Tannic acid purity identification method

Abstract

The present invention discloses a method for identifying the purity of tannic acid. The method comprises the following steps: (1) preparing a standard solution and a sample solution to be tested; the standard solution comprises a tannic acid standard, a complexing agent, and a solvent; the sample solution to be tested comprises a tannic acid sample, a complexing agent, and a solvent; the complexing agent comprises a protein or a polypeptide; the concentration of the complexing agent in the standard solution and the sample solution to be tested is 1 to 10 mg / mL; and (2) performing ultraviolet detection on the standard solution and the sample solution to be tested, establishing a standard curve, and calculating the purity. The method improves the accuracy of tannic acid purity identification, is applicable to different tannic acid application environments, and has low cost and high operability.

Description

Technical Field

[0001] The invention relates to a method for identifying the purity of tannic acid. Background Art

[0002] Tannic acid, also known as tannic acid, is a polyphenolic compound extracted from the plant Gallnut. Its numerous phenolic hydroxyl groups can complex with proteins and polypeptides to form suspensions or precipitates. Therefore, it is widely used in the pharmaceutical field, such as tannic acid ointments, tannic acid proteins, and other tannic acid drugs, as well as sustained-release drugs such as tannic acid berberine and tannic acid vasopressin. Tannic acid contains a large number of phenolic hydroxyl groups, making it easily oxidized to form black quinones. This decrease in purity directly affects its ability to bind to proteins, and thus, the quality of the drug. In production practice, tannic acid often deteriorates due to oxidation and moisture due to improper storage or prolonged storage, affecting its purity and ultimately reducing product quality.

[0003] The inspection of tannic acid purity is relatively difficult. Normally, its color and properties can be directly observed, but this method can only simply judge the quality of tannic acid, is difficult to specify a clear and quantitative standard, and has limited effect. Liquid chromatography can also be used, but the tannic acid molar absorptivity is too high, and the phenolic hydroxyl groups it contains can be closely combined with the chromatographic column, thereby polluting, blocking, and damaging the chromatographic column, resulting in extremely high costs. The ultraviolet standard curve method can be analyzed by the tannic acid maximum absorption wavelength (213nm, 276nm), but the 25 phenolic hydroxyl groups possessed by the tannic acid molecule are easily oxidized, and the molar absorptivity changes very little after partial oxidation, making it difficult to distinguish by ultraviolet method, so the analysis result will be high and inaccurate, that is, the purity caused by tannic acid oxidation is reduced, and it is impossible to detect it by ultraviolet detection. Therefore, a kind of accurate, easy-to-operate, low-cost method is needed to detect tannic acid purity. Summary of the Invention

[0004] To address the problem of inaccurate analysis results caused by difficulty in detecting the oxidation state of multiple phenolic hydroxyl groups in tannic acid when using the UV standard curve method at the wavelength of maximum absorption in the prior art, the present invention provides a method for identifying tannic acid purity. This method, through the complexation of tannic acid with different proteins or polypeptides, detects stable tannic acid complexes at specific UV wavelengths, thereby improving the accuracy of tannic acid purity identification. The method is applicable to various tannic acid application environments, is low-cost, and highly operational.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] The present invention provides a method for identifying the purity of tannic acid, which comprises the following steps:

[0007] S1. Prepare standard solution and sample solution to be tested;

[0008] The standard solution comprises a tannic acid standard, a complexing agent and a solvent;

[0009] The sample solution to be tested comprises a tannic acid sample, a complexing agent and a solvent;

[0010] The complexing agent includes a protein or a polypeptide;

[0011] The concentration of the complexing agent in the standard solution and the sample solution to be tested can be 1 to 10 mg / mL;

[0012] S2. Perform ultraviolet detection on the standard solution and the sample solution to be tested, establish a standard curve, and calculate the purity.

[0013] In the present invention, the protein or polypeptide can be defined as a protein or polypeptide that can form a complex with tannic acid. Generally, a peptide consisting of 10 to 50 amino acids is called a polypeptide; a peptide consisting of more than 50 amino acids is called a protein.

