Toad skin extract and chemical component separation method or fingerprint spectrum construction method and quality control method of preparation thereof

Abstract

The invention relates to the field of quality analysis and evaluation of Chinese patent medicines, and particularly provides a toad skin extract and a separation method of chemical components of a preparation of the toad skin extract or a construction method of a fingerprint spectrum and a quality control method. Octadecylsilane chemically bonded silica is used as a filler, a mobile phase comprises acetonitrile and water, gradient elution is carried out, a specific elution procedure is obtained through a large number of experiment screening, and the fingerprint spectrum is obtained. The method realizes effective separation of seven characteristic peaks of bufotalin, arenobufogenin, bufagin, deacetylcinobufotalin, telocinin, bufotalin and cinobufotalin, has the advantages of good peak shape, stable base line, complete separation of the measured components, short detection time, rapidness and high efficiency, provides a basis for quality detection and control of cinobufotalin tablets, and has a wide application prospect. The integrity and characteristics of cinobufagin tablets can be fully reflected, the operation is simple, and the specificity is strong.

Description

Technical Field

[0001] The invention relates to the field of quality analysis and evaluation of Chinese patent medicines, and in particular to a method for separating the chemical components of a toad skin extract and a preparation thereof, or a method for constructing a fingerprint and a quality control method. Background Art

[0002] Huachansu tablets are extracted and separated from the skin of Chinese giant toad (Bufo bufo gargarizans Cantor) or black-spotted toad (Bufomelanostictus Schneider). They are a second-class new drug independently developed in my country and a national protected traditional Chinese medicine variety.

[0003] Huachansu tablets are an anti-tumor preparation with definite clinical efficacy and few side effects. They are widely used in a variety of malignant tumors, such as liver cancer, lung cancer, pancreatic cancer, colon cancer, leukemia, etc., as well as chronic hepatitis B. Huachansu tablets are used in middle and late stage tumors to stabilize the condition or reduce the size of the tumor, enhance the patient's immunity, relieve cancer pain, and improve the patient's quality of life. When used in combination with chemotherapy drugs and radiotherapy, they increase the patient's tolerance to radiotherapy and chemotherapy, and increase the sensitivity of tumor cells to the apoptosis-inducing effects of radiotherapy and chemotherapy, which can play a role in increasing efficacy and reducing toxicity.

[0004] At present, the production implementation standard of Huachansu tablets is the drug standard of the Ministry of Health (the fourteenth volume of Chinese medicine formula preparations WS3-B-2687-97), which only has qualitative identification items and no content determination items, and fails to implement comprehensive quality control of Huachansu tablets. The prior art discloses the HPLC fingerprint study of toad skin medicinal materials from different origins, and the chromatographic conditions are: Dalian Yilite SinoChrom ODS-BP chromatographic column (4.6mm×250mm, 5um), mobile phase A is 0.5% potassium dihydrogen phosphate-0.1% glacial acetic acid (phosphoric acid adjusts pH=2.4), mobile phase B is acetonitrile, flow rate is 0.8mL / min, column temperature is 30℃, detection wavelength is 296nm, elution is carried out according to the mobile phase gradient elution program, and the common mode of HPLC fingerprint of toad skin medicinal materials is established by this method, but the detection time of this method is 85min, which is time-consuming and inefficient. Summary of the invention

[0005] The object of the present invention is to provide a method for separating the chemical components of a toad skin extract and a preparation thereof, or a method for constructing a fingerprint and a method for quality control. The method for constructing a fingerprint of the present invention is time-saving and has good peak separation in the fingerprint, thereby achieving overall quality control of cinobucina tablets. Seven substances have also been identified, namely, bufotoxin, bufotoxin, bufotoxin, deacetylcinobucina, telecinobucina, bufotoxin, and cinobucina.

