CN113512130A - Ultra-filtration classification and anticoagulant activity determination method of dextran sulfate - Google Patents
Ultra-filtration classification and anticoagulant activity determination method of dextran sulfate Download PDFInfo
- Publication number
- CN113512130A CN113512130A CN202110423927.6A CN202110423927A CN113512130A CN 113512130 A CN113512130 A CN 113512130A CN 202110423927 A CN202110423927 A CN 202110423927A CN 113512130 A CN113512130 A CN 113512130A
- Authority
- CN
- China
- Prior art keywords
- dextran sulfate
- fractionation
- ultrafiltration
- fractions
- sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/483—Physical analysis of biological material
- G01N33/487—Physical analysis of biological material of liquid biological material
- G01N33/49—Blood
- G01N33/4905—Determining clotting time of blood
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Materials Engineering (AREA)
- Polymers & Plastics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Physics & Mathematics (AREA)
- Hematology (AREA)
- Biophysics (AREA)
- Ecology (AREA)
- Sustainable Development (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Separation Using Semi-Permeable Membranes (AREA)
Abstract
The invention relates to an anticoagulant activity determination method, in particular to an ultrafiltration classification of dextran sulfate and an anticoagulant activity determination method thereof, concentrated sulfuric acid and n-butyl alcohol are measured and placed in a three-necked bottle with a drying tube and a stirring device, the mixture is stirred, cooled to minus 5 to minus 8 ℃ in an ice bath, and 5 to 7g of yeast dextran powder of 200 meshes is slowly added; after reacting for 6-8min, centrifuging, dialyzing with distilled water for 18-24h, concentrating under reduced pressure to 20-22mL, adding ethanol with volume fraction of 95%, collecting precipitate, dissolving the precipitate in water, and dialyzing for 18-24 h.
Description
Technical Field
The invention relates to an anticoagulant activity determination method, in particular to an ultrafiltration classification method of dextran sulfate and an anticoagulant activity determination method thereof.
Background
The anticoagulant is clinically used for anticoagulant treatment of cerebral intravascular coagulation syndrome, deep venous embolism, prevention of coagulation of the cardio-pulmonary system and the like, and is also an important component of a blood activating and stasis removing medicament. In vitro, anticoagulants are used in the field of biocompatible medical materials and in the field of vascular cosmetic products; is a multifunctional product worthy of wide development. At present, the anticoagulant commonly used in clinic is heparin, and different sources of heparin have the defects of unstable structure and activity, easy carrying of various animal pathogenic bacteria which are harmful to human health and the like. Therefore, the development of safe and stable heparin substitutes is attracting much attention. The invention provides an ultrafiltration classification method of dextran sulfate and a method for measuring anticoagulant activity of the dextran sulfate.
Disclosure of Invention
An object of the present invention is to solve the above-mentioned drawbacks of the background art by proposing a method for ultrafiltration fractionation of dextran sulfate and its anticoagulant activity determination.
The technical scheme adopted by the invention is as follows: a method for ultrafiltration fractionation and anticoagulation activity determination of dextran sulfate comprises the following steps:
measuring concentrated sulfuric acid and n-butyl alcohol, placing the concentrated sulfuric acid and the n-butyl alcohol into a three-necked bottle with a drying tube and a stirring device, stirring, cooling to-5 to-8 ℃ in an ice bath, and slowly adding 5 to 7g of 200-mesh yeast glucan powder;
after reacting for 6-8min, neutralizing the reaction system with NaOH, centrifuging, dialyzing with distilled water for 18-24h, concentrating under reduced pressure to 20-22mL, adding ethanol with volume fraction of 95%, standing, centrifuging, collecting precipitate, dissolving the precipitate in water, dialyzing for 18-24h, and freeze-drying the dialyzed solution to obtain dextran sulfate;
performing secondary ultrafiltration fractionation on dextran sulfate by adopting polyether sulfone membranes with molecular weight cut-offs of 4K and 10K respectively to obtain 3 fractions;
testing the intrinsic viscosity of the sample with a capillary viscometer;
administering to SD rat for 20-60min, performing intramuscular injection anesthesia with compound thiamine ketone (10ml/kg), collecting blood from heart, slowly inclining the tube for 1 time every 10s after 1-2min, and recording the liquid level motionless time when the tube is inclined, i.e. CT;
collecting blood from abdominal artery of rat, adding into test tubes containing 0.1ml of 3.8% sodium citrate, adding sample to be detected into each test tube, incubating in water bath at 37-38 deg.C for 1min, and adding 0.1ml of 0.01mol/L CaCl into each test tube2When the meter begins after the uniform mixing, the liquid level motionless time when the test tube is inclined is recorded, and the time is the RT;
the effect of 3 fractions on the anti-coagulant activity was judged by CT and RT values.
