CN108727487B - Liquid membrane extraction method of hirudin - Google Patents
Liquid membrane extraction method of hirudin Download PDFInfo
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Abstract
The invention relates to a liquid membrane extraction method of hirudin. The method comprises the following steps: s1: preparing a hirudin crude extract with the pH of 8.0-9.5; s2: adding the emulsion into the hirudin crude extract of S1, stirring, standing for layering, adding a demulsifier, standing for layering, taking down the phase extract, standing at 40-60 ℃, and cooling to obtain the hirudin; the volume ratio of the hirudin crude extract to the emulsion is 1: 4-6; the emulsion is a liquid membrane extraction system. The hirudin obtained by the liquid membrane extraction method of hirudin provided by the invention has high purity, the active recovery rate of the hirudin reaches 92.5%, the process is simple, the cost is low, and the method can be widely popularized and applied.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to a liquid membrane extraction method of hirudin.
Background
Hirudin is an acidic polypeptide extracted and separated from leech saliva, is a specific thrombin inhibitor with the strongest activity discovered so far, is relatively stable, has an isoelectric point PI =4.0, has good anticoagulation and platelet aggregation resistance effects, and is a medicament with wide development prospects in antithrombotic treatment. Research shows that hirudin can also prevent tumor cell metastasis, and hirudin can promote blood flow in tumor to enhance curative effect by combining chemotherapy and radiotherapy, so that the hirudin has wide clinical application prospect. China has abundant natural leech resources, but most of the current researches on the extraction process of hirudin can not break through the limitations of the traditional method, for example, in 1990, Stener and the like adopt the steps of gel filtration, anion exchange chromatography, reversed phase HPLC and the like to extract the hirudin from leeches, and the total activity recovery rate is only 0.011 percent. The research on the novel high-efficiency extraction process is very little, so that the hirudin cannot be widely applied.
Therefore, the development of the hirudin extraction method with high hirudin activity recovery rate has important research significance and application value.
Disclosure of Invention
The invention aims to overcome the defect of low recovery rate of hirudin extraction methods in the prior art and provides a liquid membrane extraction method of hirudin. The liquid membrane extraction method provided by the invention can realize the recycling of hirudin, and the obtained hirudin has high purity, high active recovery rate of hirudin, simple process and low cost.
In order to achieve the purpose, the invention adopts the following technical scheme:
a liquid membrane extraction method of hirudin, comprising the following steps:
s1: preparing a hirudin crude extract with the pH of 8.0-9.5;
s2: adding the emulsion into the hirudin crude extract of S1, stirring, standing for layering, adding a demulsifier, standing for layering, taking down the phase extract, standing at 40-60 ℃, and cooling to obtain the hirudin;
the volume ratio of the hirudin crude extract to the emulsion is 1: 4-6;
the emulsion is a liquid membrane extraction system which takes a mixed solution of an emulsifier and isooctane as a membrane phase, an alkaline solution as an internal phase and trioctylamine as a carrier; the mass ratio of the trioctylamine to the isooctane is 1.5-3: 100; the pH of the inner phase is 8.5-10.0; the volume ratio of the membrane phase to the internal phase in the membrane is 10: 2.0-2.5; the pH value of the inner phase is 0.5-1.5 less than that of the hirudin crude extract.
The invention provides a novel liquid membrane extraction system, wherein a membrane phase is formed by an emulsifier and isooctane, an alkaline solution is an internal phase, trioctylamine is a carrier, and hirudin extraction is realized by exploring each process condition (the mutual dosage relation of each component of the system) of the liquid membrane system and utilizing the pH difference between an internal phase and an external phase (crude hirudin extract is the external phase) and the extraction action of an extracting agent. The method realizes the recycling of the hirudin, and the obtained hirudin has high purity, high active recovery rate of the hirudin, simple process and low cost.
In the prior art, impurities are generally removed by the precipitation principle to obtain the active ingredient of hirudin. The methods for preparing the hirudin crude extract are more published at present and can be used in the invention.
