Method for detecting activity of protease preparation for external use

Abstract

The invention provides an activity detection method of an external protease preparation, which comprises the following steps: (1) dispersing the external protease preparation in a water-insoluble organic solvent to form a protease-containing dispersion system; (2) adding a buffer solution for extraction, so that the protease is transferred into the buffer solution from the water-insoluble organic solvent, and an extract containing the protease is formed; and (3) collecting and measuring the activity of the protease in the extract liquor so as to evaluate the activity level of the protease in the external protease preparation. The method disclosed by the invention can be used for testing the activity of an external preparation of protease, particularly testing protease ointments, animal collagen can be selected as a substrate, the structure of the animal collagen is consistent with that of human collagen, and the activity of the protease acting on a human body can be effectively reflected. The specific amino acid is adopted as a determination target, the titer of the high-activity protease can be accurately determined, the detection speed is high, and the method is suitable for production control.

Description

Technical Field

[0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to an activity detection method for an external protease preparation. Background Art

[0002] Proteases have shown great potential in the medical field due to their excellent biological activity, but they are limited by their poor stability in aqueous environments and difficulty in long-term storage, so they are faced with challenges in direct application as topical preparations for disease treatment. In particular, key proteases such as bromelain, papain, collagenase, trypsin and chymotrypsin are difficult to maintain their activity under normal conditions.

[0003] To solve this problem, researchers used anhydrous environment preparation technology to transform these protease preparations into ointments, effectively extending the shelf life of the preparations and thus realizing their application in the medical field. This type of ointment is not only easy to store, but also retains the therapeutic potential of proteases, opening up a new way for disease treatment.

[0004] It is particularly worth mentioning that collagenase ointment has demonstrated extraordinary efficacy in the field of burn treatment. It can efficiently perform enzymatic debridement of burn wounds and accelerate the wound healing process, so it has been widely used in many medical fields such as burns, dermatology, surgery and orthopedics. For chronic skin ulcers, especially refractory diseases such as diabetic foot ulcers and pressure sores, the clinical application of collagenase ointment has accumulated rich experience, its efficacy and safety have been widely recognized, and it has been listed as a recommended medication in many diabetic foot treatment guidelines.

[0005] However, the mismatch between the oily matrix commonly used in creams and the solubility characteristics of proteases has become an urgent problem to be solved in formulation development. Since proteases can only be dissolved in aqueous solvents, not oily matrices, it is necessary to develop an accurate and effective detection method to accurately measure the activity of proteases in formulations. The development of detection methods in this regard is directly related to the control of formulation quality and the evaluation of clinical effects, and has far-reaching significance for promoting the development and application of related protease preparations. Summary of the invention

[0006] Therefore, the purpose of the present invention is to provide an innovative and efficient method to solve the problem of activity determination caused by the incompatibility of proteases with oily matrices in topical preparations. The method constructs an extraction system to transfer the protease to an aqueous environment that is more conducive to its activity expression without changing the overall oily matrix properties of the cream, thereby achieving accurate determination of the protease activity.

[0007] The present invention provides a method for detecting the activity of an external protease preparation, the method comprising the following steps:

[0008] (1) dispersing the topical protease preparation in a water-insoluble organic solvent to form a dispersed system containing the protease;

[0009] (2) adding a buffer solution to the dispersed system for extraction, so that the protease is transferred from the water-insoluble organic solvent to the buffer solution to form an extract containing the protease;

[0010] (3) Determine the potency of the protease in the extract to calculate the activity level of the protease in the topical protease preparation.

[0011] According to the detection method provided by the present invention, the water-insoluble organic solvent in step (1) is an organic solvent with low solubility in water or almost insoluble in water. In some embodiments of the present invention, the water-insoluble organic solvent is selected from one or more of alkane solvents, aromatic hydrocarbon solvents, ether solvents and alcohol solvents.