As a preferred technical scheme of the invention: the ratio of the concentrated sulfuric acid to the n-butyl alcohol is 1: 7-9.
As a preferred technical scheme of the invention: and the second-stage ultrafiltration fractionation is divided into first-stage ultrafiltration membrane fractionation and second-stage ultrafiltration membrane fractionation.
As a preferred technical scheme of the invention: and the first-stage ultrafiltration membrane fractionation adopts a polyether sulfone membrane with the molecular weight cutoff of 10K to divide glucan sulfate into two fractions of more than 10K and less than 10K.
As a preferred technical scheme of the invention: and the second-stage ultrafiltration and fractionation adopts a polyether sulfone membrane with the molecular weight cutoff of 4K to obtain two fractions of 4-10K and below 4K.
As a preferred technical scheme of the invention: the capillary viscometer has a length of 140 plus or minus 5mm and an inner diameter of 0.5 plus or minus 0.05mm, and the volume of the measuring ball is 3.5 plus or minus 0.5 ml.
As a preferred technical scheme of the invention: the method for measuring the intrinsic viscosity of the sample by the capillary viscometry comprises the following steps: weighing dried samples, preparing a solution by using distilled water, respectively measuring the samples with the same volume and the flowing-out time of the distilled water at the temperature of 20-30 ℃ by using a capillary viscometer to calculate the relative viscosity eta, and calculating the intrinsic viscosity of the samples by a multipoint dilution and back-pushing method.
As a preferred technical scheme of the invention: the multi-point dilution backward-pushing method comprises the following steps: and (3) drawing a curve chart of the relative viscosity eta of the samples with different concentrations and the concentrations, and analyzing the intrinsic viscosity of the samples through the gentle points of the curve chart.
As a preferred technical scheme of the invention: the number of the SD rats is 160-190, and the number of the male rats and the female rats is half of the number of the male rats.
The invention adopts the sulfonation reaction of glucan to extract glucan sulfate with extremely high purity, adopts an ultrafiltration classification method to divide the concentration of the glucan sulfate, and then uses rats as samples to successfully measure the blood coagulation time and the whole blood recalcification time of the prepared glucan sulfate under the condition that different yeast glucan powders participate in the preparation and under different ice bath cooling temperatures, so that the glucan sulfate with better blood coagulation effect can be extracted under the same extraction technology.
Detailed Description
It should be noted that, in the present application, features in embodiments and embodiments may be combined with each other without conflict, and technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The preferred embodiment of the invention provides a method for ultrafiltration fractionation and anticoagulation activity determination of dextran sulfate, which comprises the following steps:
measuring concentrated sulfuric acid and n-butyl alcohol, placing in a three-necked bottle with a drying tube and a stirring device, stirring, cooling to-5 ℃ in an ice bath, and slowly adding 5g of 200-mesh yeast glucan powder;
after reacting for 6min, neutralizing the reaction system with NaOH, centrifuging, dialyzing with distilled water for 24h, concentrating under reduced pressure to 20mL, adding ethanol with volume fraction of 95%, standing, centrifuging, collecting precipitate, dissolving the precipitate in water, dialyzing for 24h, and freeze-drying the dialysate to obtain dextran sulfate;
performing secondary ultrafiltration fractionation on dextran sulfate by adopting polyether sulfone membranes with molecular weight cut-offs of 4K and 10K respectively to obtain 3 fractions;
testing the intrinsic viscosity of the sample with a capillary viscometer;
administering to SD rat for 20min, performing intramuscular injection anesthesia with compound thiamine ketone (10ml/kg), collecting blood from heart, placing in plastic test tube, slowly inclining the test tube for 1 time every 10s after 2min, and recording the liquid level motionless time when the test tube is inclined, i.e. CT;
collecting blood from abdominal artery of rat, adding into test tubes containing 0.1ml of 3.8% sodium citrate, adding sample to be detected into each test tube, incubating in water bath at 37 deg.C for 1min, and adding 0.1ml of 0.01mol/L CaCl into each test tube2After uniformly mixing, timing, and recording the liquid level immobility time when the test tube is inclined, namely RT;
the effect of 3 fractions on the anti-coagulant activity was judged by CT and RT values.
