Titer calibration method of sea sodium polysaccharide

Abstract

The invention relates to the technical field of medicines, and provides a titer calibration method of sea sodium polysaccharide. According to the method, a p-nitroaniline color development method is adopted to test the activity curve of the sodium polysaccharide, the abscissa of the activity curve of the sodium polysaccharide is the reciprocal of the weight of the sodium polysaccharide, and the ordinate of the activity curve of the sodium polysaccharide is the logarithm of the residual activity unit of the FVIII; the weight of the natriuretic polysaccharide for inhibiting the activity of iFXase (endogenous blood coagulation factor X enzyme) formed by 1IU FVIII is taken as one titer unit, and the titer of the natriuretic polysaccharide is obtained according to an activity curve of the natriuretic polysaccharide. The titer calibration method provided by the invention can be used for accurately calibrating the anticoagulant activity intensity of the sea sodium polysaccharide, and has important significance in the aspects of guaranteeing the drug curative effect, medication safety, quality control and the like.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and in particular to a method for calibrating the potency of sodium thunbergii polysaccharide. Background Art

[0002] The body wall of sea cucumber contains abundant polysaccharides, mainly including two categories: fucosylated chondroitin sulfate and fucosylated chondroitin sulfate. Among them, sea cucumber fucosylated chondroitin sulfate has rich biological activities due to its unique structure, such as lowering blood lipids, anti-tumor, anti-virus, anti-inflammatory, etc., especially good anticoagulant and anti-thrombotic activities.

[0003] The potency of a drug refers to the dose or concentration required for the drug to achieve a certain effect. It is an important indicator for measuring the strength of a drug's action, reflecting the drug's affinity for receptors and intrinsic activity. Different batches of drugs, even if produced by the same manufacturer, may have different actual activities due to factors such as production process and raw material sources. Potency calibration can accurately determine the activity strength of a drug, ensuring that in clinical use, doctors can formulate appropriate dosages based on accurate potency, so that patients can receive effective treatment.

[0004] Sodium thunbergii polysaccharide is a fucosylated osteoin sulfate natural polysaccharide extracted from the body wall of sea cucumbers. It can target the endogenous coagulation pathway and has good anticoagulant activity. Currently, there is no marketed drug with the same target. It is necessary to establish a potency calibration method for sodium thunbergii polysaccharide standards to accurately determine the activity intensity of sodium thunbergii polysaccharide. Summary of the invention

[0005] In view of this, the present invention provides a method for calibrating the potency of sodium thunbergii polysaccharide. The potency calibration method provided by the present invention can accurately calibrate the anticoagulant activity intensity of sodium thunbergii polysaccharide, which is of great significance in ensuring drug efficacy, drug safety and quality control.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] A method for calibrating the potency of sodium thunbergii polysaccharide comprises the following steps:

[0008] The activity curve of sodium hyaluronate polysaccharide was obtained by p-nitroaniline colorimetric method, wherein the abscissa of the sodium hyaluronate polysaccharide activity curve is the reciprocal of the weight of sodium hyaluronate polysaccharide, and the ordinate is the logarithm of the residual activity unit of FVIII;

[0009] The weight of the sodium hyaluronate polysaccharide that inhibits the iFXase (endogenous coagulation factor X enzyme) activity formed by 1IU FVIII is taken as 1 potency unit, and the potency of the sodium hyaluronate polysaccharide is obtained according to the sodium hyaluronate polysaccharide activity curve;

[0010] The sodium thunbergii polysaccharide is a fucosylated chondroitin sulfate polysaccharide; the weight average molecular weight of the sodium thunbergii polysaccharide is 90,000 to 130,000, the monosaccharide composition includes glucuronic acid, N-acetylgalactosamine and fucose, the molar ratio of glucuronic acid, N-acetylgalactosamine and fucose is 1:0.8 to 1.2:0.5 to 0.8; the mass percentage of sulfate groups in the sodium thunbergii polysaccharide is 25 to 40%.

[0011] Preferably, the method for obtaining the activity curve of the sodium thunbergii polysaccharide comprises:

[0012] Prepare sodium thunbergii polysaccharide working solution with gradient concentrations;

[0013] The sodium thiamethoxam polysaccharide working solution, the FVIII solution and the activation reagent solution are mixed for a first incubation to obtain a first incubation solution; the activation reagent solution comprises FIXa, FIIa, phospholipids, a buffer solution and CaCl2;

[0014] mixing the first incubation solution and the FX solution for a second incubation to obtain a second incubation solution;

[0015] The second incubation solution and the chromogenic substrate solution are mixed for a third incubation, and then a stop solution is added, and the absorbance of the resulting mixed solution at 405 nm is tested to obtain an absorbance value; the chromogenic substrate is SXa-11;

[0016] Substitute the absorbance value into the FVIII complex activity standard curve, calculate the FVIII residual activity unit, and draw the sodium hyaluronate activity curve with the reciprocal of the sodium hyaluronate weight as the abscissa and the logarithm of the FVIII residual activity unit as the ordinate; the abscissa of the FVIII complex activity standard curve is the logarithm of the FVIII concentration, and the ordinate is the absorbance value.

[0017] Preferably, the first incubation temperature is 37° C., and the incubation time is 5 to 20 minutes.

[0018] Preferably, the second incubation temperature is 37° C., and the incubation time is 1 to 10 min.

[0019] Preferably, the temperature of the third incubation is 37° C., and the incubation time is 10 to 30 minutes.

[0020] Preferably, the stop solution is a citric acid solution, and the concentration of the citric acid solution is 0.01 to 0.1 g / mL.

[0021] Preferably, the concentration range of the sodium thunbergii polysaccharide working solution is 328-500 ng / mL.

[0022] Preferably, the buffer in the activation reagent solution is Tris-HCl.

[0023] Preferably, the method for obtaining the FVIII complex activity standard curve comprises:

[0024] Prepare FVIII standard solutions with gradient concentrations;

[0025] The FVIII standard solution, water and an activation reagent solution are mixed for a fourth incubation to obtain a fourth incubation solution; the activation reagent solution comprises FIXa, FIIa, phospholipids, a buffer solution and CaCl2;

[0026] mixing the fourth incubation solution and the FX solution for a fifth incubation to obtain a fifth incubation solution;

[0027] The fifth incubation solution and the chromogenic substrate solution are mixed for the sixth incubation, and then a stop solution is added, and the absorbance of the resulting mixed solution at 405 nm is tested to obtain an absorbance value; the chromogenic substrate is SXa-11;

[0028] The FVIII complex activity standard curve was drawn with the logarithm of the FVIII concentration as the abscissa and the absorbance value as the ordinate.