[0006] To this end, the present invention discloses a method for separating the chemical components of a toad skin extract and a preparation thereof or a method for constructing a fingerprint, comprising the following steps:

[0007] (1) Preparation of test solution;

[0008] (2) The sample solution was tested by high performance liquid chromatography. The filler was octadecylsilane bonded silica gel. The mobile phase included acetonitrile and water. The gradient elution program included: 0→2 min→15 min→25 min→35 min→40 min→45 min. The volume percentage of acetonitrile in the mobile phase was 22%→22%→24%→30%→60%→22%→22%.

[0009] Further, step (1) comprises:

[0010] 1) Take the object to be tested, add a solvent to extract it, and obtain an extract;

[0011] 2) Separate the solid and liquid of the extract and take the liquid, which is the test solution;

[0012] Preferably, step (1) also satisfies any one or more of the following AE:

[0013] A. In step 1), the solvent is selected from methanol and a methanol aqueous solution having a volume percentage concentration greater than or equal to 50%;

[0014] B. In step 1), the extraction method is heating reflux extraction, and the extraction time is 90-150 min; more preferably, the extraction time is 120 min;

[0015] C. In step 1), the ratio of the mass of the analyte to the volume of the solvent is 1 g: 8-50 mL;

[0016] D. In step 2), the solid-liquid separation is centrifugation or filtration; more preferably, the centrifugal speed is 10000-15000 rpm, and the time is 5-15 min; more preferably, the specification of the filter membrane for filtration is 0.22-0.80 μm;

[0017] E. In step 1), the analyte is a piece of Sinobufacillus toad.

[0018] Furthermore, the ratio of the mass of the analyte to the volume of the solvent is 1 g:20-30 mL.

[0019] Furthermore, the chromatographic conditions of the high performance liquid chromatography in step (2) also include: a detection wavelength of 190-400 nm, a flow rate of 1.0-2.0 ml / min, a column temperature of 30-35° C., and an injection volume of 20-50 μl;

[0020] Preferably, the specifications of the chromatographic column are 4.6×250 mm, 5 μm.

[0021] Furthermore, the detection wavelength is 296 nm.

[0022] Furthermore, the construction method also includes the step of preparing a reference solution using at least one reference substance selected from the group consisting of bufotoxin, bufotoxin, bufotoxin, deacetylcintoxin, telocinobufacine, bufotoxin, and cinobufacine, and the step of detecting the reference solution by high performance liquid chromatography according to any of the construction methods to obtain a reference spectrum of the reference substance.

[0023] The present invention provides a fingerprint obtained by the above construction method, wherein the fingerprint is selected from any one of the following (1)-(3):

[0024] (1) It has 7 characteristic peaks, with peak 2 as the reference peak, and the relative retention time of each characteristic peak and the reference peak is within the range of ±5% of the specified value; the specified value is: 0.62 (peak 1), 1.00 (peak 2), 1.12 (peak 3), 1.17 (peak 4), 1.50 (peak 5), 1.53 (peak 6), 1.59 (peak 7);

[0025] (2) It has 7 characteristic peaks, with the peak corresponding to the peak of the reference substance of the ferret toxin as the reference peak, and the relative retention time of each characteristic peak and the reference peak is within the range of ±5% of the specified value, and the specified value is: 0.62 (peak 1), 1.00 (peak 2), 1.12 (peak 3), 1.17 (peak 4), 1.50 (peak 5), 1.53 (peak 6), 1.59 (peak 7);

[0026] (3) The fingerprint map is constructed according to the above construction method.

[0027] The invention also provides application of the construction method in quality control of toad skin extract and preparation thereof.

[0028] The present invention also provides a quality control method for a toad skin extract and a preparation thereof, comprising the step of comparing the fingerprint of a toad skin extract product to be tested with the fingerprint of the toad skin extract and the preparation thereof; the fingerprint of the toad skin extract product to be tested is obtained using the toad skin extract product to be tested according to the above-mentioned construction method, and the fingerprint of the toad skin extract and the preparation thereof is the fingerprint of the above-mentioned toad skin extract and the preparation thereof.