In the embodiment, the ratio of the concentrated sulfuric acid to the n-butanol is 1: 7.
In this embodiment, the second-stage ultrafiltration fractionation is divided into first-stage ultrafiltration membrane fractionation and second-stage ultrafiltration membrane fractionation.
In the first-stage ultrafiltration membrane fractionation in this embodiment, a polyethersulfone membrane with a molecular weight cut-off of 10K is used to divide dextran sulfate into two fractions, i.e., 10K and below.
In the second stage of ultrafiltration fractionation described in this example, a polyethersulfone membrane with a molecular weight cut-off of 4K was used to obtain two fractions of 4-10K and below 4K.
The capillary viscometer described in this example has a volume of 3.5ml with a 140mm length and a 0.5mm internal diameter measurement ball.
The method for measuring the intrinsic viscosity of a sample by capillary viscometry described in this example was as follows: weighing a dried sample, preparing a solution by using distilled water, respectively measuring the sample with the same volume and the distilled water outflow time at the temperature of 20 ℃ by using a capillary viscometer to calculate the relative viscosity eta, and calculating the intrinsic viscosity of the sample by a multipoint dilution and back-pushing method.
In this embodiment, the multi-point dilution backward-pushing method includes: and (3) drawing a curve chart of the relative viscosity eta of the samples with different concentrations and the concentrations, and analyzing the intrinsic viscosity of the samples through the gentle points of the curve chart.
In this example, the number of the SD rats tested was 160, and the number of the male and female rats was half.
Example 2
The preferred embodiment of the invention provides a method for ultrafiltration fractionation and anticoagulation activity determination of dextran sulfate, which comprises the following steps:
measuring concentrated sulfuric acid and n-butyl alcohol, placing in a three-necked bottle with a drying tube and a stirring device, stirring, cooling to-5 ℃ in an ice bath, and slowly adding 6g of 200-mesh yeast glucan powder;
after reacting for 6min, neutralizing the reaction system with NaOH, centrifuging, dialyzing with distilled water for 24h, concentrating under reduced pressure to 20mL, adding ethanol with volume fraction of 95%, standing, centrifuging, collecting precipitate, dissolving the precipitate in water, dialyzing for 24h, and freeze-drying the dialysate to obtain dextran sulfate;
performing secondary ultrafiltration fractionation on dextran sulfate by adopting polyether sulfone membranes with molecular weight cut-offs of 4K and 10K respectively to obtain 3 fractions;
testing the intrinsic viscosity of the sample with a capillary viscometer;
administering to SD rat for 20min, performing intramuscular injection anesthesia with compound thiamine ketone (10ml/kg), collecting blood from heart, placing in plastic test tube, slowly inclining the test tube for 1 time every 10s after 2min, and recording the liquid level motionless time when the test tube is inclined, i.e. CT;
collecting blood from abdominal artery of rat, adding into test tubes containing 0.1ml of 3.8% sodium citrate, adding sample to be detected into each test tube, incubating in water bath at 37 deg.C for 1min, and adding 0.1ml of 0.01mol/L CaCl into each test tube2After uniformly mixing, timing, and recording the liquid level immobility time when the test tube is inclined, namely RT;
the effect of 3 fractions on the anti-coagulant activity was judged by CT and RT values.
In the embodiment, the ratio of the concentrated sulfuric acid to the n-butanol is 1: 7.
In this embodiment, the second-stage ultrafiltration fractionation is divided into first-stage ultrafiltration membrane fractionation and second-stage ultrafiltration membrane fractionation.
In the first-stage ultrafiltration membrane fractionation in this embodiment, a polyethersulfone membrane with a molecular weight cut-off of 10K is used to divide dextran sulfate into two fractions, i.e., 10K and below.
In the second stage of ultrafiltration fractionation described in this example, a polyethersulfone membrane with a molecular weight cut-off of 4K was used to obtain two fractions of 4-10K and below 4K.
The capillary viscometer described in this example has a volume of 3.5ml with a 140mm length and a 0.5mm internal diameter measurement ball.
The method for measuring the intrinsic viscosity of a sample by capillary viscometry described in this example was as follows: weighing a dried sample, preparing a solution by using distilled water, respectively measuring the sample with the same volume and the distilled water outflow time at the temperature of 20 ℃ by using a capillary viscometer to calculate the relative viscosity eta, and calculating the intrinsic viscosity of the sample by a multipoint dilution and back-pushing method.