[0012] Preferably, the alkane solvent is selected from one or more of C5-C15 alkanes and C5-C15 cycloalkanes, preferably one or more of petroleum ether, pentane, cyclopentane, hexane, cyclohexane and heptane;

[0013] Preferably, the aromatic hydrocarbon solvent is selected from one or more aromatic hydrocarbons optionally monosubstituted or polysubstituted by C1-C5 alkanes or C3-C5 cycloalkanes, preferably one or more selected from benzene, toluene, xylene and ethylbenzene;

[0014] Preferably, the ether solvent is selected from one or more of AOB type ethers, wherein A and B are independently C1-C15 alkanes or C3-C15 cycloalkanes, or one or more of aromatic hydrocarbons optionally monosubstituted or polysubstituted by C1-C5 alkanes or C3-C5 cycloalkanes, preferably selected from one or more of methyl ether, methyl ethyl ether, ethyl ether, methyl tert-butyl ether and isopropyl ether;

[0015] Preferably, the alcohol solvent is selected from one or more of C4-C10 alcohols, preferably decanol and / or pentanol.

[0016] According to the detection method provided by the present invention, preferably, in step (1), based on 1 gram of the external protease preparation, the amount of the water-insoluble organic solvent can be 2-100 mL, preferably 5-50 mL.

[0017] According to the detection method provided by the present invention, preferably, the volume ratio of the buffer solution in step (2) to the water-insoluble organic solvent in step (1) can be 1: 0.5-15, preferably 1: 1-10. The number of extractions in step (2) can be 1-8 times, and the volume of the buffer solution used in each extraction process can be the same or different. After the extraction is completed, the extracts are combined.

[0018] According to the detection method provided by the present invention, the buffer solution can be selected from one or more of Tris buffer, phosphate buffer, carbonate buffer and acetate buffer.

[0019] Preferably, the pH of the buffer solution is 6.7-8.5, more preferably 6.7-7.6.

[0020] In a preferred embodiment of the present invention, the concentration of the buffer solution may be 0.001-5 mol / L, preferably 0.01-3 mol / L.

[0021] In some embodiments of the present invention, in step (3), the activity of the protease in decomposing the substrate protein can be measured as a potency indicator. Specifically, the amount of the target amino acid released from the substrate protein under the action of the protease per unit time is measured as an indicator for measuring the potency.

[0022] Specifically, when the topical protease preparation is a collagenase preparation, the amount of target amino acids such as threonine, valine, leucine, isoleucine, lysine or serine released from collagen per unit time is used as a potency indicator.

[0023] In the present invention, the protease activity unit can be defined as: at 37° C. and pH 7.5, the protease releases 1 mmol of target amino acid from the substrate protein per hour as one activity unit.

[0024] Before the test, a standard curve can be drawn as follows: weigh different amounts of target amino acid standards, prepare amino acid solutions of different concentrations in a series of test tubes, add citrate buffer and potassium cyanide-ninhydrin test solution for color reaction, and then measure the absorbance of each test tube at a specific wavelength (such as 550-580nm), and draw a standard curve with amino acid concentration as the horizontal axis and absorbance as the vertical axis.

[0025] More specifically, in some embodiments of the present invention, the method for drawing a standard curve is as follows: accurately weigh amino acids in 5-10 test tubes, dilute with water to 1-5 ml, so that the concentration of amino acids in each test tube is 0-100 μg / ml, and the amino acid concentrations in adjacent test tubes differ by 2-30 μg / ml, add equal volumes of citrate buffer and potassium cyanide-ninhydrin test solution in sequence, keep warm in a boiling water bath for 1-20 min, cool in cold water for 2-15 min, add 10-100% ethanol 1-5 mL to each tube, use the test tube without amino acids as a blank, measure the absorbance at a wavelength of 550-580 nm, and draw a standard curve.

[0026] In some embodiments of the present invention, the method for determining the titer of the protease in the extract in step (3) comprises:

[0027] (I) Prepare the following solutions in three test tubes in parallel:

[0028] Sample tube A: Add substrate protein to the buffer solution;

[0029] Substrate control tube B: add buffer solution and the same amount of substrate protein as in sample tube A;

[0030] Enzyme control tube C: add only the same volume of buffer solution as in sample tube A.

[0031] (II) After preheating the three test tubes in a water bath, add an equal amount of the extract to the sample tube A and the enzyme control tube C, keep the temperature and shake for 15-90 minutes for enzymatic hydrolysis, and then add an equal amount of anhydrous ethanol to terminate the reaction;

[0032] (III) filtering the contents of the test tubes, adding citrate buffer and potassium cyanide-ninhydrin test solution to the filtrate for color reaction, and measuring the absorbances of the three test tubes within a wavelength range of 550-580 nm, respectively, and recording them as absorbance A, absorbance B and absorbance C corresponding to the test tube numbers;

[0033] (IV) taking the difference of [absorbance A - absorbance B - absorbance C] as the net absorbance, searching the amino acid concentration value corresponding to the net absorbance on the standard curve, and calculating the potency of the protease in the extract by combining the reaction time and the amino acid concentration.

