Method and kit for identifying bear gall powder by biological enzyme method

Abstract

The invention discloses a method for identifying bear gall powder by a biological enzyme method and a kit thereof. The method comprises the following steps: preparing a reagent A, preparing a sample solution, carrying out a chromogenic reaction and judging a result. The reagent A is composed of NAD (P), oxidized mPMS, WST-1 and 7beta-HSDH enzyme, and the sample solution T is prepared by dissolving a sample to be detected. The chromogenic reaction is carried out for 5-30 minutes at the temperature of 25-35 DEG C, and the content of TUDCA in the sample is judged by observing the color change. The method disclosed by the invention is rapid (less than 30 minutes), simple and convenient to operate, accurate in result, free of expensive instruments and equipment and suitable for non-professionals to operate. The kit developed in a matched manner comprises a pre-prepared reaction reagent, a standard substance and a detailed operation guide, and can realize on-site rapid detection of instant use.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine detection, and specifically relates to a method for identifying bear bile powder by a biological enzyme method and a kit thereof. Background Art

[0002] Bear bile powder is a powder made by drying bile from the gallbladder of black bears or brown bears in the Ursidae family through tubeless drainage. Due to the limited resources of bear bile powder obtained from artificial breeding, the market often mixes pig, cattle, and sheep bile with similar appearance and Huangbai or Huanglian extract, which seriously affects the quality of bear bile powder. The current methods for identifying bear bile powder include 1) traditional identification method; 2) thin layer chromatography (TLC); 3) high performance liquid chromatography (HPLC); 4) infrared spectroscopy (IR); 5) polymerase chain reaction (PCR). Traditional identification methods mainly identify bear bile powder by hand rubbing, oral tasting, water test, and fire burning, and the results often vary greatly due to different operator experience. Thin layer chromatography has too many components in bear bile powder and poor separation effect, among which tauroursodeoxycholic acid (TUDCA) unique to bear bile powder cannot be significantly distinguished. The HPLC UV detector has weak UV absorption and low sensitivity of bile acid substances. Currently, evaporative light scattering detectors are mostly used for quantitative detection of compounds such as tauroursodeoxycholic acid and taurochenodeoxycholic acid. Infrared spectroscopy and polymerase chain reaction are two newer detection methods. Because the composition of bear bile powder is complex and the infrared absorption spectrum is close to that of other adulterants, this method relies on professional equipment and analysts; polymerase chain reaction requires the design of specific primers, and the difference in species-specific cytochrome oxidase subunit I (COI) can be used to distinguish the quality of different bear bile powders, and the different fluorescence intensities obtained by fluorescence quantitative PCR can also be used to distinguish the quality of different bear bile powders, which also has high requirements for technicians.

[0003] There are no reports on the enzymatic identification of bear bile powder. The main characteristic compound that distinguishes bear bile powder from other bile powders is tauroursodeoxycholic acid. The structure of tauroursodeoxycholic acid is different from that of other bile acid compounds in that it has a 7β-hydroxyl group. Tauroursodeoxycholic acid (TUDCA) can be converted into tauro-7-ketolithocholic acid (T-7K-LCA) by the catalysis of 7β-hydroxysteroid dehydrogenase (7β-HSDH), and an equimolar amount of reduced coenzyme I (NADH) or coenzyme II (NADPH) is generated at the same time. The content of NADH / NADPH can be quantitatively detected by using a pair of electron coupling reagents 1-methoxy-5-methylphenazine methyl sulfate (mPMS) and water-soluble tetrazolium (WST-1). Compared with the five existing identification methods, the enzymatic detection of tauroursodeoxycholic acid in bear bile powder does not require the use of any instruments and equipment, and the detection can be completed by simply observing the color change. Specifically, the fake bear bile powder does not change color during the reaction and remains colorless, while the real bear bile powder will appear yellow due to the generation of NADPH / NADH. This method is simple, economical, efficient, and easy to operate, and even non-professionals can easily use it, providing a powerful tool for the market circulation and quality evaluation of bear bile powder. Summary of the invention