In this embodiment, the multi-point dilution backward-pushing method includes: and (3) drawing a curve chart of the relative viscosity eta of the samples with different concentrations and the concentrations, and analyzing the intrinsic viscosity of the samples through the gentle points of the curve chart.
In this example, the number of the SD rats tested was 160, and the number of the male and female rats was half.
Example 3
The preferred embodiment of the invention provides a method for ultrafiltration fractionation and anticoagulation activity determination of dextran sulfate, which comprises the following steps:
measuring concentrated sulfuric acid and n-butanol, placing in a three-necked bottle with a drying tube and a stirring device, stirring, cooling to-5 ℃ in an ice bath, and slowly adding 7g of 200-mesh yeast glucan powder;
after reacting for 6min, neutralizing the reaction system with NaOH, centrifuging, dialyzing with distilled water for 24h, concentrating under reduced pressure to 20mL, adding ethanol with volume fraction of 95%, standing, centrifuging, collecting precipitate, dissolving the precipitate in water, dialyzing for 24h, and freeze-drying the dialysate to obtain dextran sulfate;
performing secondary ultrafiltration fractionation on dextran sulfate by adopting polyether sulfone membranes with molecular weight cut-offs of 4K and 10K respectively to obtain 3 fractions;
testing the intrinsic viscosity of the sample with a capillary viscometer;
administering to SD rat for 20min, performing intramuscular injection anesthesia with compound thiamine ketone (10ml/kg), collecting blood from heart, placing in plastic test tube, slowly inclining the test tube for 1 time every 10s after 2min, and recording the liquid level motionless time when the test tube is inclined, i.e. CT;
collecting blood from abdominal artery of rat, adding into test tubes containing 0.1ml of 3.8% sodium citrate, adding sample to be detected into each test tube, incubating in water bath at 37 deg.C for 1min, and adding 0.1ml of 0.01mol/L CaCl into each test tube2After uniformly mixing, timing, and recording the liquid level immobility time when the test tube is inclined, namely RT;
the effect of 3 fractions on the anti-coagulant activity was judged by CT and RT values.
In the embodiment, the ratio of the concentrated sulfuric acid to the n-butanol is 1: 7.
In this embodiment, the second-stage ultrafiltration fractionation is divided into first-stage ultrafiltration membrane fractionation and second-stage ultrafiltration membrane fractionation.
In the first-stage ultrafiltration membrane fractionation in this embodiment, a polyethersulfone membrane with a molecular weight cut-off of 10K is used to divide dextran sulfate into two fractions, i.e., 10K and below.
In the second stage of ultrafiltration fractionation described in this example, a polyethersulfone membrane with a molecular weight cut-off of 4K was used to obtain two fractions of 4-10K and below 4K.
The capillary viscometer described in this example has a volume of 3.5ml with a 140mm length and a 0.5mm internal diameter measurement ball.
The method for measuring the intrinsic viscosity of a sample by capillary viscometry described in this example was as follows: weighing a dried sample, preparing a solution by using distilled water, respectively measuring the sample with the same volume and the distilled water outflow time at the temperature of 20 ℃ by using a capillary viscometer to calculate the relative viscosity eta, and calculating the intrinsic viscosity of the sample by a multipoint dilution and back-pushing method.
In this embodiment, the multi-point dilution backward-pushing method includes: and (3) drawing a curve chart of the relative viscosity eta of the samples with different concentrations and the concentrations, and analyzing the intrinsic viscosity of the samples through the gentle points of the curve chart.
In this example, the number of the SD rats tested was 160, and the number of the male and female rats was half.
Example 4
The preferred embodiment of the invention provides a method for ultrafiltration fractionation and anticoagulation activity determination of dextran sulfate, which comprises the following steps:
measuring concentrated sulfuric acid and n-butyl alcohol, placing in a three-necked bottle with a drying tube and a stirring device, stirring, cooling to-6 ℃ in an ice bath, and slowly adding 5g of 200-mesh yeast glucan powder;
after reacting for 6min, neutralizing the reaction system with NaOH, centrifuging, dialyzing with distilled water for 24h, concentrating under reduced pressure to 20mL, adding ethanol with volume fraction of 95%, standing, centrifuging, collecting precipitate, dissolving the precipitate in water, dialyzing for 24h, and freeze-drying the dialysate to obtain dextran sulfate;
performing secondary ultrafiltration fractionation on dextran sulfate by adopting polyether sulfone membranes with molecular weight cut-offs of 4K and 10K respectively to obtain 3 fractions;
testing the intrinsic viscosity of the sample with a capillary viscometer;
administering to SD rat for 20min, performing intramuscular injection anesthesia with compound thiamine ketone (10ml/kg), collecting blood from heart, placing in plastic test tube, slowly inclining the test tube for 1 time every 10s after 2min, and recording the liquid level motionless time when the test tube is inclined, i.e. CT;
collecting blood from abdominal artery of rat, adding into test tubes containing 0.1ml of 3.8% sodium citrate, adding sample to be detected into each test tube, incubating in water bath at 37 deg.C for 1min, and adding 0.1ml of 0.01mol/L CaCl into each test tube2After uniformly mixing, timing, and recording the liquid level immobility time when the test tube is inclined, namely RT;
the effect of 3 fractions on the anti-coagulant activity was judged by CT and RT values.