[0029] The technical solution of the present invention has the following advantages:

[0030] 1. The present invention provides a method for separating the chemical components of a toad skin extract and a preparation thereof or a method for constructing a fingerprint, using octadecylsilane bonded silica gel as a filler, a mobile phase comprising acetonitrile and water, and gradient elution. A specific elution procedure is obtained through a large number of experimental screenings to obtain a fingerprint, and effective separation of seven characteristic peaks including bufotoxin, bufotoxin, bufotoxin, deacetylcinobufolin, telcinobufolin, bufotoxin, and cinobufolin is achieved. The peak shape is good, the baseline is stable, the measured components can be completely separated, the detection time is short, rapid, and efficient, which provides a basis for the quality inspection and control of toad skin extracts and preparations thereof, can fully reflect the integrity and characteristic of toad skin extracts and preparations thereof, is simple to operate, and has strong specificity.

[0031] 2. The separation method of the chemical components of the toad skin extract and its preparation provided by the present invention or the construction method of the fingerprint spectrum, after identification by the reference substance, peaks 1-7 are bufotalin, bufotalin, deacetyl cinobucinatoxin, telocinobufant, bufotalin, cinobucinatoxin, all of which are well separated and have moderate areas. The accuracy of the quality control of the toad skin extract and its preparation can be achieved in a qualitative manner by using one or more of bufotalin, bufotalin, bufotalin, deacetyl cinobucinatoxin, telocinobufant, bufotalin, cinobucinatoxin as reference. The 7 characteristic peaks determined in the fingerprint spectrum can well guarantee the accuracy of the method in the quality control of the toad skin extract and its preparation.

[0032] 3. The quality control method of the toad skin extract and its preparation provided by the present invention controls the stability of the quality of the characteristic components of the toad skin extract and its preparation as a whole, and the method is simple to operate, has high precision, good stability, good repeatability, high accuracy and fast analysis rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0034] Figure 1 It is the fingerprint of the test solution in Example 1 of the present invention.

[0035] Figure 2 It is the chromatogram of the reference substance solution in Example 1 of the present invention.

[0036] Figure 3It is an overlay of the chromatogram S2 of the reference substance solution of the bufalin reference substance and the chromatogram S1 of the test solution in Example 1 of the present invention. From top to bottom are the chromatogram S2 and the chromatogram S1.

[0037] Figure 4 It is an overlay of the chromatogram S2 of the reference substance solution of the bufotoxin in Example 1 of the present invention and the chromatogram S1 of the test solution. From top to bottom are the chromatogram S2 and the chromatogram S1.

[0038] Figure 5 It is an overlay of the chromatogram S2 of the reference substance solution of the toad taridine reference substance and the chromatogram S1 of the test solution in Example 1 of the present invention. From top to bottom are the chromatogram S2 and the chromatogram S1.

[0039] Figure 6 It is an overlay of the chromatogram S2 of the reference substance solution of deacetyl cinobufolin and the chromatogram S1 of the test solution in Example 1 of the present invention. From top to bottom are the chromatogram S2 and the chromatogram S1.

[0040] Figure 7 It is an overlay of the chromatogram S2 of the reference substance solution of telocinobufagin and the chromatogram S1 of the test solution in Example 1 of the present invention. From top to bottom are the chromatogram S2 and the chromatogram S1.

[0041] Figure 8 It is an overlay of the chromatogram S2 of the bufalin reference substance solution and the chromatogram S1 of the test solution in Example 1 of the present invention. From top to bottom are the chromatogram S2 and the chromatogram S1.

[0042] Fig. 9 It is an overlay of the chromatogram S2 of the reference substance solution of the Chinese bufotoxin in Example 1 of the present invention and the chromatogram S1 of the test solution. From top to bottom are the chromatogram S2 and the chromatogram S1.