[0029] Preferably, the concentration of the FVIII standard solution is 0.95-3 IU / mL.

[0030] The present invention provides a titer calibration method of sodium thiocyanate polysaccharide, comprising the following steps: using a p-nitroaniline colorimetric method to obtain a sodium thiocyanate polysaccharide activity curve, wherein the abscissa of the sodium thiocyanate polysaccharide activity curve is the reciprocal of the sodium thiocyanate polysaccharide weight, and the ordinate is the logarithm of the FVIII residual activity unit; the weight of sodium thiocyanate polysaccharide that inhibits the iFXase (endogenous coagulation factor X enzyme) activity formed by 1IU FVIII is taken as 1 titer unit, and the titer of sodium thiocyanate polysaccharide is obtained according to the sodium thiocyanate polysaccharide activity curve. The pharmacological effect of sodium thiocyanate polysaccharide is mainly a non-heparin-like anticoagulant drug, and its mechanism of action is to selectively inhibit the terminal rate-limiting enzyme "endogenous factor X enzyme (FIXa-FVIIIa-PL-Ca 2+The present invention performs potency calibration by measuring the activity of sea sodium polysaccharide in inhibiting iFXase (endogenous coagulation factor X enzyme). In the presence of phospholipids (PLP) and calcium ions, FVIII is activated by thrombin to form FVIIIa, FVIIIa, FIXa, phospholipids and calcium ions form iFXase (endogenous coagulation factor X enzyme), iFXase (endogenous coagulation factor X enzyme) can activate FX factor to form FXa, FXa can hydrolyze the chromogenic substrate SXa-11 to release p-nitroaniline (pNA), pNA has light absorption at 405nm, and the amount of pNA is proportional to the absorbance at 405nm. Based on the above principle, the present invention adopts p-nitroaniline colorimetric method to test the activity curve of sea sodium polysaccharide, and the inhibition of 1IU The weight of the sodium hyaluronate polysaccharide with iFXase (endogenous coagulation factor X enzyme) activity formed by FVIII is defined as 1 potency unit, and the potency of sodium hyaluronate polysaccharide is calculated by the sodium hyaluronate polysaccharide activity curve. The potency calibration method provided by the present invention can accurately calibrate the anticoagulant activity intensity of sodium hyaluronate polysaccharide, which is of great significance in ensuring drug efficacy, drug safety and quality control. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the detection mechanism of the FVIII activity assay kit;

[0032] Figure 2 This is the FVIII complex activity standard curve obtained by experimenter A in the first experiment;

[0033] Figure 3 This is the activity curve of sodium thiamethoxam polysaccharide obtained by experimenter A in the first experiment;

[0034] Figure 4 This is the FVIII complex activity standard curve obtained by experimenter A in the second experiment;

[0035] Figure 5 This is the activity curve of sodium thiamethoxam polysaccharide obtained by experimenter A in the second experiment;

[0036] Figure 6 This is the FVIII complex activity standard curve obtained by experimenter B in the first experiment;

[0037] Figure 7 This is the activity curve of sodium thunbergii polysaccharide obtained by experimenter B in the first experiment;

[0038] Figure 8 This is the FVIII complex activity standard curve obtained by experimenter B in the second experiment;

[0039] Fig. 9 This is the activity curve of sodium thunbergii polysaccharide obtained by experimenter B in the second experiment. DETAILED DESCRIPTION

[0040] First, sodium thunbergii polysaccharide will be described.

[0041] In the present invention, the sea sodium polysaccharide refers to a natural polysaccharide extracted from the body wall of a sea cucumber, and the sea cucumber includes but is not limited to one or more of the sea cucumbers of the jade foot, rough sea cucumber, sea cucumber, black sea cucumber and black milk sea cucumber, preferably the sea cucumber of the jade foot (Latin name: Holothurialeucospilota (Brandt)); the sea sodium polysaccharide is a fucosylated chondroitin sulfate polysaccharide; the weight average molecular weight of the sea sodium polysaccharide is 90,000 to 130,000, the polydispersity index is ≤2, the monosaccharide composition includes glucuronic acid, N-acetylgalactose and fucose, and the molar ratio of glucuronic acid, N-acetylgalactose and fucose is 1:0.8 to 1.2:0.5 to 0.8; the mass percentage of sulfate groups in the sea sodium polysaccharide is 25 to 40%.

[0042] The structural unit of sodium thunbergii polysaccharide is shown in Formula I:

[0043]

[0044] In formula I: R1 is H, SO3 - or fucosyl, R2 is H or SO3 - , R3 is H or SO3 - , R4 is H or fucosyl; at least one of R1 and R4 is fucosyl; X ⊕ represents a cation;

[0045] The structure of the fucosyl in R1 and R4 is shown in Formula I-1:

[0046]

[0047] In formula I-1: R5 is H or SO 3- , R6 is H or SO 3- , R7 is H or SO 3- .

[0048] The cation in formula I is a hydrogen ion, an alkali metal ion, an alkaline earth metal ion, an ammonium ion or an iron ion, specifically a hydrogen ion, a potassium ion, a sodium ion, a calcium ion, an ammonium ion or an iron ion, preferably a sodium ion; when the cation is a sodium ion, the mass percentage of the sodium ion in the sodium ion polysaccharide is 5 to 15%.

[0049] Sodium chondroitin sulfate polysaccharide is a natural fucosylated chondroitin sulfate polysaccharide extracted by the inventor from the body wall of sea cucumbers. It can target the terminal rate-limiting enzyme iFXase in the endogenous coagulation pathway, has good anticoagulant effect, and has little bleeding side effects, achieving the effect of anticoagulation and no bleeding.

[0050] The results of the evaluation of the anticoagulant activity of sodium hyaluronate polysaccharide on mice showed that sodium hyaluronate polysaccharide can significantly prolong the APTT coagulation time of mice and rats, without affecting the PT and TT coagulation times. The above research results indicate that sodium hyaluronate polysaccharide targets the intrinsic coagulation pathway and does not affect the extrinsic coagulation pathway.