In the embodiment, the ratio of the concentrated sulfuric acid to the n-butanol is 1: 7.
In this embodiment, the second-stage ultrafiltration fractionation is divided into first-stage ultrafiltration membrane fractionation and second-stage ultrafiltration membrane fractionation.
In the first-stage ultrafiltration membrane fractionation in this embodiment, a polyethersulfone membrane with a molecular weight cut-off of 10K is used to divide dextran sulfate into two fractions, i.e., 10K and below.
In the second stage of ultrafiltration fractionation described in this example, a polyethersulfone membrane with a molecular weight cut-off of 4K was used to obtain two fractions of 4-10K and below 4K.
The capillary viscometer described in this example has a volume of 3.5ml with a 140mm length and a 0.5mm internal diameter measurement ball.
The method for measuring the intrinsic viscosity of a sample by capillary viscometry described in this example was as follows: weighing a dried sample, preparing a solution by using distilled water, respectively measuring the sample with the same volume and the distilled water outflow time at the temperature of 20 ℃ by using a capillary viscometer to calculate the relative viscosity eta, and calculating the intrinsic viscosity of the sample by a multipoint dilution and back-pushing method.
In this embodiment, the multi-point dilution backward-pushing method includes: and (3) drawing a curve chart of the relative viscosity eta of the samples with different concentrations and the concentrations, and analyzing the intrinsic viscosity of the samples through the gentle points of the curve chart.
In this example, the number of the SD rats tested was 160, and the number of the male and female rats was half.
Example 5
The preferred embodiment of the invention provides a method for ultrafiltration fractionation and anticoagulation activity determination of dextran sulfate, which comprises the following steps:
measuring concentrated sulfuric acid and n-butanol, placing in a three-necked bottle with a drying tube and a stirring device, stirring, cooling to-7 ℃ in an ice bath, and slowly adding 5g of 200-mesh yeast glucan powder;
after reacting for 6min, neutralizing the reaction system with NaOH, centrifuging, dialyzing with distilled water for 24h, concentrating under reduced pressure to 20mL, adding ethanol with volume fraction of 95%, standing, centrifuging, collecting precipitate, dissolving the precipitate in water, dialyzing for 24h, and freeze-drying the dialysate to obtain dextran sulfate;
performing secondary ultrafiltration fractionation on dextran sulfate by adopting polyether sulfone membranes with molecular weight cut-offs of 4K and 10K respectively to obtain 3 fractions;
testing the intrinsic viscosity of the sample with a capillary viscometer;
administering to SD rat for 20min, performing intramuscular injection anesthesia with compound thiamine ketone (10ml/kg), collecting blood from heart, placing in plastic test tube, slowly inclining the test tube for 1 time every 10s after 2min, and recording the liquid level motionless time when the test tube is inclined, i.e. CT;
collecting blood from abdominal artery of rat, adding into test tubes containing 0.1ml of 3.8% sodium citrate, adding sample to be detected into each test tube, incubating in water bath at 37 deg.C for 1min, and adding 0.1ml of 0.01mol/L CaCl into each test tube2After uniformly mixing, timing, and recording the liquid level immobility time when the test tube is inclined, namely RT;
the effect of 3 fractions on the anti-coagulant activity was judged by CT and RT values.
In the embodiment, the ratio of the concentrated sulfuric acid to the n-butanol is 1: 7.
In this embodiment, the second-stage ultrafiltration fractionation is divided into first-stage ultrafiltration membrane fractionation and second-stage ultrafiltration membrane fractionation.
In the first-stage ultrafiltration membrane fractionation in this embodiment, a polyethersulfone membrane with a molecular weight cut-off of 10K is used to divide dextran sulfate into two fractions, i.e., 10K and below.