[0014] In step S1, the molecular weight of the protein or polypeptide may be 0.5 to 100 KDa.

[0015] In step S1, the protein is preferably trypsin, chymotrypsin, thrombin, hyaluronidase, corticotropin or bovine serum albumin; the polypeptide is preferably oxytocin, vasopressin or insulin.

[0016] In some preferred embodiments, the complexing agent is selected from chymotrypsin (25 KDa), trypsin (23 KDa) or oxytocin (1007.2 Da).

[0017] In step S1, the mass ratio of the tannic acid standard to the complexing agent in the standard solution may be (0-0.3448):1.

[0018] In step S1, the mass ratio of the tannic acid sample to the complexing agent in the sample solution to be tested may be (0-0.3448):1.

[0019] In step S1, the solvent may be water, preferably purified water or deionized water.

[0020] In step S1, the preparation of the standard solution includes: preparing a tannic acid standard solution and preparing a complexing agent solution, and mixing the tannic acid standard solution and the complexing agent solution.

[0021] The preparation of the tannic acid standard solution may include: dissolving the tannic acid standard in purified water.

[0022] The concentration series of the tannic acid standard solution are preferably 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 mg / mL.

[0023] The preparation of the complexing agent solution may include: dissolving the complexing agent in a phosphate buffer solution. The concentration of the phosphate buffer solution is preferably 0.01 to 0.2 M, for example, 0.1 M. The pH of the phosphate buffer solution is preferably 7.0.

[0024] Wherein, the mixing operation is preferably to add the tannic acid standard solution into the complexing agent solution.

[0025] Wherein, the volume ratio of the tannic acid standard solution to the complexing agent solution is preferably 1:29.

[0026] The mixing step is preferably performed on a shaker. The shaker may have a rotation speed of 150 rad / min. The shaker may have a temperature of 20 to 30° C., preferably 25° C. The mixing time may be 0.5 to 2 h.

[0027] In step S1, the preparation of the sample solution to be tested includes: preparing a tannic acid sample solution and preparing a complexing agent solution, and mixing the tannic acid sample solution and the complexing agent solution.

[0028] Wherein, the preparation of the tannic acid sample solution may include: dissolving the tannic acid sample in purified water.

[0029] Wherein, the series concentration of the tannic acid sample solution is preferably 0.1-10 mg / mL.

[0030] The preparation of the complexing agent solution may include: dissolving the complexing agent in a phosphate buffer solution. The concentration of the phosphate buffer solution is preferably 0.01 to 0.2 M, for example, 0.1 M. The pH of the phosphate buffer solution is preferably 7.0.

[0031] Wherein, the mixing operation is preferably to add the tannic acid sample solution into the complexing agent solution.

[0032] Wherein, the volume ratio of the tannic acid sample solution to the complexing agent solution is preferably 1:29.

[0033] The mixing step is preferably performed on a shaker. The shaker may have a rotation speed of 150 rad / min. The shaker may have a temperature of 20 to 30° C., preferably 25° C. The mixing time may be 0.5 to 2 h.

[0034] In step S2, the wavelength of the ultraviolet detection may be 680 nm.

[0035] In step S2, the temperature of the ultraviolet detection may be 20-25°C.

[0036] In step S2, the ultraviolet detection can be performed using a conventional ultraviolet spectrophotometer in the art, such as MAPADA UV-3100.

[0037] In step S2, the standard curve can be established according to conventional methods in the art, generally comprising: using the tannic acid concentration of the tannic acid standard solution as the abscissa and the absorbance of the standard solution obtained by ultraviolet detection as the ordinate, producing an absorbance-tannic acid concentration curve, and fitting the curve using a linear equation to obtain the standard curve. The linear equation fitting can be performed using Origin data processing software.

[0038] In step S2, the calculation of the purity can be conventional in the art, generally including: substituting the absorbance of the sample solution to be tested obtained by the ultraviolet detection into the standard curve to obtain the tannic acid determination concentration of the tannic acid sample solution, and the ratio of the tannic acid determination concentration of the tannic acid sample solution to the tannic acid concentration of the tannic acid sample solution is the purity of the tannic acid sample.