Preferably, the hirudin crude extract in S1 is prepared by the following method: mashing frozen leeches, adding a solvent to dissolve the leeches, adding an organic acid to adjust the pH value to 2-4, preserving the temperature at 65-70 ℃, and centrifuging to obtain a supernatant; adjusting the pH of the supernatant to 7-9, centrifuging, and taking the supernatant; and adjusting the supernatant to 8.0-10.5 to obtain the hirudin crude extract.
Solvents conventionally used to achieve dissolution of hirudin can be used in the present invention, such as water, ethanol, acetone, etc. More preferably, the solvent is a NaCl solution from the viewpoint of economic cost.
Conventional organic acids may be used in the present invention for adjusting the pH. More preferably, the organic acid is trichloroacetic acid from the viewpoint of the acid strength and the conditioning efficiency of the organic acid.
Preferably, the pH of the S1 hirudin crude extract is 9.0-9.5.
Preferably, the stirring temperature in S2 is 30-50 ℃.
Conventional alkaline solutions, e.g. NaOH solution, Na2CO3Solution, NaHCO3Solutions and the like can be used as the basic solution of the internal phase. Preferably, the alkaline solution of the internal phase is a NaOH solution.
Conventional emulsifiers may be used in the present invention, such as sorbitan fatty acid esters, glyceryl monostearate, and the like.
Preferably, the emulsifier in S2 is a sorbitan fatty acid ester.
More preferably, the mass ratio of the sorbitan fatty acid ester to the isooctane is 3-5: 100.
More preferably, the mass ratio of the sorbitan fatty acid ester to isooctane is 3: 100.
Preferably, the mass ratio of the trioctylamine to the isooctane is 2.5:100
The demulsifier conventional in the prior art can be applied to the present invention, and preferably, the demulsifier in S2 is acetone.
Preferably, the volume ratio of the demulsifier to the hirudin crude extract is 1: 2.
Preferably, the intramembrane volume ratio of the membrane phase to the internal phase is 10: 2.5.
Preferably, the pH of the inner phase is less than 0.5 than the pH of the crude hirudin extract.
Preferably, the intramembrane volume ratio of the membrane phase to the internal phase is 10:2.5, the mass ratio of the trioctylamine to the isooctane is 3:100, the pH of the internal phase is 10.0, and the pH of the hirudin crude extract is 9.5.
The hirudin activity recovery rate under the specific process condition is as high as 92.5%.
Preferably, the emulsion is prepared by the following method: dissolving emulsifier in isooctane, adding trioctylamine for dissolving, adding alkaline solution, and emulsifying under ultrasonic condition.
More preferably, the working conditions of the ultrasound are: phi 6 horn, power 45% W, time 10min, temperature 30 deg.C, time interval stop and start each 2 s.
Compared with the prior art, the invention has the following beneficial effects:
the liquid membrane extraction method of hirudin provided by the invention realizes the recycling of hirudin by constructing a specific liquid membrane extraction system, the obtained hirudin has high purity, the active recovery rate of the hirudin is up to 92.5%, the process is simple, the cost is low, and the liquid membrane extraction method can be widely popularized and applied.
Drawings
FIG. 1 is a graph of the relationship between the membrane-internal ratio and the recovery of hirudin ATU;
FIG. 2 is a graph showing the relationship between the pH of a feed liquid (crude extract of hirudin) and the recovery rate of hirudin ATU;
FIG. 3 is a graph of the relationship between the pH of the internal phase and the recovery of hirudin ATU;
FIG. 4 is a graph of vector concentration versus recovery of hirudin ATU;
FIG. 5 is a discontinuous polyacrylamide gel electrophoresis diagram, wherein (i) is a hirudin purified solution; ② a hirudin standard product; ③ is hirudin crude extract;
FIG. 6 is a graph of spectral scanning of a hirudin standard;
FIG. 7 is a scanning spectrum of hirudin purified solution;
FIG. 8 is a diagram showing the in vitro thrombolytic effect of hirudin, wherein (i) is physiological saline; ② hirudin crude extract; ③ hirudin purified liquid.
Detailed Description
The invention is further illustrated by the following examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples below, generally according to conditions conventional in the art or as suggested by the manufacturer; the raw materials, reagents and the like used are, unless otherwise specified, those commercially available from the conventional markets and the like. Any insubstantial changes and substitutions made by those skilled in the art based on the present invention are intended to be covered by the claims.