In the second stage of ultrafiltration fractionation described in this example, a polyethersulfone membrane with a molecular weight cut-off of 4K was used to obtain two fractions of 4-10K and below 4K.
The capillary viscometer described in this example has a volume of 3.5ml with a 140mm length and a 0.5mm internal diameter measurement ball.
The method for measuring the intrinsic viscosity of a sample by capillary viscometry described in this example was as follows: weighing a dried sample, preparing a solution by using distilled water, respectively measuring the sample with the same volume and the distilled water outflow time at the temperature of 20 ℃ by using a capillary viscometer to calculate the relative viscosity eta, and calculating the intrinsic viscosity of the sample by a multipoint dilution and back-pushing method.
In this embodiment, the multi-point dilution backward-pushing method includes: and (3) drawing a curve chart of the relative viscosity eta of the samples with different concentrations and the concentrations, and analyzing the intrinsic viscosity of the samples through the gentle points of the curve chart.
In this example, the number of the SD rats tested was 160, and the number of the male and female rats was half.
Example 6
The preferred embodiment of the invention provides a method for ultrafiltration fractionation and anticoagulation activity determination of dextran sulfate, which comprises the following steps:
measuring concentrated sulfuric acid and n-butanol, placing in a three-necked bottle with a drying tube and a stirring device, stirring, cooling to-8 ℃ in an ice bath, and slowly adding 5g of 200-mesh yeast glucan powder;
after reacting for 6min, neutralizing the reaction system with NaOH, centrifuging, dialyzing with distilled water for 24h, concentrating under reduced pressure to 20mL, adding ethanol with volume fraction of 95%, standing, centrifuging, collecting precipitate, dissolving the precipitate in water, dialyzing for 24h, and freeze-drying the dialysate to obtain dextran sulfate;
performing secondary ultrafiltration fractionation on dextran sulfate by adopting polyether sulfone membranes with molecular weight cut-offs of 4K and 10K respectively to obtain 3 fractions;
testing the intrinsic viscosity of the sample with a capillary viscometer;
administering to SD rat for 20min, performing intramuscular injection anesthesia with compound thiamine ketone (10ml/kg), collecting blood from heart, placing in plastic test tube, slowly inclining the test tube for 1 time every 10s after 2min, and recording the liquid level motionless time when the test tube is inclined, i.e. CT;
collecting blood from abdominal artery of rat, adding into test tubes containing 0.1ml of 3.8% sodium citrate, adding sample to be detected into each test tube, incubating in water bath at 37 deg.C for 1min, and adding 0.1ml of 0.01mol/L CaCl into each test tube2After uniformly mixing, timing, and recording the liquid level immobility time when the test tube is inclined, namely RT;
the effect of 3 fractions on the anti-coagulant activity was judged by CT and RT values.
In the embodiment, the ratio of the concentrated sulfuric acid to the n-butanol is 1: 7.
In this embodiment, the second-stage ultrafiltration fractionation is divided into first-stage ultrafiltration membrane fractionation and second-stage ultrafiltration membrane fractionation.
In the first-stage ultrafiltration membrane fractionation in this embodiment, a polyethersulfone membrane with a molecular weight cut-off of 10K is used to divide dextran sulfate into two fractions, i.e., 10K and below.
In the second stage of ultrafiltration fractionation described in this example, a polyethersulfone membrane with a molecular weight cut-off of 4K was used to obtain two fractions of 4-10K and below 4K.
The capillary viscometer described in this example has a volume of 3.5ml with a 140mm length and a 0.5mm internal diameter measurement ball.
The method for measuring the intrinsic viscosity of a sample by capillary viscometry described in this example was as follows: weighing a dried sample, preparing a solution by using distilled water, respectively measuring the sample with the same volume and the distilled water outflow time at the temperature of 20 ℃ by using a capillary viscometer to calculate the relative viscosity eta, and calculating the intrinsic viscosity of the sample by a multipoint dilution and back-pushing method.
In this embodiment, the multi-point dilution backward-pushing method includes: and (3) drawing a curve chart of the relative viscosity eta of the samples with different concentrations and the concentrations, and analyzing the intrinsic viscosity of the samples through the gentle points of the curve chart.
In this example, the number of the SD rats tested was 160, and the number of the male and female rats was half.