[0051] Referring to the heparin bioassay method in General Chapter 1208 of the Pharmacopoeia, the activity of sodium hyaluronate polysaccharide against factor IIa, factor Xa, ATⅢ-Ⅱa, and ATⅢ-Xa was tested. The results showed that at an experimental dose of 500 μg / mL, sodium hyaluronate polysaccharide showed no activity against factor IIa, factor Xa, and ATⅢ-Ⅱa, and only had a weak effect on ATⅢ-Xa (inhibition rate of 36.7%). The activity of the rate-limiting enzyme of the endogenous coagulation pathway, "intrinsic factor coagulation factor X enzyme (iFXase)", was tested by ELISA. The results showed that the test drug could significantly inhibit the rate-limiting enzyme iFXase of the endogenous coagulation pathway, IC 50 The value is 207.5ng / mL; the above experiment further clarified from the mechanism that sodium thiazolinone polysaccharide can selectively inhibit the activity of iFXase, the rate-limiting enzyme in the intrinsic coagulation pathway, and target the intrinsic coagulation pathway.

[0052] The calibration method of the present invention is specifically a method for calibrating the potency of a sodium hyaluronate standard. The potency calibration method of the present invention is described in detail below.

[0053] The present invention provides a method for calibrating the potency of sodium thunbergii polysaccharide, comprising the following steps:

[0054] The activity curve of sodium hyaluronate polysaccharide was obtained by p-nitroaniline colorimetric method, wherein the abscissa of the sodium hyaluronate polysaccharide activity curve is the reciprocal of the weight of sodium hyaluronate polysaccharide, and the ordinate is the logarithm of the residual activity unit of FVIII;

[0055] The weight of sodium hyaluronate polysaccharide that inhibits the activity of iFXase (endogenous coagulation factor X enzyme) formed by 1IU FVIII is taken as 1 potency unit, and the potency of sodium hyaluronate polysaccharide is obtained according to the activity curve of sodium hyaluronate polysaccharide.

[0056] In the present invention, the method for obtaining the activity curve of the sodium thunbergii polysaccharide comprises:

[0057] Prepare sodium thunbergii polysaccharide working solution with gradient concentrations;

[0058] The sodium thiamethoxam polysaccharide working solution, the FVIII solution and the activation reagent solution are mixed for a first incubation to obtain a first incubation solution; the activation reagent solution comprises FIXa, FIIa, phospholipids, a buffer solution and CaCl2;

[0059] mixing the first incubation solution and the FX solution for a second incubation to obtain a second incubation solution;

[0060] The second incubation solution and the chromogenic substrate solution are mixed for a third incubation, and then a stop solution is added, and the absorbance of the resulting mixed solution at 405 nm is tested to obtain an absorbance value; the chromogenic substrate is SXa-11;

[0061] Substitute the absorbance value into the FVIII complex activity standard curve, calculate the FVIII residual activity unit, and draw a standard curve with the reciprocal of the weight of sodium hyaluronate as the abscissa and the logarithm of the FVIII residual activity unit as the ordinate; the abscissa of the FVIII complex activity standard curve is the logarithm of the FVIII concentration, and the ordinate is the absorbance value.

[0062] The kit used in the present invention for testing the activity curve of sodium thiazolyl glycol by a p-nitroaniline colorimetric method is a FVIII activity assay kit (specification: BIOPHEN FVllI:C), wherein the reagents in the FVIII activity assay kit include R1, R2, R3 and R4, wherein R1 is FX, R2 is an activation reagent, and the components include FIXa, FIIa, phospholipids, a buffer and CaCl2, wherein the buffer is Tris-HCl; R3 is a colorimetric substrate SXa-11, and R4 is a Tris-BSA Buffer, which is used for diluting a FVIII standard solution.

[0063] In the present invention, the preparation method of each solution used in the p-nitroaniline color development method is as follows:

[0064] Ultrapure water is added to R1, R2 and R3 respectively to obtain FX solution, activation reagent solution and chromogenic substrate solution; the concentration of the FX solution is preferably 40-60nM, more preferably 50nM, the concentration of FIXa in the activation reagent solution is preferably 40-80nM, more preferably 60nM, the concentration of FIIa is preferably 30-60nM, more preferably 47nM, the concentration of phospholipid is preferably 0.1-0.6mg / mL, more preferably 0.32mg / mL, the concentration of CaCl2 is preferably 5-15mM, more preferably 12mM; the concentration of the chromogenic substrate solution is preferably 4-12mM, more preferably 8.4mM; R4 is used to dissolve and dilute FVIII to obtain a FVIII solution.

[0065] The present invention first prepares a gradient concentration of sodium thunbergii polysaccharide working solution. In the present invention, the concentration range of the sodium thunbergii polysaccharide working solution is preferably 328-500 ng / mL, preferably 328.05-500 ng / mL, specifically 328.05 ng / mL, 364.5 ng / mL, 405 ng / mL, 450 ng / mL, 500 ng / mL; the solvent for preparing the sodium thunbergii polysaccharide working solution is preferably water, and the water is preferably double distilled water (ddH2O).

[0066] After obtaining the sodium hyaluronate working solution, the present invention mixes the sodium hyaluronate working solution, FVIII solution and activation reagent solution for the first incubation to obtain the first incubation solution; the components of the activation reagent solution include FIXa, FIIa, phospholipids, buffer and CaCl2. In the present invention, the concentration of the FVIII solution is preferably 3IU / mL; the volume ratio of the sodium hyaluronate working solution and the FVIII solution is preferably 1:1; the volume ratio of the sodium hyaluronate working solution and the activation reagent solution is preferably 1:1; the temperature of the first incubation is preferably 37°C, and the incubation time is preferably 5 to 20 minutes, specifically 15 minutes. During the first incubation process, in the presence of phospholipids (PLP) and calcium ions, FVIII is activated by FIIa to form FVIIIa, and FVIIIa, FIXa, phospholipids and calcium ions form endogenous factor X enzyme (iFXase).

[0067] After obtaining the first incubation solution, the present invention mixes the first incubation solution and the FX solution for a second incubation to obtain a second incubation solution. In the present invention, the volume ratio of the sodium thiamethoxam working solution and the FX solution is preferably 1:1; the temperature of the second incubation is 37°C, and the incubation time is 1 to 10 minutes, preferably 1 minute. During the second incubation process, iFXase (endogenous coagulation factor X enzyme) activates FX to form FXa. At the same time, due to the presence of sodium thiamethoxam polysaccharide, the activity of part of iFXase (endogenous coagulation factor X enzyme) is inhibited and FX cannot be activated.