[0004] The purpose of the present invention is to provide a method for identifying bear bile powder by bioenzymatic method, which can quickly and accurately identify bear bile powder and its adulterants through specific enzyme reactions. The purpose of the present invention is also to provide a kit for identifying bear bile powder by enzymatic method, which is easy to operate, suitable for use by non-professionals, and can quickly identify the authenticity and quality of bear bile powder. The above purpose is achieved through the following method and principle: NADP, oxidized mPMS, WST-1, 7β-HSDH enzyme powders are dissolved in buffer to obtain reagent A, and the sample or standard to be tested is fully dissolved in the same buffer to obtain sample solution T or standard solution S. After mixing the sample solution T or standard solution S with reagent A for a period of time, the color change is observed. Yellow color indicates that the sample contains tauroursodeoxycholic acid, and the darker the color, the higher the tauroursodeoxycholic acid content. The color development principle is shown in FIG. Figure 1 .

[0005] The specific method of the present invention is as follows:

[0006] 1) Preparation of Reagent A

[0007] Prepare NADP, oxidized mPMS, and WST-1 at concentrations of 10 mM, 1 mM, and 10 mM, respectively, and then prepare 7β-HSDH enzyme solution at a concentration of 1-5 mg / ml. Mix the prepared NADP, oxidized mPMS, WST-1, and 7β-HSDH enzyme solution at a ratio of 1:1:1:1 to obtain a 25× mixed solution, and dilute the 25× mixed solution to 1× with buffer Tris-HCl or PBS to obtain reagent A.

[0008] 2) Preparation of sample solution T

[0009] Weigh the bile powder sample to be tested and dissolve it with methanol to obtain a 100× sample solution of 5 mg / ml. Then dilute the mother sample solution 100 times with Tris-HCl or PBS to obtain the sample solution T to be tested. At this time, the concentration of the bile powder is 0.05 g / L.

[0010] 3) Color reaction

[0011] Depending on the reaction volume, the color development reaction can be carried out in a 96-well plate, 1.5 ml / 2 ml EP tube or small test tube. Take an appropriate amount of sample solution T and add it to 10 times the volume of reagent A. Incubate at 25-35 ℃ for 5-30 min and observe the color change of the reaction solution.

[0012] 4) Standard product setting

[0013] In order to more accurately determine whether the sample T to be tested is adulterated with pseudo-bile powder, 2-3 groups of standard samples need to be set. According to the 11th volume of the Ministry of Health's drug standards for bear bile powder (WS3-09 (B-09)-96 (Z)), the quality standard for qualified bear bile powder is tauroursodeoxycholic acid content >23%. The basis for setting the concentration of standard sample S to 23 μM is as follows: the content of tauroursodeoxycholic acid in the sample to be tested is 23%, and when the sample to be tested is prepared with a concentration of 0.05 g / L, the concentrations of tauroursodeoxycholic acid and tauroursodeoxycholic acid standard samples are consistent, which is 23%×0.05 g / L / 499.68 g / mol≈23 μM (the molecular weight of sulfoursodeoxycholic acid is 499.68 g / mol). The tauroursodeoxycholic acid concentration of standard solution S2 is 35 μM (the tauroursodeoxycholic acid concentration of S2 solution is 1.5 times that of S1, and the concentration of tauroursodeoxycholic acid in gold bile powder is even higher). The color difference between S1 and S2 standards and the reference after color development can be used to visually determine the quality of bear bile powder.

[0014] 5) Result determination

[0015] The color reaction method of standard solutions S1 and S2 is consistent with that of sample solution T. When sample solution T is colorless after color reaction, it means that the sample is counterfeit bile powder; when the yellow color of sample solution T is lighter than that of S1 after color development, it means that the sample is adulterated with counterfeit products; when the yellow color of sample solution T is between that of standard solutions S1 and S2 after color development, it is ordinary bear bile powder; when the yellow color of sample solution T is darker than that of standard solution S2 after color development, it is gold bile powder.