EXAMPLE 1 preparation of hirudin crude extract and emulsion
(1) Preparation of hirudin crude extract
Adding 0.9% NaCl into the crushed sample with volume 5 times of that of the frozen leech, stirring for 30min at 250r/min by using 3 times of the volume for the first time, centrifuging for 10min at 4800r/min, and collecting the liquid phase. Adding the rest NaCl solution into the precipitate, stirring at 250r/min for 30min, centrifuging at 4800r/min for 10min, removing precipitate, and collecting liquid phase. Combining the two extraction liquids, adjusting the pH value to 2.0 by trichloroacetic acid with the mass fraction of 10%, preserving the heat at 65-70 ℃ for 30min, centrifuging at 4800r/min for 10min, and collecting supernatant; adjusting pH to 9.0 with 1 mol/L NaOH, centrifuging at 4800r/min for 10min to obtain supernatant as crude extractive solution, and adjusting pH to desired value; the activity of the crude hirudin extract was determined to be 1 ATU/mL.
(2) Preparation of emulsions
Emulsification conditions: ultrasonic emulsification is selected for the emulsification in the experiment, and the working conditions are as follows: phi 6 horn, power 45% W, time 10min, temperature 30 deg.C, time interval stop and start each 2 s.
Preparation: taking a proper amount of isooctane, adding 3% of sorbitan fatty acid ester according to mass fraction, dissolving the sorbitan fatty acid ester according to the emulsification condition, adding trioctylamine according to certain mass fraction according to the combination, and preparing a mixed solution according to the emulsification condition. Then respectively adding corresponding amount of internal phase NaOH solution with corresponding pH value into the combined membranes, uniformly mixing, and ultrasonically emulsifying into uniform single liquid at a certain power.
Example 2 Effect of ratio in Membrane on recovery of hirudin ATU
A crude extract of hirudin with a pH of 9.0 was obtained by the preparation method provided in example 1.
When the emulsion is prepared according to the preparation method of the emulsion provided in example 1, the amount of the carrier trioctylamine is 2.0% by mass of isooctane, and the internal phase is a NaOH solution with pH of 9.5, then the internal phase NaOH solutions with the same pH are respectively added according to the membrane internal ratio (i.e. the volume ratio of the membrane phase to the internal phase) of 10.0:2.0, 10.0:2.5, 10.0:3.0, 10.0:3.5, 10.0:4.0, mixed uniformly, ultrasonically emulsified into a uniform single liquid at a certain power, and then extracted.
The whole process is as follows:
(1) preparing a hirudin crude extract with pH of 9.0;
(2) adding hirudin crude extract into 5mL of emulsion at a ratio of 1:6, stirring in 30 deg.C constant temperature water bath at 200r/min for 30min, taking out, and standing for 30 min. After separation, the emulsion was taken out and added with about 1/2 volumes of acetone to break the emulsion by mixing. Standing for layering, taking down the phase extract, and placing in a constant temperature water bath at 60 deg.C for 30 min. After cooling to room temperature, 2mol/L HCl was added to adjust the pH to 5.0, and the recovery was calculated by measuring ATU.
Here, the recovery rate was determined by a thrombin titration method modified from Chenghuayou et al (Chenghuayou, Chenchen Dynasty, Liyunity, etc.. Thrombin titration method [ J ] Biotechnology, 2002,12 (6): 24-25) on the basis of the binding ratio of hirudin to thrombin being 1:1(mol/mol) and 1:5 (g/g). The international unit NIH for thrombin, the activity of hirudin is expressed in antithrombin activity units ATU, one ATU being equal to the amount of hirudin which neutralizes an NIH thrombin. The content is calculated according to the following formula:
D=20d
wherein D is the number of ATU per 1 mL of the test solution (the test solution is the crude hirudin extract in this experiment), and D is the number of units containing ATU per 50. mu.L of the test solution.