[0043] Fig.10 It is an overlay of the chromatograms of 10 batches of test solution and the chromatogram of the cinobacillus reference solution in Example 2 of the present invention; wherein, S1: the chromatogram of the reference solution; S2-S11 are chromatograms of the test solution prepared from different batches of cinobacillus tablets, from top to bottom, they are chromatograms S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11. The batch numbers of cinobacillus tablets are: S2: 220901; S3: 220902; S4: 220903; S5: 220904; S6: 220905; S7: 220906; S8: 220907; S9: 220908; S10: 220909; S11: 220910.

[0044] Fig.11It is the chromatogram of the test solution in Comparative Example 1 of the present invention.

[0045] Fig.12 It is the chromatogram of the test solution in Comparative Example 2 of the present invention.

[0046] Fig.13 It is the chromatogram of the test solution in Experimental Example 4 of the present invention at -35°C.

[0047] Fig.14 It is the chromatogram of the test solution in Experimental Example 8 of the present invention at -270nm.

[0048] Fig.15 It is the chromatogram of the test solution in Experimental Example 8 of the present invention at -280nm.

[0049] Fig.16 It is the chromatogram of the test solution in Experimental Example 8 of the present invention at -296nm.

[0050] Fig.17 It is the chromatogram of the test solution in Experimental Example 8 of the present invention at -310nm.

[0051] Fig.18 It is the chromatogram of the test solution in Experimental Example 9 of the present invention -20 μL.

[0052] Fig.19 It is the chromatogram of the test solution in Experimental Example 9 of the present invention - 25 μL.

[0053] Fig. 20 It is the chromatogram of the test solution in Experimental Example 9 of the present invention - 35 μL.

[0054] Figure symbols: Peak 1: bufotolin; Peak 2: bufotolin; Peak 3: bufotolin; Peak 4: deacetylcintolin; Peak 5: telcintolin; Peak 6: bufotolin; Peak 7: cintolin. DETAILED DESCRIPTION

[0055] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0056] If the specific experimental steps or conditions are not specified in the examples, the operations or conditions of the conventional experimental steps described in the literature in this field can be carried out. If the manufacturer of the reagents or instruments used is not specified, they are all conventional reagent products that can be obtained commercially. The Huachansu tablets used below are commercially available, and the active ingredient is dry toad skin extract.

[0057] Example 1

[0058] This embodiment provides a method for separating the chemical components of a toad skin extract and a preparation thereof or a method for constructing a fingerprint, the construction method comprising the following steps:

[0059] (1) Preparation of test solution:

[0060] Weigh 2 g of Huachansu tablet powder (passed through No. 4 sieve), accurately weigh and place in a 100 ml round-bottom flask, add appropriate amount of methanol, heat and reflux for 120 minutes, take out, let stand at room temperature, add methanol to the scale, shake well, transfer to a centrifuge tube, centrifuge at 15000 rpm for 5 minutes, and take the supernatant.

[0061] (2) Preparation of reference substance solution:

[0062] Take appropriate amount of toad venom, bufotoxin, toad venom, deacetyl cinobucoid, telecinobucoid, toad venom, and cinobucoid reference substances, weigh them accurately, and add methanol to dissolve them respectively to make individual reference substance solutions containing 20.6 μg of toad venom, 21.4 μg of bufotoxin, 19.8 μg of toad venom, 20.1 μg of deacetyl cinobucoid, 19.1 μg of telecinobucoid, 21.3 μg of toad venom, and 19.4 μg of cinobucoid per 1 ml, as reference substance solutions.

[0063] Take 2 ml each of the reference substance solutions of toad venom, bufotoxin, bufotoxin, deacetylcinobufolin, telocinobufagin, bufotoxin, and cinobufolin, and mix them to serve as single reference substance mixed solutions.

[0064] (3) Determination method: Accurately pipette 25ul of reference substance solution, single reference substance solution and test substance solution respectively and inject into liquid chromatograph. The chromatographic conditions of liquid chromatograph are as follows:

[0065] Mobile phase A was acetonitrile, mobile phase B was water, and gradient elution was performed according to the gradient elution program shown in Table 1 below; the detection wavelength was 296 nm; the column temperature was 30°C; the flow rate was 1.0 ml / min; the chromatographic column was Agilent SB-C18, the specifications of the chromatographic column were 4.6 mm×250 mm, and the filler particle size was 5 μm.