[0016] The present invention also provides a kit for implementing the above method, which comprises: (1) NADP mother solution, the concentration is 10 mM, the volume is 1 ml; (2) oxidized mPMS mother solution, the concentration is 1 mM, the volume is 1 ml; (3) WST-1 mother solution, the concentration is 10 mM, the volume is 1 ml; (4) 7β-HSDH Rt -V3 enzyme solution, concentration of 1-5 mg / ml, volume of 1 ml; (5) buffer Tris-HCl or PBS, concentration of 100 mM, pH7.5, total volume of 100 mL; (6) standard solutions S1 and S2, tauroursodeoxycholic acid concentrations of 23 μM and 35 μM, respectively, volume of 200 μl each; (7) methanol, sample diluent, purity of chromatographic grade, total volume of 50 ml; (8) 1 instruction manual. The specific steps are as follows: 1) Mix the mother solution NADP, oxidized mPMS, WST-1, and 7β-HSDH enzyme solution in a ratio of 1:1:1:1 to obtain a 25× mixed solution, and dilute the 25× mixed solution to 1× with buffer Tris-HCl or PBS to obtain reagent A. Note that the prepared reagent A needs to be used up in time or stored in a dark place.

[0017] 2) Weigh the bile powder sample to be tested and dissolve it in methanol to obtain a 100× sample solution of 5 mg / ml. Then dilute the mother sample solution 100 times with Tris-HCl or PBS to obtain the sample solution T to be tested.

[0018] 3) Take an appropriate volume of the sample T to be tested and put it into the container, and take the same volume of the standard samples S1 and S2 and put them into the container.

[0019] 4) Add 200 μL of reagent A to the container and mix well.

[0020] 5) Incubate at 25-35℃ for 5-30 min and observe the color change of the reaction solution.

[0021] 6) Result determination: When the sample solution T is colorless after the color reaction, it means that the sample is a fake bile powder; when the sample solution T is lighter in yellow than S1 after the color reaction, it means that the sample is adulterated with a fake product; when the sample solution T is between the standard solution S1 and S2 after the color reaction, it is ordinary bear bile powder; when the sample solution T is darker in yellow than the standard solution S2 after the color reaction, it is gold bile powder. When it is difficult to determine with the naked eye, it can be further determined by diluting the sample into different gradients and comparing the color reaction with the standard, or measuring the light absorption value at 450nm.

[0022] Beneficial effects: 1) A simple visual colorimetric determination standard is established: Compared with the existing methods, which are time-consuming (>1 h), expensive equipment, and require high professional and technical personnel, the method of the present invention is fast (<30 min), easy to operate, and accurate. 2) A matching ready-to-use kit is provided: The kit contains all the reagents and standards required to implement the method, which can achieve rapid and accurate identification of the authenticity and quality evaluation of bear bile powder. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The reaction equation for coupling mPMS / WST-1 reaction of 7β-HSDH enzyme catalyzing T-UDCA to produce T-7K-LCA.

[0024] Figure 2 These are the color reaction results of different concentrations of TUDCA standard products, pig, cattle, and sheep counterfeits, Jiangxi Bangtai transformed bear bile powder, and commercially available Qingganling artificial bear bile powder (96-well plate).

[0025] Figure 3 After color development with different TUDCA concentrations (0-64 μM), 450 The absorbance value of (standard curve).

[0026] Figure 4 The color reaction results of TUDCA standard, converted bear bile powder and adulterated products in 1.5 ml EP tubes. DETAILED DESCRIPTION

[0027] In order to further illustrate the content of the present invention, the following examples are listed for illustration.