[0068] After obtaining the second incubation solution, the present invention mixes the second incubation solution and the chromogenic substrate solution for the third incubation, then adds the stop solution, tests the absorbance of the obtained mixed solution at 405nm, and obtains the absorbance value. In the present invention, the chromogenic substrate is SXa-11; the volume ratio of the sea sodium polysaccharide working solution and the chromogenic substrate solution is preferably 1:1; the temperature of the third incubation is preferably 37°C, and the incubation time is preferably 10 to 30min, specifically 15min; the stop solution is preferably a citric acid solution, and the concentration of the citric acid solution is preferably 0.01 to 0.1g / mL, specifically 0.02g / mL; the volume ratio of the sea sodium polysaccharide working solution and the stop solution is preferably 2:3. The instrument used to test the absorbance is preferably an enzyme marker. During the third incubation process, FXa hydrolyzes the chromogenic substrate SXa-11 to release p-nitroaniline (pNA), pNA has light absorption at 405nm, and the amount of pNA is proportional to the absorbance value at 405nm.

[0069] After obtaining the absorbance value, the present invention substitutes the absorbance value into the FVIII complex activity standard curve, calculates the FVIII residual activity unit, and draws the sodium hyaluronate activity curve with the reciprocal of the sodium hyaluronate weight as the abscissa and the logarithm of the FVIII residual activity unit as the ordinate; in the present invention, after obtaining the sodium hyaluronate activity curve, it also includes obtaining a fitting equation according to the sodium hyaluronate activity curve.

[0070] In the present invention, the abscissa of the FVIII complex activity standard curve is the logarithm of the FVIII concentration, and the ordinate is the absorbance.

[0071] In the present invention, the method for obtaining the FVIII complex activity standard curve preferably comprises:

[0072] Prepare FVIII standard solutions with gradient concentrations;

[0073] The FVIII standard solution, water and an activation reagent solution are mixed for a fourth incubation to obtain a fourth incubation solution; the activation reagent solution comprises FIXa, FIIa, phospholipids, a buffer solution and CaCl2;

[0074] mixing the fourth incubation solution and the FX solution for a fifth incubation to obtain a fifth incubation solution;

[0075] The fifth incubation solution and the chromogenic substrate solution are mixed for the sixth incubation, and then a stop solution is added, and the absorbance of the resulting mixed solution at 405 nm is tested to obtain an absorbance value; the chromogenic substrate is SXa-11;

[0076] The FVIII complex activity standard curve was drawn with the logarithm of the FVIII concentration as the abscissa and the absorbance value as the ordinate.

[0077] In the present invention, the kit used for testing the FVIII complex activity standard curve is the same as the kit used for testing the activity curve of sodium thiamethoxam. The preparation methods of FX solution, activation reagent solution and chromogenic substrate solution are also the same as those in the sodium thiamethoxam activity curve test, which will not be repeated here.

[0078] The present invention first prepares a FVIII standard solution with a gradient concentration. In the present invention, the concentration range of the FVIII standard solution is preferably 0.95 to 3 IU / mL, preferably 0.94922 IU / mL to 3 IU / mL, and the concentration of the FVIII standard solution with a gradient concentration is specifically 3 IU / mL, 2.25 IU / mL, 1.6875 IU / mL, 1.265625 IU / mL and 0.94922 IU / mL; in a specific embodiment of the present invention, it is preferred to use the R4 reagent in the kit to dissolve FVIII to prepare a 4 IU / mL mother solution, and then use the R4 reagent to dilute the mother solution step by step to obtain each gradient FVIII standard solution.

[0079] After obtaining the FVIII standard solution, the present invention mixes the FVIII standard solution, water and the activation reagent solution for a fourth incubation to obtain a fourth incubation solution; the temperature of the fourth incubation is preferably 37° C., and the incubation time is preferably 5 to 20 minutes, specifically 15 minutes; the specific operating conditions of the fourth incubation are the same as those of the first incubation, except that the sodium hyaluronate working solution is replaced with water, and the FVIII solution is replaced with a FVIII standard solution with a gradient concentration.

[0080] After obtaining the fourth incubation solution, the present invention mixes the fourth incubation solution and the FX solution for a fifth incubation to obtain a fifth incubation solution. In the present invention, the temperature of the fifth incubation is preferably 37° C., and the incubation time is preferably 1 to 10 minutes, specifically 1 minute. The specific operating conditions of the fifth incubation are the same as those of the second incubation, and will not be repeated here.

[0081] After obtaining the fifth incubation solution, the present invention mixes the fifth incubation solution and the chromogenic substrate solution for the sixth incubation, then adds the stop solution, tests the absorbance of the obtained mixed solution at 405nm, and obtains the absorbance value; the chromogenic substrate is SXa-11. In the present invention, the temperature of the sixth incubation is preferably 37°C, and the incubation time is preferably 10 to 30 minutes, specifically 15 minutes; the specific operating conditions of the sixth incubation are the same as those of the third incubation, and will not be repeated here.

[0082] After obtaining the absorbance value, the present invention uses the logarithm of FVIII concentration as the abscissa and the absorbance value as the ordinate to draw a FVIII complex activity standard curve. In a specific embodiment of the present invention, after obtaining the FVIII complex activity standard curve, it also includes obtaining a fitting equation according to the standard curve.

[0083] In the present invention, the absorbance value obtained in the sea sodium polysaccharide activity curve test is substituted into the FVIII complex activity standard curve, and the FVIII concentration corresponding to the absorbance value can be calculated. The FVIII concentration is the residual FVIII concentration in the sea sodium polysaccharide activity curve test, and the residual FVIII activity unit is further calculated according to the residual FVIII concentration. The sea sodium polysaccharide activity curve is drawn according to the logarithm of the residual FVIII activity unit and the corresponding absorbance value, and then the sea sodium polysaccharide weight that inhibits the iFXase (endogenous coagulation factor X enzyme) activity formed by 1IU FVIII is calculated according to the sea sodium polysaccharide activity curve, which is 1 potency unit of the sea sodium polysaccharide. For convenience of expression, the potency of the sea sodium polysaccharide is finally expressed as the number of potency units contained per unit mass of polysaccharide, and the unit mass described in the present invention is 1 mg. In a specific embodiment of the present invention, the potency of the sea sodium polysaccharide is tested 4 times, and the average value of the 4 tests is used as the potency of the sea sodium polysaccharide.