[0039] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0040] The reagents and raw materials used in the present invention are commercially available.

[0041] The positive progress effect of the present invention is:

[0042] The present invention proposes a method for identifying the purity of tannic acid. A standard curve is prepared by detecting the absorbance of a suspension formed by complexing tannic acid with different proteins or polypeptides, thereby obtaining the purity of tannic acid. The suspension is relatively stable, which improves the accuracy of tannic acid purity identification and is suitable for different tannic acid application environments, with an RSD of ≤2% under different application environments. The method is highly operable and low-cost, can be carried out simultaneously with industrial production, and data results are fed back in real time. At the same time, it helps to analyze actual production deviations, find process optimization directions, and provide data support for process improvements. In pharmaceutical industrial production, it can become a rapid detection method that accurately determines the purity of tannic acid, which is of great significance to the production and quality of medicines. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the absorbance-tannic acid concentration standard curve in Example 1 and Example 2.

[0044] Figure 2 This is the absorbance-tannic acid concentration standard curve in Example 3 and Example 4.

[0045] Figure 3 This is the absorbance-tannic acid concentration standard curve in Example 5 and Example 6.

[0046] Figure 4 This is the binding curve of tannic acid standard and chymotrypsin in Comparative Example 1.

[0047] Figure 5 This is the binding curve of tannic acid standard and chymotrypsin in Comparative Example 2.

[0048] Figure 6 This is the chromatogram of the 10 μg / mL tannic acid standard in Comparative Example 3.

[0049] Figure 7 This is the standard curve of integrated area of ​​tannic acid standard eluted through the chromatographic column in Comparative Example 3 - tannic acid concentration. DETAILED DESCRIPTION

[0050] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0051] The reagents used in the following examples are shown in Table 1. All purchased reagents were used directly after purchase without further purification unless otherwise specified.

[0052] Among them, the tannic acid sample No. 1 used in the following examples is the remaining product after 2 years of use, the tannic acid sample No. 2 is the remaining product after 1 year of use, and the tannic acid sample No. 3 is a newly purchased product.

[0053] The UV detection data in the following examples were all measured by an UV spectrophotometer, and the instrument model used was MAPADA UV-3100.

[0054] Table 1 Relevant information of the reagents used in the examples of the present invention

[0055]

[0056]

[0057] Example 1

[0058] Prepare a 5mg / mL standard high concentration chymotrypsin solution with 0.1M phosphate buffer (pH=7.0). Prepare 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10mg / mL tannic acid standard solutions with purified water. After complete dissolution, take 100μL of the tannic acid standard solution and add it dropwise to 2900μL of the standard chymotrypsin solution. Incubate on a shaker at 25℃ for 30min at a speed of 150rad / min to obtain standard solutions of different concentrations. Take samples of the above standard solutions and measure the ultraviolet absorbance at 680nm to make an absorbance-tannic acid concentration standard curve, such as Figure 1 a.

[0059] Use origin data processing software and select linear fitting as follows:

[0060] A=kx

[0061] Where A is the absorbance of the standard solution, x is the tannic acid concentration of the tannic acid standard solution, and k is the linear coefficient.

[0062] Dissolve the tannic acid sample in water to prepare a 5 mg / mL tannic acid sample solution. Take 100 μL of the sample and add it dropwise into 2900 μL of the above standard chymotrypsin solution. After complexation according to the above steps, obtain the sample solution to be tested. Take a sample and measure the ultraviolet absorbance A at 680 nm. Repeat 5 times to obtain A 1～5 , put it into the above standard curve, and get the tannic acid sample concentration of the tannic acid sample solution as x 1～5 Take the average value x as the concentration of the tannic acid sample and calculate the purity of the tannic acid sample by the following formula:

[0063] y=x / c*100%

[0064] Wherein, y is the purity of the tannic acid sample, and c is the tannic acid concentration of the tannic acid sample solution.

[0065] Purity data of three groups of tannic acid samples were obtained according to the above method. The relevant data are shown in Tables 2-4.