[0028] Example 1: Quantification of TUDCA content in bear bile powder by ELISA

[0029] 7β-HSDH mutant with high enzyme activity disclosed in Chinese patent CN113416717A Rt -V3 is freeze-dried to form freeze-dried powder, and the enzyme activity is still greater than 90% when stored at -20°C for more than 2 years, indicating that it has very good long-term stability and is applied to the enzymatic detection of the bear bile powder of the present invention.

[0030] (1) The preparation of relevant reagents in reagent A is as follows:

[0031] Preparation of Tris-HCl (100 mM, pH 7.5) buffer: weigh 1.21 g of tris (hydroxymethyl)aminomethane (Tris) and dissolve it in about 80 ml of purified water. Stir magnetically until completely dissolved, then adjust the pH to 7.5 with concentrated hydrochloric acid, and then dilute to 100 ml in a 100 ml volumetric flask.

[0032] Preparation of NADP or NADP-Na2 stock solution (10 mM, 100×): accurately weigh 37.2 mg NADP or 39.4 mg NADP-Na2 on an analytical balance and dissolve it in about 4 ml of purified water, then dilute to 5 ml mark in a 5 ml volumetric flask.

[0033] Preparation of oxidized mPMS mother solution (1 mM, 100×): accurately weigh 3.4 mg of 1-methoxy-5-methylphenazine methyl sulfate (oxidized mPMS) on an analytical balance and dissolve it in about 8 ml of purified water, then dilute to 10 ml mark with a 10 ml volumetric flask.

[0034] Preparation of WST-1 mother solution (10 mM, 100×): 32.6 mg of water-soluble WST-1 sodium salt (C 19 H 12 IN4NaO8S2) was dissolved in about 4 ml of purified water, and then the volume was made up to 5 ml mark with a 5 ml volumetric flask.

[0035] 7β-HSDH enzyme solution (1 mg / ml, 100×): 10 mg 7β-HSDH was accurately weighed on an analytical balance Rt -V3 freeze-dried enzyme powder was dissolved in about 8 ml of purified water, and then the volume was made up to 10 ml mark in a 10 ml volumetric flask.

[0036] Preparation of reagent A: 100 μl each of the above-mentioned NADP or NADP-Na2 mother solution (10 mM, 100×), oxidized mPMS mother solution (1 mM, 100×), WST-1 mother solution (10 mM, 100×), and 7β-HSDH enzyme solution (1 mg / ml, 100×) were mixed evenly to obtain 400 μl of reagent A (25×).

[0037] (2) Preparation of TUDCA gradient standard solution (0-64 μM)

[0038] 3.2 mg TUDCA standard powder was accurately weighed on an analytical balance and dissolved in about 80 ml of water, then diluted to 100 ml with a 100 ml volumetric flask to obtain a TUDCA standard solution with a concentration of 64 μM, which is standard point 7 in Table 1; then diluted with Tris-HCl (100 mM, pH 7.5) by 0.1, 0.2, 0.4, 0.6, and 0.8 times, respectively, to obtain 6.4, 12.8, 25.6, 38.4, and 51.2 μM TUDCA standard solutions, which are standard points 2-6 in Table 1, and the blank control is standard point 1.

[0039] (3) Preparation of sample solution

[0040] An analytical balance was used to accurately weigh 5 mg of pig, cattle, and sheep bile powder, as well as the transformed bear bile powder (Batch YF-20231003) from Jiangxi Bangtai Green Biosynthesis Industrial Park Co., Ltd. and the commercially available Qingganling bear bile powder, and dissolved in about 80 ml of Tris-HCl (100 mM, pH 7.5), and then diluted to 100 ml with a 100 ml volumetric flask.

[0041] (4) Color reaction

[0042] According to the dosage shown in Table 1 and Table 2, reagent A, Tris-HCl, TUDCA or the sample to be tested were added to the 96-well plate in sequence. Three replicates were set for each sample. After gently shaking horizontally for 10-15 seconds, the plate was incubated at 35°C for 20 minutes (the color change was as shown in Table 1). Figure 2 Then, the sample was taken out and put into the microplate reader for detection, and the detection wavelength was set to 450 nm and the box temperature was set to 35 °C. The raw data obtained were shown in Table 3.