The present invention performed experiments on CT (rat clotting time) and RT (whole blood recalcification time) values of 3 fractions F1 (molecular weight cut-off > 10K), F2 (molecular weight cut-off 4K-10K), F3 (molecular weight cut-off < 4K) in the above 6 examples, respectively, and the results are shown in the following table:
as can be seen from the data in the table above, in the range of 5-7g yeast glucan powder, every 1g yeast glucan powder is added, the CT value is obviously increased, the RT value is also obviously increased, and every 1 ℃ reduction in ice bath cooling in the range of-5 to-8 ℃, the CT value is obviously increased, but the RT value is shortened.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (9)
1. A method for ultrafiltration fractionation and anticoagulation activity determination of dextran sulfate comprises the following steps:
measuring concentrated sulfuric acid and n-butyl alcohol, placing the concentrated sulfuric acid and the n-butyl alcohol into a three-necked bottle with a drying tube and a stirring device, stirring, cooling to minus 5 to minus 8 ℃ in an ice bath, and slowly adding 5 to 7g of 200-mesh yeast glucan powder;
after reacting for 6-8min, neutralizing the reaction system with NaOH, centrifuging, dialyzing with distilled water for 18-24h, concentrating under reduced pressure to 20-22mL, adding ethanol with volume fraction of 95%, standing, centrifuging, collecting precipitate, dissolving the precipitate in water, dialyzing for 18-24h, and freeze-drying the dialysate to obtain dextran sulfate;
performing secondary ultrafiltration fractionation on dextran sulfate by adopting polyether sulfone membranes with molecular weight cut-offs of 4K and 10K respectively to obtain 3 fractions;
testing the intrinsic viscosity of the sample with a capillary viscometer;
administering to SD rat for 20-60min, performing intramuscular injection anesthesia with compound thiamine ketone (10ml/kg), collecting blood from heart, slowly inclining the tube for 1 time every 10s after 1-2min, and recording the liquid surface motionless time when the tube is inclined, i.e. CT;
collecting blood from abdominal artery of rat, adding into test tubes containing 0.1ml of 3.8% sodium citrate, adding sample to be tested into each test tube, incubating in water bath at 37-38 deg.C for 1min, and adding 0.1ml of 0.01mol/L CaCl into each test tube2After uniformly mixing, timing, and recording the liquid level immobility time when the test tube is inclined, namely RT;
the effect of 3 fractions on the anti-coagulant activity was judged by CT and RT values.
2. The method for ultrafiltration fractionation of dextran sulfate according to claim 1, characterized in that: the ratio of the concentrated sulfuric acid to the n-butyl alcohol is 1: 7-9.
3. The method for ultrafiltration fractionation of dextran sulfate according to claim 1, characterized in that: and the second-stage ultrafiltration fractionation is divided into first-stage ultrafiltration membrane fractionation and second-stage ultrafiltration membrane fractionation.
4. The method for ultrafiltration fractionation of dextran sulfate according to claim 3, characterized in that: and the first-stage ultrafiltration membrane is used for classifying dextran sulfate into two fractions of more than 10K and less than 10K by adopting a polyether sulfone membrane with the molecular weight cutoff of 10K.
5. The method for ultrafiltration fractionation of dextran sulfate according to claim 3, characterized in that: and the second-stage ultrafiltration and fractionation adopts a polyether sulfone membrane with the molecular weight cutoff of 4K to obtain two fractions of 4-10K and below 4K.
6. The method for ultrafiltration fractionation of dextran sulfate according to claim 1, characterized in that: the capillary viscometer has a length of 140 plus or minus 5mm and an inner diameter of 0.5 plus or minus 0.05mm, and the volume of the measuring ball is 3.5 plus or minus 0.5 ml.
7. The method for ultrafiltration fractionation of dextran sulfate according to claim 1, characterized in that: the method for measuring the intrinsic viscosity of the sample by the capillary viscometry comprises the following steps: weighing dried samples, preparing a solution by using distilled water, respectively measuring the samples with the same volume and the flowing-out time of the distilled water at the temperature of 20-30 ℃ by using a capillary viscometer to calculate the relative viscosity eta, and calculating the intrinsic viscosity of the samples by a multipoint dilution and back-pushing method.
8. The method for ultrafiltration fractionation of dextran sulfate according to claim 7, characterized in that: the multi-point dilution backward-pushing method comprises the following steps: and (3) drawing a curve chart of the relative viscosity eta of the samples with different concentrations and the concentrations, and analyzing the intrinsic viscosity of the samples through the gentle points of the curve chart.