[0066] Example 2

[0067] The protein solution in Example 1 was changed to a 1 mg / mL standard low concentration chymotrypsin solution. The complexation condition was changed to incubation on a shaker at 25°C for 2 h. The rest of the operation was the same as in Example 1. The standard curve was obtained as shown in FIG. Figure 1 b.

[0068] The same three groups of tannic acid samples were treated under the conditions of Example 1 and then added to the standard protein solution for complexation. The absorbance was repeatedly measured and incorporated into the standard curve. The remaining operations were the same as in Example 1. The relevant data are shown in Tables 2-4.

[0069] Example 3

[0070] The protein solution in Example 1 was changed to a 5 mg / mL standard high concentration trypsin solution. The remaining operations were the same as in Example 1, and the standard curve was obtained as shown in FIG. Figure 2 a.

[0071] The same three groups of tannic acid samples were treated under the conditions of Example 1 and then added to the standard protein solution for complexation. The absorbance was repeatedly measured and incorporated into the standard curve. The remaining operations were the same as in Example 1. The relevant data are shown in Tables 2-4.

[0072] Example 4

[0073] The protein solution in Example 1 was changed to a 1 mg / mL standard low concentration trypsin solution. The complexation condition was changed to incubation on a shaker at 25°C for 2 h. The rest of the operation was the same as in Example 1. The standard curve was obtained as shown in FIG. Figure 2 b.

[0074] The same three groups of tannic acid samples were treated under the conditions of Example 1 and then added to the standard protein solution for complexation. The absorbance was repeatedly measured and incorporated into the standard curve. The remaining operations were the same as in Example 1. The relevant data are shown in Tables 2-4.

[0075] Example 5

[0076] The protein solution in Example 1 was changed to a 5 mg / mL standard high concentration oxytocin solution. The remaining operations were the same as in Example 1, and a standard curve was obtained as shown in FIG. Figure 3 a.

[0077] The same three groups of tannic acid samples were treated under the conditions of Example 1 and then added to the standard protein solution for complexation. The absorbance was repeatedly measured and incorporated into the standard curve. The remaining operations were the same as in Example 1. The relevant data are shown in Tables 2-4.

[0078] Example 6

[0079] The protein solution in Example 1 was changed to a 1 mg / mL standard low concentration oxytocin solution. The complexation condition was changed to incubation on a shaker at 25°C for 2 h. The rest of the operation was the same as in Example 1. The standard curve was obtained as shown in FIG. Figure 3 b.

[0080] The same three groups of tannic acid samples were treated under the conditions of Example 1 and then added to the standard protein solution for complexation. The absorbance was repeatedly measured and incorporated into the standard curve. The remaining operations were the same as in Example 1. The relevant data are shown in Tables 2-4.

[0081] Table 2 Purity data of tannic acid sample No. 1 in different embodiments

[0082]

[0083] Table 3 Purity data of tannic acid sample No. 2 in different embodiments

[0084]

[0085] Table 4 Purity data of tannic acid sample No. 3 in different embodiments

[0086]

[0087] As shown in Tables 2-4, the RSD of the purity of the same group of tannic acid samples determined by the standard curve method under different application environments was ≤2%. This indicates that this method is not limited by the protein concentration and system used to determine the purity of tannic acid and has good accuracy and repeatability.

[0088] Comparative Example 1

[0089] The protein solution in Example 1 was changed to a 20 mg / mL standard high concentration chymotrypsin solution. The remaining operations were the same as in Example 1. Figure 4 a. The obtained binding curve cannot be fitted linearly, and the subsequent tannic acid purity determination cannot be performed.

[0090] The protein solution in Example 1 was changed to a 0.5 mg / mL standard low-concentration chymotrypsin solution. The complexation condition was changed to incubation on a shaker at 25°C for 2 h. The remaining operations were the same as in Example 1. Figure 4 b. The obtained binding curve cannot be fitted linearly, and the subsequent tannic acid purity determination cannot be performed.

[0091] Comparative Example 2

[0092] The tannic acid standard solution in Example 1 was changed to 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 mg / mL, and the remaining operations were the same as in Example 1. Figure 5 As shown, the obtained binding curve cannot be fitted linearly, and the subsequent tannic acid purity determination cannot be performed.