[0066] Table 1 Gradient elution program

[0067] Time (min) Volume percentage of mobile phase A Volume percentage of mobile phase B 0～2 22 78 2～15 22→24 78→76 15～25 24→30 76→70 25～35 30→60 70→40 35～40 60→22 40→78 40～45 22 78

[0068] The fingerprints of the reference solution and the test solution obtained by detection are as follows: Figure 1-2 The data are shown in Table 2. Figure 1-2As shown in Table 2, the chromatogram obtained by gradient elution in this embodiment can effectively separate seven components, i.e., bufotoxin, bufotoxin, deacetylcinobufolin, telocinobufolin, bufotoxin, and cinobufolin, and then the contents of these components can be determined simultaneously. The method of the present invention has a short detection cycle, is fast, and has high efficiency. The method is easy to operate and has strong specificity. Therefore, the method of this embodiment can be used for the overall quality control of cinobufolin tablets.

[0069] Compare the spectrum of the single reference solution with the spectrum of the test solution ( Figure 3-9 ), it can be seen from the figure that the peaks in the spectrum of the single reference substance solution correspond to the peaks in the spectrum of the test solution, and it is concluded that peaks 1-7 in the spectrum of the test solution are toad venom, toad venom, toad venom, deacetyl cinobucinatoxin, far cinobucinatoxin, toad venom, and cinobucinatoxin.

[0070] Table 2 Test results

[0071]

[0072] Example 2

[0073] This embodiment provides a fingerprint spectrum and a method for constructing and controlling the quality of the fingerprint spectrum, comprising: taking 10 batches of Cinobufacini tablets (batch numbers: 220901, 220902, 220903, 220904, 220905, 220906, 220907, 220908, 220909, 220910) and preparing and testing the test solution according to methods (1) and (3) in Example 1.

[0074] The test results are shown in Table 3. Fig.10 , the test results of the test solution prepared from 10 batches of cinobucin tablets were analyzed, and the fingerprint similarity evaluation software "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Committee was used to generate the reference fingerprint. The 7 chromatographic peaks with good separation of the fingerprint were confirmed by HPLC and LC / MS / MS for the identification and identification of characteristic peaks. According to the results of peak identification and reference positioning, peak 1 was confirmed to be bufotoxin, peak 2 was bufotoxin, peak 3 was bufotalidin, peak 4 was deacetyl cinobucin, peak 5 was telocinobufagin, peak 6 was bufotoxin, and peak 7 was cinobucin. Taking peak 2 as the reference peak, the relative retention times of each characteristic peak and the reference peak were calculated to be 0.62 (peak 1), 1.00 (peak 2), 1.12 (peak 3), 1.17 (peak 4), 1.50 (peak 5), 1.53 (peak 6), and 1.59 (peak 7).

[0075] Table 3 Relative retention time results of fingerprints of 10 batches of Huachansu tablets

[0076]

[0077]

[0078] In summary, when the fingerprint of the present invention is used for overall quality control, the chromatogram of the tested Huachansu tablets can be compared with the constructed fingerprint. If the four characteristic peaks corresponding to the same toad venom, toad venom, toad venom and toad venom in the fingerprint appear in the chromatogram of the tested Huachansu tablets; preferably, the chromatogram of the tested Huachansu tablets is compared with the fingerprint, which includes at least 7 characteristic peaks, and the peak corresponding to the reference peak is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative standard deviation of the relative retention time of each characteristic peak and the relative retention time of each characteristic peak in the fingerprint is within ±5%, then the quality of the Huachansu tablets is qualified, otherwise it is unqualified. In this way, rapid detection and judgment of the quality of Huachansu tablets can be achieved.

[0079] Comparative Example 1

[0080] This comparative example provides a method for constructing a fingerprint of Huachansu tablets, which differs from Example 1 only in the gradient elution procedure. The gradient elution procedure of this comparative example is shown in Table 4 below. Fig.11 As can be seen from the figure, the peak separation effect in the spectrum obtained by the gradient elution program of this comparative example is poor.