[0043] It can be observed with the naked eye from the color development results that as the concentration of TUDCA increases, the yellow color becomes darker and darker; pig, cattle and sheep bile powder is colorless, while Jiangxi Bangtai converted bear bile powder and Qingganling bear bile powder are yellow, and the color of the converted bear bile powder is slightly darker than that of Qingganling bear bile powder.

[0044] Table 1: TUDCA standard curve drawing

[0045]

[0046] Table 2: Sample solution color reaction configuration system

[0047]

[0048] Table 3: Absorbance values ​​at 450 nm read by the microplate reader

[0049]

[0050] The TUDCA quantitative standard curve was obtained by plotting the 7 standard points of TUDCA and the absorbance value at 450 nm. Figure 3 As shown, the linear correlation equation y=0.0034x+0.0469 (R 2>0.99). The pig, cattle and sheep bile powders contained 2.0, 2.4 and 2.7 μM TUDCA, respectively, which were lower than the detection limit of 3 μM (LOD = 3 μM calculated based on 3 times the standard deviation of the blank sample OD value), equivalent to no TUDCA. The converted bear bile powder (YF-20231003 batch) and Qingganling bear bile powder contained 45.1±7.68 μM and 34.8±6.6 μM TUDCA, respectively, which were almost consistent with the HPLC method detection values ​​of 46.2 and 34.4 in Table 4 of Example 2. According to the setting of the standard product and the dilution of the sample to be tested to 0.05 g / L, it can be seen that the percentage value of the bear bile powder content is consistent with the concentration value of tauroursodeoxycholic acid in the bear bile powder. The linear relationship and method mutual verification show that the enzyme labeling method can quantitatively detect the content of TUDCA in bear bile powder, as low as 3 μM (the concentration of qualified bear bile powder detected by the above method should be greater than 23 μM).

[0051] Example 2: HPLC method to detect TUDCA content of samples

[0052] The samples in Table 2 of Example 1 were tested by HPCL method. The specific method is referenced to Li Xiaoqiong, Liu Yanting, Ye Caifa, et al. One-test-multiple-evaluation method for determining the content of various conjugated bile acids in bear bile powder [J]. Strait Pharmacy, 2016, 28(3):4. DOI:CNKI:SUN:HAIX.0.2016-03-025. The sample quantitative conversion adopts the external standard method, and the standard sample is configured with taurine ursodeoxycholic acid to 0.5 mg / ml. The samples are all configured to 2 mg / ml. The concentration of the test product is converted according to the peak area ratio, and the final calculated content of TUDCA in the samples in Table 2 is shown in Table 4 below, which is highly consistent with the enzyme labeling method detection data in Example 1.

[0053] Table 4: HPLC method to detect the content of TUDCA in different bile powder samples

[0054]

[0055] Example 3: 1.5 ml EP tube colorimetric identification of bear bile powder grade and adulteration

[0056] In order to directly observe the color to distinguish bear bile powder and its adulterants, as well as the difference in quality of different bear bile powders, two sets of standard products S1 (23 μM corresponds to the bear bile powder quality standard TUDCA 23%) and S2 (35 μM corresponds to the gold bile powder threshold) are set as references. After the color reaction of the test sample with S1 and S2, the color is compared to know the grade of the bear bile powder or whether it is an adulterant. The standard product configuration method refers to Example 1. The transformed bear bile powder YF-202308012 (No. T1), YF-202309001 (No. T2), YF-202309002 (No. T3), and YF-202309004 (No. T4) of Jiangxi Bangtai Green Biosynthesis Industrial Park Co., Ltd. were randomly selected, and the samples were processed according to the method for preparing the sample solution to be tested in Example 1; the preparation of adulterated samples is shown in Table 5, and 3 proportions of adulterated bear bile powder are set.