[0093] Comparative Example 3

[0094] Tannic acid standards were added to purified water to prepare reference stock solutions at concentrations of 2, 4, 6, 8, and 10 μg / mL, respectively. Tannic acid samples 1-3 were prepared as test solutions at 5 μg / mL. Purified water was used as the blank solvent. The chromatographic column was a Zorbax C18 (250 mm × 4.6 mm, 5 μm); the mobile phase was acetonitrile (A)-0.1% phosphoric acid (B), with a gradient elution (0-4 min, 2%-5% A; 4-5 min, 5%-8% A; 5-6 min, 8%-15% A; 6-15 min, 15% A; 15-25 min, 15%-23% A; 25-30 min, 23% A); the flow rate was 1.0 mL / min; the detection wavelength was 276 nm; the column temperature was 25°C; and the injection volume was 10 μL. The theoretical plate number is not less than 5000. The chromatogram of 10 μg / mL tannic acid standard is shown in Figure 6 The concentration of tannic acid standard was used as the horizontal axis and the corresponding integrated area obtained by elution was used as the vertical axis to prepare the following Figure 7 The integrated area-tannic acid concentration standard curve is shown. Tannic acid samples 1-3 were tested under the same conditions, and the obtained integrated areas were substituted into the standard curve equation. The corresponding data obtained by calculation are shown in Table 5.

[0095] Table 5 HPLC measurement of tannic acid purity

[0096]

[0097] As is known in the art, HPLC, as one of the commonly used quantitative analysis techniques, is also commonly used in applications such as substance purity identification. Regarding the identification of tannic acid purity, although the prior art does not record the use of HPLC as a standard method for tannic acid purity identification, the results obtained by this method for tannic acid purity detection still have an accuracy acceptable in the art.

[0098] Combining the data in Table 5 with those in Tables 2-4, it can be seen that the data obtained by the method of the present invention are similar to those obtained by HPLC, indicating that the method of the present invention has good accuracy. However, the method of the present invention is also more cost-effective and easier to operate.

[0099] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with this patent can make slight changes or modifications to equivalent embodiments using the above technical content without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the solution of the present invention.

Claims

1. A method for identifying the purity of tannic acid, characterized in that: The tannic acid purity identification method comprises the following steps: (1) preparing a standard solution and a sample solution to be tested; The standard solution comprises a tannic acid standard, a complexing agent and a solvent; The sample solution to be tested comprises a tannic acid sample, a complexing agent and a solvent; The complexing agent includes a protein or a polypeptide; the protein is trypsin or chymotrypsin; the polypeptide is oxytocin; The concentration of the complexing agent in the standard solution and the sample solution to be tested is 1 to 10 mg / mL; (2) performing ultraviolet detection on the standard solution and the sample solution to be tested, wherein the wavelength of the ultraviolet detection is 680 nm; Establish a standard curve and calculate the purity.

2. The method for identifying the purity of tannic acid according to claim 1, wherein In step (1), the molecular weight of the protein or polypeptide is 0.5 to 100 KDa.

3. The method for identifying the purity of tannic acid according to claim 1, wherein In step (1), the mass ratio of the tannic acid standard to the complexing agent in the standard solution is (0-0.3448):1; And / or, in step (1), the mass ratio of the tannic acid sample to the complexing agent in the sample solution to be tested is (0-0.3448):1; And / or, in step (1), the solvent is water.

4. The method for identifying the purity of tannic acid according to claim 1, wherein In step (1), the solvent is purified water or deionized water.

5. The method for identifying the purity of tannic acid according to claim 1, wherein In step (1), the preparation of the standard solution includes: preparing a tannic acid standard solution and preparing a complexing agent solution, and mixing the tannic acid standard solution and the complexing agent solution.

6. The method for identifying the purity of tannic acid according to claim 5, wherein: The preparation of the tannic acid standard solution comprises: dissolving the tannic acid standard in purified water; and / or, the series of concentrations of the tannic acid standard solution is 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 mg / mL; And / or, the preparation of the complexing agent solution comprises: dissolving the complexing agent in a phosphate buffer solution.