[0034] The buffer solution in the protease titer determination method can also be selected from one or more of Tris buffer, phosphate buffer, carbonate buffer and acetate buffer, and can be the same as or different from the buffer solution used in the extraction process.

[0035] In some embodiments of the present invention, the protease may be selected from one or more of bromelain, papain, collagenase, trypsin and chymotrypsin.

[0036] In a preferred embodiment of the present invention, the protease is collagenase, and the substrate protein is collagen, for example, it can be animal collagen commonly used in the art, such as extracts of pig skin, cow skin, beef tendon, etc.

[0037] Preferably, the temperature of the water bath in step (II) may be 35-40°C, preferably 37°C.

[0038] More specifically, in some embodiments of the present invention, the operation of determining the titer of the protease in the extract is as follows:

[0039] Sample tube A: Add 10-30 mg of substrate protein and 1-5 ml of buffer solution into the test tube;

[0040] Substrate control tube B: Add 10-30 mg of substrate protein and 1-5 mL of buffer solution to the test tube;

[0041] Enzyme control tube C: Add 1-5 mL of buffer solution to the test tube.

[0042] Preheat the sample tube A, substrate control tube B and enzyme control tube C in a 35-40°C water bath for 0.5-8 min, add 0.1-5 mL of extract to the sample tube A and enzyme control tube C, respectively, keep warm in a 37°C water bath and shake for 15-90 min, add 0.1-3 mL of anhydrous ethanol to the sample tube A, substrate control tube B and enzyme control tube C, respectively, and cool to room temperature, filter, take 0.1-2 mL of the filtrate, add water to 1-5 mL, add equal volumes of citrate buffer and potassium cyanide-ninhydrin test solution in sequence, keep warm in a boiling water bath for 1-20 min, cool in cold water for 2-15 min, add 1-5 mL of 10-100% ethanol to each tube, measure the absorbance at a wavelength of 550-580 nm, subtract the absorbance of the substrate control tube B and enzyme control tube C from the absorbance of the sample tube A, find the concentration of the amino acid from the standard curve, and calculate the titer.

[0043] The activity detection method of the topical protease preparation provided by the present invention can be used for the activity test of the topical protease preparation, especially the test of the ointment. The detection method of the present invention can select animal collagen as a substrate, which is consistent with the structure of human collagen and can effectively reflect the activity of the protease acting on the human body. The present invention adopts specific amino acids as the determination target, can accurately determine the titer of the highly active protease, has a fast detection speed, and is suitable for production control. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0045] Figure 1 The linear relationship curve between leucine concentration and absorbance in Example 1 is shown. DETAILED DESCRIPTION

[0046] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention.

[0047] Example 1

[0048] (1) Take 2 g of collagenase ointment (produced by Yuanda Life Sciences (Anshan) Co., Ltd.) and add 25 mL of petroleum ether to disperse and dissolve;

[0049] (2) Add 0.05 mol / L Tris buffer (pH = 7.6) and extract four times with the volume of each time being 30 ml, 30 ml, 15 ml and 15 ml respectively. After combining the extraction solutions, dilute them to 100 ml with Tris buffer (pH = 7.6).

[0050] (3) Determine the titer of collagenase.

[0051] Standard curve drawing: accurately weigh leucine, dilute with water to 1mL solution containing 50μg as reference stock solution; accurately measure 0mL, 0.1mL, 0.2mL, 0.3mL, 0.4mL, 0.5mL, 0.6mL of reference stock solution in different test tubes, add water to 1mL respectively, then add 1mL of citrate buffer and 1mL of potassium cyanide-ninhydrin test solution in sequence, keep warm in boiling water bath for 15min, cool in cold water for 10min, add 3mL of 60% ethanol to each tube, use 0ml tube as blank, and measure absorbance at 570nm wavelength. See Table 1 for specific test data. Draw the standard curve with leucine concentration as the horizontal axis and absorbance as the vertical axis, as shown in Figure 1 shown.