[0084] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0085] Example 1 Extraction of sodium thunbergii polysaccharide

[0086] After the body wall of the sea cucumber (Holothurialeucospilota (Brandt)) was carefully separated from other tissues, it was immediately immersed in acetone and stored at 4°C for 24 hours. The dried tissue (500g) was ground into powder, suspended in 10L of 0.1mol / L sodium acetate buffer (pH 6) containing 50g papain, incubated at 65°C for 24 hours, and then the incubation liquid was centrifuged at low temperature (4000rpm, 4°C centrifugation for 30min) to obtain the enzymatic supernatant. 6mol / L hydrochloric acid aqueous solution was added to the enzymatic supernatant to adjust the pH to 2.5±0.5, reacted for 2 hours under stirring, and centrifuged at low temperature (4000rpm, 4°C centrifugation for 30min) to obtain the acid hydrolysis supernatant. Add 40wt% sodium hydroxide aqueous solution to the acid hydrolysis supernatant to adjust the pH value to 7.0±0.5. Under stirring, add 95vol% ethanol in a volume of 1 times the supernatant to the reaction system. After maintaining at -10°C for 24h, centrifuge at low temperature (4000rpm, centrifuge at 4°C for 30min) to collect the formed precipitate. The precipitate is dissolved in 500mL of distilled water, and 1L95vol% ethanol is added. After maintaining at -10°C for 24h, centrifuge at low temperature (4000rpm, centrifuge at 4°C for 30min) to collect the formed precipitate. Add 500mL95vol% ethanol to the precipitate for washing, centrifuge at low temperature (4000rpm, centrifuge at 4°C for 30min), collect the solid, dissolve in 500mL of distilled water, and freeze-dry to obtain 4g of crude sea sodium polysaccharide.

[0087] 4g of crude sodium polysaccharide was placed on a DEAE cellulose column (10cm×4cm) pre-equilibrated with 0.1mol / L HAc-NaAc buffer (pH 6), and washed with 5L of HAc-NaAc buffer (pH 6) containing 0.5mol / L NaCl and 2L of HAc-NaAc buffer (pH 6) containing 1mol / L NaCl, respectively, with a column flow rate of 50mL / min, and 500mL of each fraction was collected. Each fraction was detected by molecular weight and molecular weight distribution HPLC method, and the chromatographic peak fraction with a weight average molecular weight of 90,000 to 130,000 was collected, and 1 volume of 95vol% ethanol of the effluent was added, and after maintaining at -10°C for 24h, low-temperature centrifugation (4000rpm, 4°C centrifugation for 30min) was performed to collect the formed precipitate. The precipitate is dissolved in distilled water and concentrated to 1 / 2 of the original volume with an ultrafiltration membrane with a molecular weight cutoff of 10,000, water is added to the original volume, ultrafiltration is performed to 1 / 2 volume, water is added to the original volume, and then concentrated to 1 / 2 of the original volume, water is added to the original volume, and then concentrated to 1 / 2 of the original volume, and then water is added to the original volume. , Concentrate to 1 / 2 of the original volume, collect the concentrate, freeze-dry, and obtain 2.5g of pure sea sodium polysaccharide (cation is sodium). According to the above operation, multiple batches of parallel samples are continuously prepared, and the samples of 202401 batches are applied to subsequent embodiments. After testing, the sample weight-average molecular weight of 202401 batches is 108791, the PDI value is 1.32, the molar ratio of glucuronic acid, N-acetylgalactosamine and fucose is 1: 1.04: 0.67, the mass percentage of sodium ions is 9.24%, and the mass percentage of sulfate esters determined by ion chromatography is 27.3%.

[0088] Example 2 Establishment of the titer calibration method of sodium hyaluronate polysaccharide standard

[0089] The pharmacological effect of sodium hyaluronate is mainly a non-heparin-like anticoagulant drug, and its mechanism of action is to selectively inhibit the terminal rate-limiting enzyme of the endogenous coagulation pathway, "intrinsic factor X enzyme (Intrinsic tenase, FIXa-FVIIIa-PL-Ca 2+ complex,iFXase)".

[0090] Since FVIII can be easily obtained as a standard, the FVIII activity assay kit (FVIII: C) was used to determine the potency of this product by measuring its ability to inhibit the activity of iFXase (endogenous coagulation factor X enzyme). Under the assay conditions, the amount of sodium hyaluronate polysaccharide that can inhibit the activity of iFXase (endogenous coagulation factor X enzyme) formed by 1IU FVIII is defined as 1 potency unit of this product.

[0091] The detection mechanism of the FVIII activity assay kit is as follows: In the presence of phospholipids (PLP) and calcium ions, FVIII is activated by thrombin to form FVIIIa. FVIIIa, FIXa phospholipids and calcium ions form an enzyme complex (iFXase (endogenous coagulation factor X enzyme)), thereby activating factor FX. The generated factor FXa hydrolyzes the chromogenic substrate SXa-11 to release p-nitroaniline (pNA). pNA absorbs light at 405nm, and the amount of pNA is proportional to the absorbance value at 405nm (OD405). The content of p-nitroaniline is detected by an enzyme reader at 405nm to reflect the activity of the enzyme complex. The logarithm of the FVIII concentration is proportional to OD405. The mass of sodium thiamethoxam polysaccharide that inhibits the activity of the enzyme complex formed by 1IU FVIII is defined as 1 potency unit of this product. Figure 1 Schematic diagram of the detection mechanism of the FVIII activity assay kit.

[0092] The specific calibration method is as follows:

[0093] 1. Instruments and Materials

[0094] The instruments and reagents required for the experiment are shown in Tables 1-2.

[0095] Table 1 Instruments required for the experiment

[0096]

[0097] Table 2 Drugs and reagents required for the experiment

[0098]

[0099] 2. Experimental methods

[0100] 2.1 FVIII complex activity standard curve

[0101] 1) Preparation of kit solution:

[0102] 2.5 mL of ultrapure water was added to R1 (FX), R2 (FIXa, FIIa, phospholipids, Tris-HCl, CaCl2), and R3 (SXa-11) respectively to obtain R1 (FX) solution, R2 (activation reagent) solution, and R3 (chromogenic substrate) solution; the concentration of R1 solution was 50 nM; the concentration of FIXa in R2 solution was 60 nM, the concentration of FIIa was 47 nM, the concentration of phospholipids was 0.32 mg / mL, and the concentration of CaCl2 was 12 mM; the concentration of R3 solution was 8.4 mM. R4 (Tris-BSA Buffer) was used to dilute the FVIII standard solution.

[0103] 2) Preparation of FVIII standard solution:

[0104] Take FVIII (4.9 IU / vial), quantitatively draw 1.225 mL of R4 reagent to dissolve, and prepare a 4 IU / mL FVIII mother solution; take 1000 μL of 4 IU / mL FVIII mother solution, add 333 μL of R4, and prepare a 3 IU / mL FVIII standard solution; take 600 μL of 3 IU / mL FVIII standard solution, add 200 μL of R4, and prepare a 2.25 IU / mL FVIII standard solution; take 600 μL of 2.25 IU / mL FVIII standard solution, add 20 Take 600 μL of 1.6875 IU / mL FVIII standard solution and add 200 μL of R4 to prepare a 1.265625 IU / mL FVIII standard solution; take 600 μL of 1.265625 IU / mL FVIII standard solution and add 200 μL of R4 to prepare a 0.94922 IU / mL FVIII standard solution.