Recovery rate of ATU
。
The membrane-in-membrane ratio refers to the volume of the membrane phase (organic solvent) in the liquid membrane emulsion during the preparation of the emulsionV m) And initial internal phase volume (V i) The ratio of the two components is a structural parameter of the liquid film, has great influence on the performance of the liquid film, and the size of the ratio in the film can influence the stability and the permeability of the liquid film. The recovery relationships at different membrane internal ratios are shown in figure 1. As can be seen from FIG. 1, the recovery rate of hirudin ATU in the liquid membrane emulsion increases from 10.0:2.0 to 10.0: 2.5; when the membrane internal ratio is increased from 10.0:2.5 to 10.0:3.0, the recovery rate of hirudin ATU is in a descending trend; when the membrane internal ratio is increased from 10.0:3.0 to 10.0:3.5, the ATU recovery rate of hirudin is basically unchanged; however, when the membrane internal ratio is increased to 10.0:4.0, the recovery rate of ATU of hirudin is in a descending trend; it can be seen that the recovery of ATU is highest when the in-film ratio is 10.0: 2.5. This is because the larger the membrane ratio, the more stable the liquid membrane, which facilitates the extraction of hirudin. However, the larger the membrane internal ratio, the larger the liquid membrane thickness, and simultaneously the viscosity is increased, the resistance to mass transfer is increased, and the permeability of the liquid membrane is reduced, which has adverse effect on hirudin extraction. Of course, if the membrane internal ratio is too small, the membrane is unstable, and the membrane is easily broken, thereby reducing the extractionEfficiency. Therefore, the in-film ratio of 10.0:2.5 is a relatively stable state of the liquid film, and is suitable in terms of the amount of the raw material used, which is a preferable condition.
EXAMPLE 3 Effect of pH of feed solution (crude extract of hirudin) on recovery of ATU of hirudin
Crude extracts of hirudin having pH values of 10.5, 10.0, 9.5, 9.0, 8.5, 8.0 were obtained by the preparation method provided in example 1.
When the emulsion is prepared according to the preparation method of the emulsion provided in the embodiment 1, the amount of the carrier trioctylamine is 2.0% of the mass fraction of isooctane, the internal phase is a NaOH solution with the pH of 9.5, then the internal phase NaOH solutions with the same pH are respectively added according to the membrane internal ratio (i.e. the volume ratio of the membrane phase to the internal phase) of 10.0:2.5, mixed uniformly, ultrasonically emulsified into a uniform single liquid at a certain power, and then extracted.
The whole process is as follows:
(1) preparing hirudin crude extract with pH of 10.5, 10.0, 9.5, 9.0, 8.5, and 8.0;
(2) adding hirudin crude extract into 5mL of emulsion at a ratio of 1:6, stirring in 30 deg.C constant temperature water bath at 200r/min for 30min, taking out, and standing for 30 min. After separation, the emulsion was taken out and added with about 1/2 volumes of acetone to break the emulsion by mixing. Standing for layering, taking down the phase extract, and placing in a constant temperature water bath at 60 deg.C for 30 min. After cooling to room temperature, 2mol/L HCl was added to adjust the pH to 5.0, and the recovery was calculated by measuring ATU.
The feed liquid is hirudin crude extract, and as can be seen from the above figure, in the liquid membrane emulsion, when the pH of the feed liquid is increased from 8.0 to 8.5, the ATU recovery rate of hirudin is basically unchanged; when the pH value of the feed liquid is increased from 8.5 to 9.0, the ATU recovery rate of the hirudin is in an increasing trend; when the pH value of the feed liquid is increased from 9.0 to 9.5, the ATU recovery rate of the hirudin also tends to rise, but the rising speed tends to be gentle; when the pH value of the feed liquid is increased from 9.5 to 10.0, the ATU recovery rate of the hirudin obviously shows a descending trend; when the pH of the feed solution is increased from 10.0 to 10.5, the ATU recovery rate of hirudin decreases more rapidly than before. It can be seen that the maximum recovery of ATU from hirudin, i.e. the highest permeability of the liquid membrane, is obtained when the pH of the feed solution is 9.5. From the principle of transmission, as the pH value of the feed liquid increases, the hirudin is negatively charged, and the target hirudin is exchanged with anions in the liquid membrane through the transportation of carrier cations, so that the target is transferred to the inner phase. Therefore, as the pH of the feed solution increases, the transport efficiency of hirudin increases, but only negatively charged hirudin can be transported into the internal phase, and the concentration gradient of the two phases is switched as the anions in the internal phase are continuously transferred to the membrane phase. Therefore, as the pH of the feed solution increases, the concentration of the inner phase hirudin decreases and the extraction yield decreases.