[0052] Table 1

[0053] Serial number 1 2 3 4 5 6 7 Control solution added ml 0 0.1 0.2 0.3 0.4 0.5 0.6 Leucine μg / ml 0 4.90 9.80 14.71 19.61 24.51 29.41 <![CDATA[Absorbance A 570nm > 0 0.103 0.235 0.326 0.453 0.548 0.665

[0054] The equation of the linear regression curve is y = 0.0226x + 0.0004, R 2 = 0.9988.

[0055] The leucine concentration was in the range of 4.90-29.41 μg / ml, and there was a good linear relationship between the leucine concentration and the absorbance value.

[0056] The preparation method of the relevant solution is as follows:

[0057] Citrate buffer (pH 5.0): Dissolve 8.4 g of citric acid in an appropriate amount of water, add about 80 ml of sodium hydroxide (1 mol / L), and dilute to 200 ml with water.

[0058] Potassium cyanide-ninhydrin solution: Take 1 ml of potassium cyanide aqueous solution (0.5 mg / ml), add it to 10 ml of 5% ninhydrin ethylene glycol methyl ether solution, and dilute it to 50 ml with ethylene glycol methyl ether.

[0059] The specific measurement process is as follows:

[0060] Collagenase solution: Take 2 ml of the extract in step (2) above and dilute it to 5 ml with Tris buffer (pH = 7.6) for later use (the extract in step (2) can also be used directly without dilution).

[0061] (I) Prepare the following solutions in three test tubes in parallel:

[0062] Sample tube A: Add 20 mg of bovine collagen and 1 mL of Tris buffer (pH = 7.6) into the test tube;

[0063] Substrate control tube B: add 20 mg of bovine collagen and 1.5 mL of Tris buffer (pH = 7.6) to the test tube;

[0064] Enzyme control tube C: Add 1 mL of Tris buffer (pH=7.6) to the test tube.

[0065] (II) After preheating the three test tubes in a 37°C water bath for 5 min, add 0.5 mL of the collagenase solution to sample tube A and enzyme control tube C, incubate at 37°C with shaking for 1 h to carry out enzymatic hydrolysis reaction, and then add 0.5 mL of anhydrous ethanol to each of the three test tubes and cool to room temperature.

[0066] (III) Filter the contents of the three test tubes separately, and take 0.5 mL of the filtrate from each tube. Add water to 2.5 mL, shake well, take 1 mL and add 1 mL of citrate buffer (pH 5.0) and 1 mL of potassium cyanide-ninhydrin test solution in sequence. Keep warm in a boiling water bath for 15 min, cool in cold water for 10 min, add 3 mL of 60% ethanol to each tube, and measure the absorbance at a wavelength of 570 nm.

[0067] (IV) The net absorbance is obtained by subtracting the absorbance of the substrate control tube B and the enzyme control tube C from the absorbance of the sample tube A. The concentration of leucine is found from the standard curve and the potency of the collagenase ointment is calculated using the following formula:

[0068]

[0069] The absorbance of sample tube A was 0.451, the absorbance of substrate control tube B was 0.102, the absorbance of enzyme control tube was 0.051, the molecular weight of leucine was 131.2 g / mol, and the potency of collagenase ointment was calculated to be 251 units / gram.

[0070] Example 2

[0071] Take 2g of collagenase ointment, add 25mL of ether to disperse and dissolve, add 1mol / L Tris buffer (pH=7.0) to extract 7 times, each volume is 20ml, combine the extraction solutions and make up to 150ml with Tris buffer (pH=7.0), and measure the titer of the aqueous solution. Except that Tris buffer (pH=7.6) is replaced by Tris buffer (pH=7.0), other operations are the same as Example 1, and the titer of the ointment is calculated to be 245 units / gram.

[0072] Example 3

[0073] Take 2 g of bromelain ointment (produced by Yuanda Life Sciences (Anshan) Co., Ltd.), add 50 mL of heptane to disperse and dissolve, add 100 mL of 0.02 mol / L phosphate buffer (pH=8.0), extract once, and measure the titer of the aqueous solution. Other operations are the same as those in Example 1. The titer of the ointment is calculated to be 135 units / gram.

