1.
3. The simple and efficient method for recovering heparin sodium for blood purification according to claim 1, wherein the content of heparin sodium in the waste liquid is 50% in step S1.
4. The simple and efficient method for recovering heparin sodium for blood purification as set forth in claim 3, wherein the coupling process is a coupling reaction of an aldehyde group or an epoxy group on a carrier with an amino group on the heparin sodium.
5. The simple and efficient method for recovering heparin sodium for blood purification as set forth in claim 1, wherein the volume ratio of the waste liquid to the ethanol in step S2 is 1mL: 0.2-3 mL.
6. The simple and efficient method for recovering heparin sodium for blood purification as set forth in claim 1, wherein the volume ratio of the waste liquid to the ethanol is 1mL: 0.5-1.5 mL.
7. The simple and efficient method for recovering heparin sodium for blood purification according to claim 1, wherein the volume ratio of the waste liquid to the ethanol is 1mL:0.9 mL.
8. The simple and efficient method for recovering heparin sodium for blood purification according to claim 1, wherein the purity of the ethanol is 95% in step S2.
9. The simple and efficient method for recovering heparin sodium for blood purification as set forth in claim 1, wherein the preset time for the centrifugation in step S2 is 5-30 min.
10. The simple and efficient method for recovering heparin sodium for blood purification according to claim 1, wherein step S2 is performed at 0 to 40 ℃.
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| CN1844166A (en) * | 2006-05-11 | 2006-10-11 | 中国农业大学 | Method for simultaneous extraction of sodium heparin and antibacterial peptide of pig intestinal mucosa |
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