[0057] Table 5: Adulterated Bear Bile Powder

[0058]

[0059] The above samples S1, S2, T1, T2, T3, T4, and the adulterated samples pig 1, pig 2, pig 3, cattle 1, cattle 2, cattle 3, sheep 1, sheep 2, and sheep 3 in the table were prepared with the reaction system according to the conditions shown in Table 2 and subjected to color development reaction. After mixing by pipetting, the mixture was incubated in a constant temperature incubator at 35°C for 20 minutes. The color development reaction results were as follows: Figure 4 As shown. It is not difficult to see from the picture that compared with the standard products S1 and S2, the yellow of T1, T2, and T3 is darker than S1, and T1 and T2 are darker than S2, T3 is close to S2, and T4 is close to S1, indicating that the content of the four batches of converted bear bile powder is qualified, and T1 and T2 can be gold bile powder; while the other nine groups of bear bile powder mixed with pig, cattle, and sheep bile powder (slightly yellow can be seen by the naked eye) are lighter in color than S1, indicating that they are unqualified bear bile powder. Therefore, the authenticity and quality of bear bile powder can be identified through the EP tube color reaction.

[0060] Example 4: Preparation and use of the kit

[0061] The above-mentioned Examples 1-2 verified the accuracy of the visual detection method through experimental data. This example provides a kit for identifying bear bile powder by enzymatic method, the components of which include: (1) Component 1: NADP mother solution, with a concentration of 10 mM and a volume of 1 ml; (2) Component 2: oxidized mPMS mother solution with a concentration of 1 mM and a volume of 1 ml; (3) Component 3: WST-1 mother solution with a concentration of 10 mM and a volume of 1 ml; (4) Component 4: 7β-HSDH enzyme solution with a concentration of 1-5 mg / ml and a volume of 1 ml; (5) Component 5: buffer Tris-HCl or PBS with a concentration of 100 mM, pH 7.5, 100 mL; (6) Component 6: TUDCA standard solution with a concentration of 23 μM and 35 μM, with a volume of 200 μl each; (7) Component 7: methanol as a diluent for the sample to be tested, with a purity of chromatographic grade and a total volume of 50 ml; (8) Component 8: 1 instruction manual.

[0062] Instructions for use of the kit: 1) Preparation of working solution: Take 10 μL of each component 1-4, add 960 μL of component 5, mix well to obtain 1 mL of detection working solution; 2) Sample treatment: Take 5 mg of the sample to be tested, add 1 mL of component 7, vortex to dissolve, take 10 μL and dilute to 1 mL with component 5; 3) Detection: Take 20 μL of sample solution, add 200 μL of working solution, and react at 35°C for 20 minutes;

[0063] 4) Standard sample processing: Take 20 μL of standard samples S1 and S2 and process them simultaneously with the test samples; 5) Result interpretation: Compare the results with the color of the standard sample. When the sample solution T is colorless after the color reaction, it means that the sample is a fake bile powder; when the sample solution T is lighter in yellow than S1 after the color reaction, it means that the sample is adulterated with a fake; when the sample solution T is between the standard solution S1 and S2 after the color reaction, it is a general bear bile powder; when the sample solution T is darker in yellow than the standard solution S2 after the color reaction, it is a gold bile powder.

[0064] Kit stability test: The components of the kit were stored at -20°C for 12 months and then tested. The results showed that the activity retention rate of each component was >90%, which met the usage requirements.

[0065] Notes on the kit: Components 1-4 need to be kept frozen. If reagent A is not used up, it needs to be kept in a dark place and refrigerated. Use it up within 3 days.