[0074] Example 4

[0075] Take 2g of trypsin ointment (produced by Yuanda Life Science (Anshan) Co., Ltd.), add 10mL of methyl tert-butyl ether to disperse and dissolve, add 0.5mol / L carbonate buffer (pH=6.8) and extract three times with the volume of each time being 10mL, 20mL, and 10mL respectively, combine the aqueous solutions and dilute to 50ml with carbonate buffer (pH=6.8), and measure the titer of the aqueous solution. Except that the Tris buffer is replaced by carbonate buffer (pH=6.8) in the method, other operations are the same as in Example 1, and the titer of the ointment is calculated to be 187 units / gram.

[0076] Example 5

[0077] Take 2g of chymotrypsin ointment (produced by Yuanda Life Science (Anshan) Co., Ltd.), add 100mL of decanol to disperse and dissolve, add 3mol / L acetate buffer (pH=8.5) to extract twice, each volume is 150mL and 80mL respectively, combine the aqueous solution and dilute to 250ml with acetate buffer (pH=8.5), and measure the potency of the aqueous solution. Other operations are the same as in Example 1, and the potency of the ointment is calculated to be 262 units / gram.

[0078] Example 6

[0079] In the water-insoluble organic solvent screening experiment, the petroleum ether in Example 1 was replaced with the solvents listed in Table 2, and other operations remained unchanged. The measured potency values ​​are shown in Table 2 below.

[0080] Table 2

[0081]

[0082] Compared with the petroleum ether used as the extraction solvent in Example 1, the above solvents have smaller differences in the measured ointment potency, with an RSD% of 1.86%. Therefore, it can be inferred that the extraction effects of the solvents are consistent and there is no significant difference.

[0083] Comparative Example 1

[0084] The extraction solvent ether in Example 2 was replaced with the solvent in Table 3, and other operations remained unchanged. The measured potency values ​​are shown in Table 3 below.

[0085] Table 3

[0086] Solvents Measured value (unit / g) Chloroform 176 Ethyl acetate 135

[0087] Taking the titer of the ointment in Example 2 as the standard and chloroform as the extraction solvent, the titer of the ointment was measured to be 176 units / gram, which is only 71.8% of the titer of the ointment in Example 2 with ether as the extraction solvent. Taking ethyl acetate as the extraction solvent, the titer was measured to be 135 units / gram, which is only 55.1% of the titer of the ointment in Example 2 with ether as the extraction solvent. It can be seen that chloroform and ethyl acetate are not suitable as extraction solvents for the titer detection of protease ointment preparations such as collagenase.

[0088] Comparative Example 2

[0089] In this comparative example, the activity was detected by directly extracting with buffer without adding an organic solvent, for comparison with Example 2.

[0090] Take 2 g of collagenase ointment and directly add 1 mol / L Tris buffer (pH=7.0) to extract 7 times, each time with a volume of 20 ml. Other operations are the same as in Example 2. The ointment potency is calculated to be 95 units / gram.

[0091] The protease in the protease preparation cannot be completely extracted by using only a buffer solution instead of an organic solvent for dissolution and dispersion, and its potency is only 38.8% of the potency determination result of the ointment in Example 2.

[0092] Comparative Example 3

[0093] This comparative example adopts a non-extraction method for determination, that is, the ointment is dissolved in a water-soluble organic solvent, and then the activity is detected by adding a buffer solution and a mixed extraction with the water-soluble organic solvent, for comparison with Example 1.

[0094] Take 2 g of collagenase ointment, add 25 mL of the solvent listed in Table 4 to dissolve, then add 75 mL of 0.05 mol / L Tris buffer (pH=7.6). Other operations are the same as those in Example 1. The ointment potency is calculated as shown in Table 4 below.

[0095] Table 4

[0096] Solvents Measured value (unit / g) Measured value / measured value of Example 1*100% Methanol 102 40.6 Ethanol 113 45.0 Propanol 110 43.8 glycerin 105 41.8 Propylene glycol 109 43.4 Ethylene glycol 99 39.4 DMF 87 34.7 DMSO 89 35.5

[0097] The results showed that although the ointment was first dissolved with an organic solvent, the organic solvent used was miscible with water and could not be extracted, so the protease in the ointment could not be extracted, and its titer was only less than 46% of the titer measured in Example 1. It can be seen that water-soluble organic solvents cannot be used as extraction solvents for titer detection in ointments.

[0098] Comparative Example 4

[0099] This comparative example is based on 1 gram of external protease preparation, and the amount of water-insoluble organic solvent used is not within the preferred range of the present invention. Activity detection is carried out for comparison with Example 4.