9. The method for ultrafiltration fractionation of dextran sulfate according to claim 1, characterized in that: the number of the SD rats is 160-190, and the number of the male rats and the female rats is half.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110423927.6A CN113512130A (en) | 2021-04-20 | 2021-04-20 | Ultra-filtration classification and anticoagulant activity determination method of dextran sulfate |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110423927.6A CN113512130A (en) | 2021-04-20 | 2021-04-20 | Ultra-filtration classification and anticoagulant activity determination method of dextran sulfate |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113512130A true CN113512130A (en) | 2021-10-19 |
Family
ID=78061471
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110423927.6A Pending CN113512130A (en) | 2021-04-20 | 2021-04-20 | Ultra-filtration classification and anticoagulant activity determination method of dextran sulfate |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113512130A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB873995A (en) * | 1959-03-31 | 1961-08-02 | Ohio Commw Eng Co | Sulfuric acid esters of dextranic acid |
| WO2017008768A1 (en) * | 2015-07-14 | 2017-01-19 | 青岛海大海洋生物医药销售有限公司 | Glucan and preparation method thereof, and application in preparation of immune-enhancing and antitumor medicine and functional food |
-
2021
- 2021-04-20 CN CN202110423927.6A patent/CN113512130A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB873995A (en) * | 1959-03-31 | 1961-08-02 | Ohio Commw Eng Co | Sulfuric acid esters of dextranic acid |
| WO2017008768A1 (en) * | 2015-07-14 | 2017-01-19 | 青岛海大海洋生物医药销售有限公司 | Glucan and preparation method thereof, and application in preparation of immune-enhancing and antitumor medicine and functional food |
Non-Patent Citations (2)
| Title |
|---|
| 师少飞 等: "葡聚糖硫酸酯的超滤分级及其抗凝血活性研究", 《时珍国医国药》 * |
| 王庆华 等: "酵母葡聚糖硫酸酯的制备及抗凝血活性", 《食品研究与开发》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107106595B (en) | Novel dextran sulfate | |
| WO2017032277A1 (en) | Bovine lung enoxaparin sodium, preparation method therefor, and application thereof | |
| Kopeček et al. | New types of synthetic infusion solutions. I. Investigation of the effect of solutions of some hydrophilic polymers on blood | |
| CN103336110B (en) | Whole blood quality control material and preparation method thereof | |
| Le Grimellec et al. | 3 H-Inulin and electrolyte concentrations in Bowman's capsule in rat kidney: Comparison with artificial ultrafiltration | |
| CN112180095A (en) | Cardiovascular and cerebrovascular and diabetes related four-high-index composite quality control product and preparation method thereof | |
| CN105037577A (en) | Procoagulant blackberry seed polysaccharide, and extraction separation method and application thereof | |
| CN105693935B (en) | A kind of hydroxyethyl cellulose modified object of bisexual ion purification and preparation method thereof and purposes | |
| CN103285031A (en) | Application of depolymerized holothurian glycosaminolycan in preparation of medicine for preventing and treating thromboembolism diseases | |
| CN105237657A (en) | Preparation method for low-molecular heparin originated from new species | |
| CN113512130A (en) | Ultra-filtration classification and anticoagulant activity determination method of dextran sulfate | |
| Samachson et al. | Comparative ultrafiltration of calcium and strontium in serum. | |
| Hildebrand et al. | Distribution and Particle Size of Type A Botulinum Toxin in Body Fluids of Intravenously Injected Rabbits. | |
| CN106432548A (en) | Thiol-ene click chemistry based preparation and characterization of fatty acid modified heparin | |
| WO2023123994A1 (en) | Sodium carboxymethyl starch and injection thereof | |
| JP2024505364A (en) | Method and use thereof for detecting the content of carboxylated glycosaminoglycan derivatives in a sample | |
| CN110441435A (en) | A kind of measuring method of oversulfated chondroitin sulfate in sodium heparin content | |
| Ege et al. | The distribution of glucose between human blood plasma and red corpuscles and the rapidity of its penetration | |
| Ricketts | Chemistry of dextran and its derivatives | |
| CN106749710B (en) | Narrow-distribution hydroxyethyl starch and application thereof | |
| JP2010082068A (en) | Blood circuit and circulation system for evaluation and inspection of blood compatibility | |
| Sutton | Studies on blood coagulation and the effect of digitalis | |
| CN111122802A (en) | Method for measuring release curve of propofol fat emulsion injection | |
| CN109521201A (en) | A kind of source of people whole blood matrix quality-control product for portable glucose meter | |
| Mitchell et al. | Histamine and granulocytes in the umbilical cord blood of infants at birth |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20211019 |