[0105] 3) Preparation of reaction termination solution:

[0106] Weigh 1 g of citric acid and add 50 mL of ddH2O to prepare a 0.02 g / mL citric acid stop solution.

[0107] 4) Determination of FVIII complex activity standard curve:

[0108] (1) Add 20 μL of ddH2O to each well of a 96-well plate, for a total of 5 wells;

[0109] (2) Add 20 μL of FVIII standard solution of different concentrations to the above test wells in turn, and add 20 μL of R2 solution to each well, and incubate at 37°C for 15 min;

[0110] (3) Add 20 μL R1 solution to each well and incubate at 37°C for 1 min;

[0111] (4) Add 20 μL of R3 solution to each well, incubate at 37°C for 15 min, add 30 μL of stop solution, and place in a microplate reader to measure the absorbance at 405 nm (OD405).

[0112] (5) A standard curve of FVIII complex activity was prepared with the logarithm of FVIII concentration as the horizontal axis and OD405 as the vertical axis.

[0113] 2.2 Polysaccharide activity titer calibration

[0114] 1) Preparation of kit solution:

[0115] Add 2.5 mL of ultrapure water to R1 (FX), R2 (FIXa, FIIa, phospholipids, Tris-HCl, CaCl2), and R3 (SXa-11) to obtain R1 (FX) solution, R2 (activation reagent) solution, and R3 (chromogenic substrate) solution. R4 (Tris-BSA Buffer) is used to dilute the FVIII standard solution. The preparation method and concentration are the same as those of the kit solution for the FVIII complex activity standard curve item in 2.1.

[0116] 2) Preparation of 3IU FVIII solution:

[0117] Take FVIII (4.9 IU / vial), quantitatively aspirate 1.225 mL of R4 reagent to dissolve, and prepare 4 IU / mL FVIII; take 1000 μL of 4 IU / mL FVIII, add 333 μL of R4, and prepare 3 IU / mL FVIII solution.

[0118] 3) Preparation of polysaccharide mother solution and working solution

[0119] Accurately weigh 25 mg of polysaccharide reference substance and dissolve it in 10 mL of ddH2O to prepare 2.5 mg / mL polysaccharide mother solution 1; aspirate 100 μL of mother solution 1, add 9900 μL of ddH2O, and dilute to 25 μg / mL polysaccharide mother solution 2; aspirate 100 μL of mother solution 2, add 4900 μL of ddH2O, and dilute to 500 ng / mL polysaccharide working solution 1; aspirate 900 μL of polysaccharide working solution 1, add 100 μL of ddH2O, and dilute to 500 ng / mL. Aspirate 900 μL of polysaccharide working solution 2, add 100 μL of ddH2O, and dilute to 450 ng / mL polysaccharide working solution 2; aspirate 900 μL of polysaccharide working solution 2, add 100 μL of ddH2O, and dilute to 405 ng / mL polysaccharide working solution 3; aspirate 900 μL of polysaccharide working solution 3, add 100 μL of ddH2O, and dilute to 364.5 ng / mL polysaccharide working solution 4; aspirate 900 μL of polysaccharide working solution 4, add 100 μL of ddH2O, and dilute to 328.05 ng / mL polysaccharide working solution 5.

[0120] 4) Preparation of reaction termination solution

[0121] Weigh 1 g of citric acid and add 50 mL of ddH2O to prepare a 0.02 g / mL citric acid stop solution.

[0122] 5) Polysaccharide activity assay

[0123] (1) Add 20 μL of ddH2O to the blank test wells in a 96-well plate, and add 20 μL of polysaccharide working solutions of different concentrations to the polysaccharide test wells in sequence;

[0124] (2) Add 20 μL of 3 IU / mL FVIII solution to the above test wells in turn, and add 20 μL of R2 to each well, and incubate at 37°C for 15 min;

[0125] (3) Add 20 μL R1 solution to each well and incubate at 37°C for 1 min;

[0126] (4) Add 20 μL of R3 solution to each well, incubate at 37°C for 15 min, add 30 μL of stop solution, and place in a microplate reader to measure the absorbance at 405 nm (OD405).

[0127] (5) Substitute the absorbance value into the FVIII complex activity standard curve to calculate the residual FVIII concentration. For the convenience of calculation, a regression equation is drawn with the reciprocal of the polysaccharide sample volume magnified 50 times as the horizontal axis and the logarithm of the FVIII residual unit magnified 50 times as the vertical axis.

[0128] (6) The initial concentration of FVIII was 3 IU / mL. The amount of FVIII and polysaccharide solution added was 20 μL. For the convenience of calculation, the data was amplified 50 times and calculated based on the amount of FVIII added being 1 mL, that is, the amount of FVIII added to the reaction system was 3 IU. The amount of sodium hyaluronate required to inhibit 1 IU and leave 2 IU residual was calculated to be 1 potency unit of sodium hyaluronate.

[0129] 3. Experimental results:

[0130] The following experimental data was independently tested twice by two experimental analysts, and the experimental results are as follows:

[0131] 3.1 Results of the first experiment by experimenter A

[0132] 1) FVIII complex activity standard curve

[0133] The results of the activity test of FVIII complex at different unit concentrations are shown in Table 3.

[0134] Table 3 FVIII complex enzyme activity results

[0135] FVIII (IU / mL) 0.94921875 1.265625 1.6875 2.25 3 OD405 0.895 1.005 1.243 1.442 1.643 Log(FVIII) -0.022633692 0.102305045 0.227243782 0.352182518 0.477121255

[0136] According to the experimental data, a standard curve was drawn with the logarithm of FVIII concentration as the horizontal axis and the absorbance OD405 as the vertical axis. The results are as follows: Figure 2 shown.

[0137] 2) Polysaccharide activity test results

[0138] The test results of polysaccharide activity at different concentrations are shown in Table 4. Based on the measured experimental data, the absorbance of the polysaccharide working solution was substituted into the FVIII complex enzyme activity standard curve to calculate the logarithm of the FVIII residual concentration. The results are shown in Table 4.

[0139] Table 4 Polysaccharide test activity results

[0140]

[0141] The initial concentration of FVIII was 3 IU / mL, and the amount of FVIII and polysaccharide solution added was 20 μL. Based on the amount of FVIII added to the reaction system being 3 IU FVIII, the data needs to be amplified 50 times. The results are shown in Table 5. The results of the first experiment by experimenter A.