[0066] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Therefore, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for identifying bear bile powder by bioenzymatic method, characterized in that: The following steps are involved: (1) Prepare reagent A: dissolve NADP, oxidized mPMS, WST-1, and 7β-HSDH enzyme powder in buffer to obtain reagent A; (2) Prepare sample solution: fully dissolve the sample or standard in buffer to obtain sample solution T or standard solution S; (3) Color reaction: mix sample solution T or standard solution S with reagent A, incubate for 5-30 minutes, and observe the color change; (4) Result determination: if the reaction solution changes from colorless to yellow, it indicates that the sample contains tauroursodeoxycholic acid.

2. The method according to claim 1, characterized in that The preparation method of the reagent A comprises: (1) preparing 100× mother solutions of NADP, oxidized mPMS and WST-1, with concentrations of 10 mM, 1 mM and 10 mM, respectively; (2) preparing 100× 7β-HSDH enzyme solution, with a concentration of 1-5 mg / ml; (3) mixing 100× mother solutions of NADP, oxidized mPMS, WST-1 and 7β-HSDH enzyme solution in a ratio of 1:1:1:1 to obtain a 25× mixed solution; (4) diluting the 25× mixed solution to 1× with a buffer solution of Tris-HCl or PBS to obtain reagent A.

3. The method according to claim 1, characterized in that: The preparation method of the sample solution T comprises: (1) Weigh the bile powder sample to be tested and dissolve it in methanol to obtain a 100× sample solution of 5 mg / ml; (2) Dilute the 100× sample solution 100 times with Tris-HCl or PBS to obtain the sample solution T to be tested.

4. The method according to claim 1, characterized in that: The color development reaction is carried out in a 96-well plate, a PCR tube, a 1.5 ml / 2 ml EP tube or a small test tube, the reaction temperature is 25-35° C., and the reaction time is 5-30 minutes.

5. The method according to claim 1, characterized in that: The result determination includes: (1) setting standard solutions S1 and S2, and the concentrations of the standard tauroursodeoxycholic acid are 23 μM and 35 μM, respectively; (2) if the sample solution T is colorless after color development, it is determined to be pseudo-bile powder; (3) if the yellow color of the sample solution T is lighter than that of S1 after color development, it is determined to be bear bile powder adulterated with counterfeit products; (4) if the yellow color of the sample solution T is between that of S1 and S2 after color development, it is determined to be ordinary bear bile powder; (5) if the yellow color of the sample solution T is darker than that of S2 after color development, it is determined to be gold bile powder.

6. The method according to claim 1, characterized in that The buffer is Tris-HCl or PBS buffer with a pH value of 7.0-7.

5.

7. The method according to claim 1, characterized in that The 7β-HSDH enzyme is 7β-HSDH Rt -V3 mutant, its enzyme activity remains above 90% after being stored at -20℃ for more than 2 years.

8. A kit for implementing the method according to any one of claims 1 to 7, characterized in that: The system includes the following components: (1) NADP stock solution, with a concentration of 10 mM and a volume of 1 ml; (2) oxidized mPMS stock solution, with a concentration of 1 mM and a volume of 1 ml; (3) WST-1 stock solution, with a concentration of 10 mM and a volume of 1 ml; (4) 7β-HSDH Rt -V3 enzyme solution, concentration is 1-5 mg / ml, volume is 1 ml; (5) Buffer solution Tris-HCl or PBS, concentration is 100 mM, pH 7.5, total volume is 100 mL; (6) Standard solutions S1 and S2, tauroursodeoxycholic acid concentrations are 23 μM and 35 μM, respectively, volume is 200 μl each; (7) Methanol, diluent for the sample to be tested, purity is chromatographic grade, total volume is 50 ml; (8) 1 instruction manual.

9. The kit according to claim 8, characterized in that The kit is used for rapidly identifying the content of tauroursodeoxycholic acid in bear bile powder, with a reaction time of 5-30 minutes and a reaction temperature of 25-35°C.

10. The kit according to claim 8, characterized in that The test kit is suitable for operation by non-professionals, and the authenticity and quality of the bear bile powder can be determined by observing the color change.

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