[0100] Take 2 g of trypsin ointment (produced by Yuanda Life Science (Anshan) Co., Ltd.), add 2 mL of methyl tert-butyl ether and disperse and dissolve it. Other operations are the same as those in Example 4. The potency of the ointment is calculated to be 59 units / gram.

[0101] The amount of the water-insoluble organic solvent used was not within the range, and the protease preparation could not be completely extracted, and its potency was only 31.6% of the ointment potency measurement result of Example 4.

[0102] Comparative Example 5

[0103] The volume ratio of the buffer solution to the water-insoluble organic solvent in this comparative example is not within the preferred range of the present invention, and an activity test is performed for comparison with Example 3.

[0104] Take 2 g of bromelain ointment (produced by Yuanda Life Sciences (Anshan) Co., Ltd.), add 50 mL of heptane to disperse and dissolve, add 10 mL of 0.02 mol / L phosphate buffer (pH 8.0), extract once, and perform other operations as in Example 3. The ointment potency is calculated to be 88 units / gram.

[0105] The ratio of the amount of the buffer solution to the water-insoluble organic solvent is not within the preferred range of the present invention, and the protease preparation cannot be completely extracted, and its potency is only 65.2% of the ointment potency test result of Example 3.

[0106] Comparative Example 6

[0107] The pH value of the buffer solution in this comparative example is not within the preferred range of the present invention, and an activity test is performed for comparison with Example 2.

[0108] Take 2 g of collagenase ointment, add 25 mL of ether to disperse and dissolve, add 1 mol / L Tris buffer (pH=6.5) to extract 7 times, and perform other operations as in Example 2. The ointment potency is calculated to be 75 units / gram.

[0109] Take 2 g of collagenase ointment, add 25 mL of ether to disperse and dissolve, add 1 mol / L Tris buffer (pH=9.0) to extract 7 times, and perform other operations as in Example 2. The ointment potency is calculated to be 183 units / gram.

[0110] The pH of the buffer solution is not within the preferred range of the present invention, and the pH values ​​are 6.5 and 9.0, respectively. The buffer solution with this pH value cannot completely extract the protease in the protease preparation, and the titers thereof are 30.6% and 74.7% of the titer determination results of the ointment in Example 2, respectively.

[0111] Solution stability test

[0112] The absorbance of the sample measurement solution of Example 4 was measured at a wavelength of 570 nm at 0, 5, 10, 15, 20, 30, 45, and 60 min, respectively. The results are shown in Table 5 below.

[0113] Table 5

[0114] Time (min) 0 5 10 15 20 30 45 60 average value RSD% Example 4 0.54 0.541 0.538 0.535 0.530 0.521 0.516 0.519 0.530 1.96

[0115] The results show that the absorbance of the sample measurement solution is basically stable within 60 minutes, and the stability of the measurement using this solution is good.

[0116] Recovery rate experiment

[0117] About 2 g of each of the three collagenase ointments of 200 units / g (80%), 250 units / g (100%), and 300 units / g (120%) were accurately weighed and the potency was determined according to the method of Example 1. The measured potency was divided by the labeled potency to calculate the recovery rate. The results are shown in Table 6 below.

[0118] Table 6

[0119]

[0120] It can be seen from the above data that the recovery rate of the ointment potency determination measured by the detection method provided by the present invention is 96.24%, and the RSD% is 2.03%. This method is a biochemical reaction. After the protease is extracted from the ointment, it acts on the substrate protein to generate a polypeptide. This method can be used to determine the potency of the protease preparation.

Claims

1. A method for detecting the activity of an external protease preparation, the method comprising the following steps: (1) dispersing the topical protease preparation in a water-insoluble organic solvent to form a dispersed system containing the protease; (2) adding a buffer solution to the dispersed system for extraction, so that the protease is transferred from the water-insoluble organic solvent to the buffer solution to form an extract containing the protease; (3) Determine the potency of the protease in the extract to calculate the activity level of the protease in the topical protease preparation.