EXAMPLE 5 Effect of internal phase pH on hirudin ATU recovery
A crude extract of hirudin with a pH of 9.0 was obtained by the preparation method provided in example 1.
When the emulsion is prepared according to the preparation method of the emulsion provided in example 1, the amount of the carrier trioctylamine is 2.0% by mass of isooctane, the internal phase is a NaOH solution with pH of 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, then NaOH internal phase solutions with the same pH are respectively added according to the membrane internal ratio (i.e. the volume ratio of the membrane phase to the internal phase) of 10.0:2.5, mixed uniformly, ultrasonically emulsified into a uniform single liquid at a certain power, and then extracted.
The whole process is as follows:
(1) a crude extract of hirudin having a pH of 9.0 was obtained by the preparation method provided in example 1;
(2) adding hirudin crude extract into 5mL of emulsion at a ratio of 1:6, stirring in 30 deg.C constant temperature water bath at 200r/min for 30min, taking out, and standing for 30 min. After separation, the emulsion was taken out and added with about 1/2 volumes of acetone to break the emulsion by mixing. Standing for layering, taking down the phase extract, and placing in a constant temperature water bath at 60 deg.C for 30 min. Cooled to room temperature, adjusted to pH 5.0 by addition of 2mol/LHCl and the recovery calculated by determination of ATU.
The relationship between the recovery of hirudin ATU and the pH of the internal phase in each extraction system after extraction is shown in FIG. 3. As can be seen in FIG. 3, the ATU recovery of hirudin increased with increasing pH of the inner phase from 8.0 to 9.5; however, as the pH of the inner phase increased from 9.5 to 10.5, the ATU recovery of hirudin decreased. It can be seen that the maximum recovery of ATU of hirudin is obtained when the pH of the inner phase is 9.5. This is because hirudin is transported from the external phase to the internal phase by the cationic carrier due to the difference in the concentrations of the external and internal phases, and when the pH of the external phase is constant, the cationic carrier transports more hirudin to the internal phase as the pH of the internal phase increases; however, when the pH of the inner phase is increased to a certain degree, the pH of the inner phase and the pH of the outer phase reach an equilibrium state, and the pH of the outer phase is reduced along with the increase of the pH of the inner phase, so that the hirudin of the inner phase is transported into the outer phase under the action of a cationic carrier, and the concentration of the hirudin of the inner phase is reduced, thereby influencing the ATU recovery rate of the hirudin of the inner phase.
EXAMPLE 5 Effect of vector concentration on recovery of hirudin ATU
A crude extract of hirudin with a pH of 9.0 was obtained by the preparation method provided in example 1.
When the emulsion is prepared according to the preparation method of the emulsion provided in example 1, the amount of the carrier trioctylamine is 1%, 1.5%, 2.0%, 2.5%, 3.0% by mass of isooctane, and the internal phase is a NaOH solution with pH of 9.5, then the internal phase NaOH solutions with the same pH are respectively added according to the membrane internal ratio (i.e. the volume ratio of the membrane phase to the internal phase) of 10.0:2.5, mixed uniformly, ultrasonically emulsified into a uniform single liquid at a certain power, and then extracted.
The whole process is as follows:
(1) a crude extract of hirudin having a pH of 9.0 was obtained by the preparation method provided in example 1;
(2) adding hirudin crude extract into 5mL of emulsion at a ratio of 1:6, stirring in 30 deg.C constant temperature water bath at 200r/min for 30min, taking out, and standing for 30 min. After separation, the emulsion was taken out and added with about 1/2 volumes of acetone to break the emulsion by mixing. Standing for layering, taking down the phase extract, and placing in a constant temperature water bath at 60 deg.C for 30 min. Cooled to room temperature, adjusted to pH 5.0 by addition of 2mol/LHCl and the recovery calculated by determination of ATU.