7. The method for identifying the purity of tannic acid according to claim 6, wherein: The preparation of the complexing agent solution comprises: dissolving the complexing agent in a phosphate buffer solution; wherein the concentration of the phosphate buffer solution is 0.01-0.2M.

8. The method for identifying the purity of tannic acid according to claim 7, wherein: The concentration of the phosphate buffer is 0.1 M; And / or, the pH of the phosphate buffer is 7.

0.

9. The method for identifying the purity of tannic acid according to claim 5, wherein: The mixing operation is to add the tannic acid standard solution into the complexing agent solution.

10. The method for identifying the purity of tannic acid according to claim 9, wherein: The volume ratio of the tannic acid standard solution to the complexing agent solution is 1:29; And / or, the mixing step is performed using a shaker; And / or, the mixing time is 0.5 to 2 hours.

11. The method for identifying the purity of tannic acid according to claim 10, wherein: The rotation speed of the shaking table is 150 rad / min; And / or, the temperature of the shaking table is 20-30°C.

12. The method for identifying the purity of tannic acid according to claim 11, wherein: The temperature of the shaker was 25°C.

13. The method for identifying the purity of tannic acid according to claim 5, wherein: In step (2), the establishment of the standard curve includes: taking the tannic acid concentration of the tannic acid standard solution as the horizontal coordinate and the absorbance of the standard solution obtained by ultraviolet detection as the vertical coordinate, making an absorbance-tannic acid concentration curve, and using linear equation fitting to obtain the standard curve.

14. The method for identifying the purity of tannic acid according to claim 13, wherein: The linear equation fitting was performed using origin data processing software.

15. The method for identifying the purity of tannic acid according to claim 1, wherein: In step (1), the preparation of the sample solution to be tested includes: preparing a tannic acid sample solution and preparing a complexing agent solution, and mixing the tannic acid sample solution and the complexing agent solution.

16. The method for identifying the purity of tannic acid according to claim 15, wherein: The preparation of the tannic acid sample solution comprises: dissolving the tannic acid sample in purified water; and / or, the concentration of the tannic acid sample solution is 0.1 to 10 mg / mL; And / or, the preparation of the complexing agent solution comprises: dissolving the complexing agent in a phosphate buffer solution.

17. The method for identifying the purity of tannic acid according to claim 16, wherein: The concentration of the phosphate buffer is 0.01 to 0.2 M; And / or, the pH of the phosphate buffer is 7.

0.

18. The method for identifying the purity of tannic acid according to claim 17, wherein: The concentration of the phosphate buffer is 0.1M.

19. The method for identifying the purity of tannic acid according to claim 15, wherein: In step (1), the mixing operation is to add the tannic acid sample solution to the complexing agent solution.

20. The method for identifying the purity of tannic acid according to claim 19, wherein: The volume ratio of the tannic acid sample solution to the complexing agent solution is 1:29; And / or, the mixing step is performed using a shaker; And / or, the mixing time is 0.5 to 2 hours.

21. The method for identifying the purity of tannic acid according to claim 20, wherein: The rotation speed of the shaking table is 150 rad / min; And / or, the temperature of the shaking table is 20-30°C.

22. The method for identifying the purity of tannic acid according to claim 21, wherein: The temperature of the shaker was 25°C.

23. The method for identifying the purity of tannic acid according to claim 15, wherein: In step (2), the calculation of the purity includes: substituting the absorbance of the sample solution to be tested obtained by the ultraviolet detection into the standard curve to obtain the tannic acid determination concentration of the tannic acid sample solution, and the ratio of the tannic acid determination concentration of the tannic acid sample solution to the tannic acid concentration of the tannic acid sample solution is the purity of the tannic acid sample.

24. The method for identifying the purity of tannic acid according to any one of claims 1 to 23, wherein: In step (2), the temperature of the ultraviolet detection is 20-25°C; And / or, in step (2), the ultraviolet detection is performed using an ultraviolet spectrophotometer.

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