2. The detection method according to claim 1, wherein: The water-insoluble organic solvent is selected from one or more of an alkane solvent, an aromatic hydrocarbon solvent, an ether solvent and an alcohol solvent; Preferably, the alkane solvent is selected from one or more of C5-C15 alkanes and C5-C15 cycloalkanes, preferably one or more of petroleum ether, pentane, cyclopentane, hexane, cyclohexane and heptane; Preferably, the aromatic hydrocarbon solvent is selected from one or more aromatic hydrocarbons optionally monosubstituted or polysubstituted by C1-C5 alkanes or C3-C5 cycloalkanes, preferably one or more selected from benzene, toluene, xylene and ethylbenzene; Preferably, the ether solvent is selected from one or more of AOB type ethers, wherein A and B are independently C1-C15 alkanes or C3-C15 cycloalkanes, or one or more of aromatic hydrocarbons optionally monosubstituted or polysubstituted by C1-C5 alkanes or C3-C5 cycloalkanes, preferably selected from one or more of methyl ether, methyl ethyl ether, ethyl ether, methyl tert-butyl ether and isopropyl ether; Preferably, the alcohol solvent is selected from one or more of C4-C10 alcohols, preferably decanol and / or pentanol.

3. The detection method according to claim 1, wherein in step (1), based on 1 gram of the external protease preparation, the amount of the water-insoluble organic solvent is 2-100 mL, preferably 5-50 mL.

4. The detection method according to claim 1, wherein: The volume ratio of the buffer solution in step (2) to the water-insoluble organic solvent in step (1) is 1:0.5-15, preferably 1:1-10; Preferably, the number of extractions in step (2) is 1-8 times, and the volume of buffer solution used in each extraction is the same or different.

5. The detection method according to any one of claims 1 to 4, wherein the buffer solution in step (2) is selected from one or more of Tris buffer, phosphate buffer, carbonate buffer and acetate buffer; Preferably, the pH of the buffer solution is 6.7-8.5, more preferably 6.7-7.

6.

6. The detection method according to any one of claims 1 to 5, wherein The concentration of the buffer solution is 0.001-5 mol / L, preferably 0.01-3 mol / L.

7. The detection method according to claim 1, wherein: The method for determining the potency of the protease in the extract in step (3) is to determine the decomposition activity of the protease on the substrate protein; Preferably, the amount of target amino acid released from the substrate protein under the action of the protease per unit time is measured as an indicator of potency; Preferably, the external enzyme preparation is a collagenase preparation, and the target amino acid is selected from one or more of threonine, valine, leucine, isoleucine, lysine and serine.

8. The detection method according to claim 7, wherein: Step (3) further comprises drawing a standard curve according to the following method: weighing different amounts of target amino acid standards, preparing amino acid solutions of different concentrations in a series of test tubes, adding citrate buffer and potassium cyanide-ninhydrin test solution for color reaction, then measuring the absorbance of each test tube at a wavelength of 550-580 nm, and drawing a standard curve with amino acid concentration as the horizontal axis and absorbance as the vertical axis.

9. The detection method according to claim 7 or 8, wherein: The method for determining the titer of the protease in the extract in step (3) comprises: (I) Prepare the following solutions in three test tubes in parallel: Sample tube A: Add substrate protein to the buffer solution; Substrate control tube B: add an equal amount of substrate protein to the buffer solution as in sample tube A; Enzyme control tube C: add only the same volume of buffer solution as in sample tube A. (II) After preheating the three test tubes in a water bath, add an equal amount of the extract to the sample tube A and the enzyme control tube C, keep the temperature and shake for 15-90 minutes for enzymatic hydrolysis, and then add an equal amount of anhydrous ethanol to terminate the reaction; (III) filtering the contents of the test tubes, adding citrate buffer and potassium cyanide-ninhydrin test solution to the filtrate for color reaction, and then measuring the absorbances of the three test tubes within a wavelength range of 550-580 nm, and recording them as absorbance A, absorbance B and absorbance C corresponding to the test tube numbers; (IV) The value of absorbance A - absorbance B - absorbance C is taken as the net absorbance, the amino acid concentration value corresponding to the net absorbance is found on the standard curve, and the potency of the protease in the extract is calculated based on the reaction time and the amino acid concentration.

10. The detection method according to any one of claims 1 to 9, wherein: The protease is selected from one or more of bromelain, papain, collagenase, trypsin and chymotrypsin; Preferably, the protease is collagenase, and the substrate protein is collagen, preferably animal collagen, more preferably an extract of pig skin, cow skin and / or beef tendon.

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