The carrier plays a role of transporting the object in the extraction process. As can be seen from FIG. 4, the ATU recovery rates of hirudin all tended to increase when the carrier concentration in the emulsion membrane system increased from 1.0% to 2.5%; however, when the concentration of the carrier in the emulsion membrane system was increased from 2.5% to 3.0%, the recovery of ATU from hirudin tended to decrease. This may be the case because the extraction process is controlled by a combination of chemical reactions between hirudin and the carrier and diffusion processes after its bindingAnd (5) preparing. When the concentration of the trioctylamine carrier is equal to[24]When the recovery rate is lower, the recovery rate is mainly controlled by the chemical reaction between hirudin and the carrier, and the higher the carrier concentration is in a certain range, the more sufficient the reaction is, the higher the recovery rate is; if the carrier concentration is close to saturation, recovery is mainly controlled by the complex diffusion process. At this time, steric hindrance by the carrier at a high concentration prevents the complex from entering the membrane, resulting in a decrease in recovery rate. For economic reasons, a carrier concentration of 2.5% is most suitable for use in the membrane.
EXAMPLE 6 crude extract extraction orthogonal test
The membrane internal ratio, the carrier amount, the internal phase pH and the feed liquid pH cross influence the hirudin ATU recovery rate of a liquid membrane system, and in order to comprehensively examine the influence of the factors, according to the single-factor experiment result, the hirudin crude extract is subjected to L treatment9（34) And optimizing hirudin extraction process conditions of the emulsion membrane system by using an orthogonal table. The experimental arrangement is shown in table 1 and the results are shown in tables 2 and 3.
TABLE 1 emulsion liquid membrane system hirudin extraction experimental factors and levels
TABLE 2 emulsion membrane system hirudin back extraction orthogonal test and results
TABLE 3 emulsion membrane system hirudin back extraction orthogonal test analysis of variance
The order of factors affecting the extraction was found to be the carrier concentration (%)>Internal phase pH>Ratio of membrane to membrane>The pH of the external phase and the optimal extraction process conditions are A1B2C1D2I.e. internal phase pH 10.0, carrier concentration 25%, membrane internal ratio 10:2.0, external phase pH 9.5.
EXAMPLE 7 identification of hirudin purification solution
The following conditions were followed: the hirudin purified solution is prepared by extracting a feed liquid with the pH of 9.5 by a liquid membrane system with the membrane-internal ratio of 10:2.0, the carrier amount of 2.5 and the internal phase pH of 10.0, and testing the performances such as purity and the like.
(1) Discontinuous polyacrylamide gel electrophoresis
Polyacrylamide gel electrophoresis is abbreviated as PAGE (Polyacrylamide geleletrophoresis). A common electrophoresis technique using polyacrylamide gel as a support medium. PAGE is classified into two major groups, continuous and discontinuous, according to the presence or absence of a concentration effect. The experiment here applies to discontinuous systems. The discontinuous system consists of electrode buffer solution, concentrated gel and separating gel. The concentrated gel is macroporous gel prepared by catalytic polymerization of AP, and the gel buffer is Tris-HCl with pH6.7. The separation gel is a small-pore gel prepared by AP catalytic polymerization, and the gel buffer is Tris-HCl with the pH value of 8.9. The electrode buffer was Tris-glycine buffer, pH 8.3. In the discontinuous system, due to the discontinuity of buffer solution ion components, pH, gel concentration and potential gradient, the charged particles not only have charge effect, molecular sieve effect, but also have concentration effect in the electric field, so that the definition and resolution of the separation band are better than those of continuous electrophoresis. The results of the discontinuous polyacrylamide gel electrophoresis performed in the order of the hirudin standard, the crude hirudin extract and the purified hirudin solution are shown in FIG. 5.
The hirudin purified solution subjected to electrophoretic analysis by using discontinuous polyacrylamide gel and a hirudin standard substance show uniform strips, and the purified sample is proved to be electrophoretically pure. The lower part of the final hirudin crude extract has a plurality of strips, because the polyacrylamide gel has a net structure and has a molecular sieve effect, the protein can be separated according to the difference of the molecular size of the protein. During electrophoresis, proteins with small molecular weights run down quickly, while proteins with large molecular weights run up slowly to form bands as shown in the upper graph.