[0142] Table 5 Potency determination data table

[0143]

[0144] According to the FVIII addition amount in the reaction system of 3IU, the weight of sodium hyaluronate polysaccharide required to inhibit 1IU and residual 2IU is defined as 1 potency unit of sodium hyaluronate polysaccharide (i.e. 1IU). According to the results in Table 5, the logarithm of residual activity unit × 50 is used as the ordinate and 1 / polysaccharide sample amount × 50 is used as the abscissa to establish a regression equation. The results are as follows Figure 3 shown.

[0145] according to Figure 3 The regression equation in the formula is calculated based on the residual activity unit 2IU (i.e., inhibition of 1IU), Log 2IU = 0.301029996, and the inverse of the sample amount (X) is calculated to be 7.191548 μg. -1 , that is, the sample size is 0.139052115 μg. For the convenience of expression, the potency of sodium hyaluronate polysaccharide is expressed as the number of potency units contained in each unit mass (mg) of polysaccharide. After calculation, the potency of sodium hyaluronate polysaccharide standard is 7192 IU / mg.

[0146] 3.2 Experimenter A - Results of the Second Experiment

[0147] 1) FVIII complex activity standard curve

[0148] The activity test results of FVIII complex at different unit concentrations are shown in Table 6.

[0149] Table 6 FVIII complex enzyme activity results

[0150]

[0151] According to the experimental data, a standard curve was drawn with the logarithm of FVIII concentration as the horizontal axis and the absorbance OD405 as the vertical axis. The results are as follows: Figure 4 shown.

[0152] 2) Polysaccharide activity test results

[0153] The activity test results of polysaccharides at different unit concentrations are shown in Table 7. According to the experimental data, the absorbance of the polysaccharide working solution was substituted into the FVIII complex enzyme activity curve to calculate the logarithm of the FVIII residual concentration. The results are shown in Table 7.

[0154] Table 7 Polysaccharide activity test results

[0155] Polysaccharide working solution (μg / mL) 0.32805 0.3645 0.405 0.45 0.5 OD405 0.418 0.328 0.27 0.227 0.182 LogFVIII concentration -0.34244 -0.40268 -0.441499 -0.47028 -0.5004

[0156] The initial concentration of FVIII was 3 IU / mL, and the amount of FVIII and polysaccharide solution added was 20 μL. According to the amount of FVIII added to the reaction system of 3 IU FVIII, the data needs to be amplified 50 times. The results are shown in Table 8. The results of the second experiment of experimenter A.

[0157] Table 8 Potency determination data table

[0158]

[0159]

[0160] According to the FVIII addition amount in the reaction system of 3IU, the weight of sodium hyaluronate polysaccharide required to inhibit 1IU and residual 2IU is defined as 1 potency unit of sodium hyaluronate polysaccharide (i.e. 1IU). According to the results in Table 8, a regression equation was established with the logarithm of residual activity unit × 50 as the ordinate and 1 / polysaccharide sample amount × 50 as the abscissa. The results are as follows Figure 5 shown.

[0161] The residual activity unit is 2IU, Log 2IU = 0.301029996, which is substituted into the regression equation to calculate the reciprocal of the sample amount to be 7.465604μg. -1 , that is, the sample size is 0.133948 μg. For the convenience of expression, the potency of sodium hyaluronate polysaccharide is expressed as the number of potency units per unit mass (mg) of polysaccharide. After calculation, the potency of the polysaccharide reference substance is 7466 IU / mg.

[0162] 3.3 Experimenter B - Results of the first experiment

[0163] 1) FVIII complex activity standard curve

[0164] The results of the activity test of FVIII complex at different unit concentrations are shown in Table 9.

[0165] Table 9 FVIII complex enzyme activity results

[0166] FVIII (IU / mL) 0.94921875 1.265625 1.6875 2.25 3 OD405 0.711 0.891 1.165 1.487 1.749 Log(FVIII) -0.022633692 0.102305045 0.227243782 0.352182518 0.477121255

[0167] According to the experimental data, a standard curve was drawn with the logarithm of FVIII concentration as the horizontal axis and the absorbance OD405 as the vertical axis. The results are as follows: Figure 6 shown.

[0168] 2) Polysaccharide activity test results

[0169] The activity test results of polysaccharides at different unit concentrations are shown in Table 10. According to the experimental data, the absorbance of the polysaccharide working solution was substituted into the FVIII complex enzyme activity curve to calculate the logarithm of the FVIII residual concentration. The results are shown in Table 10.

[0170] Table 10 Polysaccharide test activity results

[0171] Polysaccharide working solution (ug / mL) 0.32805 0.3645 0.405 0.45 0.5 OD405 value 0.373 0.326 0.238 0.211 0.166 LogFVIII concentration -0.1597 -0.18167 -0.2228144 -0.23544 -0.25647

[0172] The initial concentration of FVIII was 3 IU / mL, and the amount of FVIII and polysaccharide solution added was 20 μL. According to the amount of FVIII added to the reaction system of 3 IU FVIII, the data needs to be amplified 50 times. The results are shown in Table 11. The results of the first experiment of experimenter B.

[0173] Table 11 Potency determination data table

[0174]

[0175] According to the FVIII addition amount in the reaction system of 3IU, the weight of sodium hyaluronate polysaccharide required to inhibit 1IU and residual 2IU is defined as 1 potency unit of sodium hyaluronate polysaccharide (i.e. 1IU). According to the results in Table 11, a regression equation was established with the logarithm of residual activity unit × 50 as the ordinate and 1 / polysaccharide sample amount × 50 as the abscissa. The results are as follows Figure 7 shown.

[0176] The residual activity unit is 2IU, Log 2IU = 0.301029996, which is substituted into the regression equation to calculate the reciprocal of the sample amount to be 7.906336μg. -1 , that is, the sample size is 0.126481 μg. For the convenience of expression, the potency of sodium hyaluronate polysaccharide is expressed as the number of potency units per unit mass (mg) of polysaccharide. After calculation, the potency of the polysaccharide reference substance is 7906 IU / mg.

[0177] 3.4 Experimenter B - Results of the Second Experiment

[0178] 1) FVIII complex activity standard curve

[0179] The results of the activity test of FVIII complex at different unit concentrations are shown in Table 12.

[0180] Table 12 FVIII complex enzyme activity results

[0181] FVIII (IU / mL) 0.94921875 1.265625 1.6875 2.25 3 OD405 0.783 0.953 1.134 1.356 1.473 Log(FVIII) -0.022633692 0.102305045 0.227243782 0.352182518 0.477121255

[0182] According to the experimental data, a standard curve was drawn with the logarithm of FVIII concentration as the horizontal axis and the absorbance OD405 as the vertical axis. The results are as follows: Figure 8 shown.