(2) Ultraviolet spectral analysis
In the ultraviolet absorption spectrum analysis, the electronic transition is accompanied with the transition of the vibration rotation energy level, so that the absorbance of light with different wavelengths is different. Can be used for structural identification and quantitative analysis. The ultraviolet spectra of the hirudin standard and purified solution are shown in FIGS. 6 and 7.
The wavelength distribution of the ultraviolet spectrum is determined by the energy difference between transition energy levels of the generation bands, reflects the distribution condition of the energy levels in the molecule, and is the basis of material qualification. From the two spectral graphs, the maximum absorption wavelengths of the spectral scanning curves of the hirudin standard product and the purified liquid are both 204nm, and the absorption curve shapes of the hirudin standard product and the purified liquid are similar to reflect that the substance molecules contained in the hirudin standard product and the purified liquid are similar.
(3) Hirudin in vitro thrombolysis experiment
The mouse thrombus is respectively placed in normal saline, hirudin crude extract and hirudin purified solution for 24h at 37 ℃, as shown in figure 8, the thrombus strips soaked in the hirudin crude extract are partially dissolved, the thrombus strips in the hirudin purified solution are completely dissolved, and the thrombus strips soaked in the normal saline are unchanged and still keep a solidification state, which shows that the hirudin has a thrombolytic effect and the hirudin purified solution has an obvious thrombolytic effect.
From the performance tests, the method realizes the recycling of hirudin by constructing a specific liquid membrane extraction system, and the obtained hirudin has high purity, high antithrombin activity yield, simple process and low cost, and can be widely popularized and applied.
Claims (1)
1. A liquid membrane extraction method of hirudin is characterized by comprising the following steps:
s1: preparing a hirudin crude extract;
s2: adding the emulsion into the hirudin crude extract of S1, stirring, standing for layering, adding a demulsifier, standing for layering, taking down the phase extract, standing at 40-60 ℃, and cooling to obtain the hirudin;
the volume ratio of the hirudin crude extract to the emulsion is 1: 4-6;
the emulsion is a liquid membrane extraction system which takes a mixed solution of an emulsifier and isooctane as a membrane phase, an alkaline solution as an internal phase and trioctylamine as a carrier;
the emulsifier is sorbitan fatty acid ester;
the volume ratio of the membrane phase to the inner phase in the membrane is 10:2.5, the mass ratio of the trioctylamine to the isooctane is 3:100, the pH of the inner phase is 10.0, and the pH of the hirudin crude extract is 9.5;
the emulsion is prepared by the following method: dissolving emulsifier in isooctane, adding trioctylamine for dissolving, adding alkaline solution, and emulsifying under ultrasonic condition.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286098A (en) * | 2011-08-18 | 2011-12-21 | 周维官 | Production method of natural hirudin |
| CN103663598A (en) * | 2013-09-27 | 2014-03-26 | 苏州国环环境检测有限公司 | Process for treating phenol wastewater through liquid membrane |
| WO2014064703A1 (en) * | 2012-10-28 | 2014-05-01 | Peritech Pharma Ltd. | Pharmaceutical liquid adhesive compositions for treatment of anorectal disorders |
| CN106928347A (en) * | 2017-03-13 | 2017-07-07 | 佛山市爱的生物科技有限公司 | A kind of separation method of hirudin |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286098A (en) * | 2011-08-18 | 2011-12-21 | 周维官 | Production method of natural hirudin |
| WO2014064703A1 (en) * | 2012-10-28 | 2014-05-01 | Peritech Pharma Ltd. | Pharmaceutical liquid adhesive compositions for treatment of anorectal disorders |
| CN103663598A (en) * | 2013-09-27 | 2014-03-26 | 苏州国环环境检测有限公司 | Process for treating phenol wastewater through liquid membrane |
| CN106928347A (en) * | 2017-03-13 | 2017-07-07 | 佛山市爱的生物科技有限公司 | A kind of separation method of hirudin |
Non-Patent Citations (1)
| Title |
|---|
| 以D2EHPA 为流动载体的乳状液膜萃取菲牛蛭中的水蛭素实验研究;方富永等;《中国中药杂志》;20121015;第37卷(第20期);参见对比文件第3056页最后一段，第3057页第2段，第2节，第3.2.2节，第3.3节，第4节 * |
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