[0183] 2) Polysaccharide activity test results

[0184] The test results of polysaccharide activity at different unit concentrations are shown in Table 13. According to the experimental data, the absorbance of the polysaccharide working solution was substituted into the FVIII complex enzyme activity standard curve to calculate the logarithm of the FVIII residual concentration. The results are shown in Table 13.

[0185] Table 13 Polysaccharide test activity results

[0186] Polysaccharide working solution (ug / mL) 0.32805 0.3645 0.405 0.45 0.5 OD405 0.446 0.357 0.291 0.227 0.203 LogFVIII concentration -0.25893 -0.3213 -0.36755 -0.4124 -0.42922

[0187] The initial concentration of FVIII was 3 IU / mL, and the amount of FVIII and polysaccharide solution added was 20 μL. According to the amount of FVIII added to the reaction system of 3 IU FVIII, the data needs to be amplified 50 times. The results are shown in Table 14. The results of the second experiment of experimenter B.

[0188] Table 14 Potency determination data table

[0189]

[0190] According to the FVIII addition amount in the reaction system of 3IU, the weight of sodium hyaluronate polysaccharide required to inhibit 1IU and residual 2IU is defined as 1 potency unit of sodium hyaluronate polysaccharide (i.e. 1IU). According to the results in Table 14, a regression equation was established with the logarithm of residual activity unit × 50 as the ordinate and 1 / polysaccharide sample amount × 50 as the abscissa. The results are as follows Fig. 9 shown.

[0191] Calculated according to the residual activity unit 2IU, Log 2IU = 0.301029996, substituted into the regression equation, the reciprocal of the sample amount was calculated to be 6.463757μg -1 , the sample size is 0.154709 μg. For the convenience of expression, the potency of sodium hyaluronate polysaccharide is expressed as the number of potency units per unit mass (mg) of polysaccharide. After calculation, the potency of the polysaccharide reference substance is 6464 IU / mg.

[0192] 3.5 Results of titer determination of sodium hyaluronate polysaccharide

[0193] Based on the results of four experiments conducted by two researchers, the potency of sodium hyaluronate polysaccharide was 7192 IU / mg, 7466 IU / mg, 7906 IU / mg, and 6464 IU / mg, with an average of 7257 IU / mg. Based on this, the potency of sodium hyaluronate polysaccharide was determined to be 7257 IU / mg.

[0194] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for calibrating the potency of sodium thunbergii polysaccharide, characterized in that: The following steps are involved: The activity curve of sodium hyaluronate polysaccharide was obtained by p-nitroaniline colorimetric method, wherein the abscissa of the sodium hyaluronate polysaccharide activity curve is the reciprocal of the weight of sodium hyaluronate polysaccharide, and the ordinate is the logarithm of the residual activity unit of FVIII; The weight of the sodium hyaluronate polysaccharide that inhibits the iFXase activity formed by 1 IU of FVIII was taken as 1 potency unit, and the potency of the sodium hyaluronate polysaccharide was obtained according to the sodium hyaluronate polysaccharide activity curve; The sodium thunbergii polysaccharide is a fucosylated chondroitin sulfate polysaccharide; the weight average molecular weight of the sodium thunbergii polysaccharide is 90,000 to 130,000, the monosaccharide composition includes glucuronic acid, N-acetylgalactosamine and fucose, the molar ratio of glucuronic acid, N-acetylgalactosamine and fucose is 1:0.8 to 1.2:0.5 to 0.8; the mass percentage of sulfate groups in the sodium thunbergii polysaccharide is 25 to 40%.

2. The potency calibration method according to claim 1, characterized in that: The method for obtaining the activity curve of sodium thunbergii polysaccharide comprises: Prepare sodium thunbergii polysaccharide working solution with gradient concentrations; The sodium thiamethoxam polysaccharide working solution, the FVIII solution and the activation reagent solution are mixed for a first incubation to obtain a first incubation solution; the activation reagent solution comprises FIXa, FIIa, phospholipids, a buffer solution and CaCl2; mixing the first incubation solution and the FX solution for a second incubation to obtain a second incubation solution; The second incubation solution and the chromogenic substrate solution are mixed for a third incubation, and then a stop solution is added, and the absorbance of the resulting mixed solution at 405 nm is tested to obtain an absorbance value; the chromogenic substrate is SXa-11; Substitute the absorbance value into the FVIII complex activity standard curve, calculate the FVIII residual activity unit, and draw the sodium hyaluronate activity curve with the reciprocal of the sodium hyaluronate weight as the abscissa and the logarithm of the FVIII residual activity unit as the ordinate; the abscissa of the FVIII complex activity standard curve is the logarithm of the FVIII concentration, and the ordinate is the absorbance value.

3. The potency calibration method according to claim 2, characterized in that: The first incubation temperature is 37° C., and the incubation time is 5 to 20 minutes.

4. The potency calibration method according to claim 2, characterized in that: The second incubation temperature is 37° C., and the incubation time is 1 to 10 minutes.

5. The potency calibration method according to claim 2, characterized in that: The temperature of the third incubation is 37° C., and the incubation time is 10 to 30 minutes.

6. The potency calibration method according to claim 2, characterized in that: The stop solution is a citric acid solution, and the concentration of the citric acid solution is 0.01-0.1 g / mL.

7. The potency calibration method according to claim 2, characterized in that: The concentration range of the sodium thunbergii polysaccharide working solution is 328-500 ng / mL.

8. The potency calibration method according to claim 2, characterized in that: The buffer in the activation reagent solution is Tris-HCl.

9. The potency calibration method according to claim 2, characterized in that: The method for obtaining the FVIII complex activity standard curve comprises: Prepare FVIII standard solutions with gradient concentrations; The FVIII standard solution, water and an activation reagent solution are mixed for a fourth incubation to obtain a fourth incubation solution; the activation reagent solution comprises FIXa, FIIa, phospholipids, a buffer and CaCl2; Mixing the fourth incubation solution and the FX solution for a fifth incubation to obtain a fifth incubation solution; The fifth incubation solution and the color substrate solution are mixed for the sixth incubation, and then a stop solution is added, and the absorbance of the obtained mixed solution at 405 nm is tested to obtain an absorbance value; the color substrate is SXa-11; The FVIII complex activity standard curve was drawn with the logarithm of the FVIII concentration as the abscissa and the absorbance value as the ordinate.

10. The potency calibration method according to claim 9, characterized in that: The concentration of the FVIII standard solution is 0.95-3 IU / mL.

Images