[0081] Table 4 Gradient elution program

[0082]

[0083]

[0084] Comparative Example 2

[0085] This comparative example provides a method for constructing a fingerprint of Huachansu tablets. The only difference from Example 1 is that the mobile phase is different. The mobile phase A of this comparative example is methanol and the mobile phase B is water. Fig.12 As can be seen from the figure, the components cannot be separated well using the comparative example method.

[0086] Experimental Example 1

[0087] This experimental example investigates the solvents in the preparation method of the test solution. The preparation method of the test solution is basically the same as the preparation method in Example 1, the only difference is the extraction solvent. The extraction solvents in this experimental example are pure methanol, 50% methanol aqueous solution or 70% methanol aqueous solution. The prepared test solution is tested according to the liquid phase conditions in Example 1 and the ratio of the peak area of ​​each peak to the sample weight (A / g) is calculated.

[0088] The calculation results are shown in Table 5. As shown in Table 5, when the extraction solvent is pure methanol, the measured values ​​of the peak areas of the peaks in the spectrum corresponding to the test solution are relatively high. After comprehensive consideration, pure methanol is selected as the extraction solvent for Huachansu Tablets.

[0089] Table 5 Calculation results - A / g

[0090]

[0091] Experimental Example 2

[0092] This experimental example investigates the ratio (solid-liquid ratio) of the mass of the object to be tested in the preparation method of the test solution to the volume of the solvent. Specifically, take the powder of Huachansu tablets (3g, 2g, 1g, 1g) respectively, accurately weigh and place in round-bottom flasks of different volumes (25mL, 25mL, 25mL, 50mL), add appropriate amount of methanol, heat to reflux for 120 minutes, take out, place at room temperature, add methanol to make up to the scale, the solid-liquid ratio is 3:25, 1:12.5, 1:25, 1:50, shake well, then transfer to a centrifuge tube, centrifuge at 15000rpm for 5min, and take the supernatant. The prepared test solution is detected according to the liquid phase conditions in Example 1 and the ratio (A / g) of the peak area of ​​each peak to the sample weight is calculated.

[0093] The calculation results are shown in Table 6. As can be seen from Table 6, the results of the peak areas of each peak under different conditions are slightly different. Compared with 3:25, 1:12.5 and 1:50, when the solid-liquid ratio (m:v) is 1:25, the measured values ​​of the peak areas of each peak in the spectrum corresponding to the test solution are relatively high. Therefore, the optimal solid-liquid ratio is determined to be 1:25.

[0094] Table 6 Calculation results - A / g

[0095]

[0096] Experimental Example 3

[0097] This experimental example investigates the extraction time in the preparation method of the test solution. The preparation method of the test solution is basically the same as the preparation method in Example 1, the only difference is the extraction time, which is 90min, 120min, and 150min in this experimental example. The prepared test solution is tested according to the liquid phase conditions in Example 1 and the ratio of the peak area of ​​each peak to the sample weight (A / g) is calculated.

[0098] The calculation results are shown in Table 7. As can be seen from Table 7, the results of the peak areas of each peak under different conditions are slightly different. Compared with 90min and 150min, when the extraction time is 120min, the measured values ​​of the peak areas of each peak in the spectrum corresponding to the test solution are relatively high. Therefore, the optimal extraction time is selected as 120min.

[0099] Table 7 Calculation results - A / g

[0100]

[0101] Experimental Example 4

[0102] This experimental example investigated the column temperature. The liquid chromatography conditions of this experimental example are basically the same as those of Example 1, except that the column temperature is different, and the column temperature of this experimental example is 35° C. The test solution prepared according to the preparation method of Example 1 was tested according to the liquid chromatography conditions of this experimental example.

[0103] Test results see Fig.13 .Depend on Fig.13 It can be seen that when the column temperature is 35°C, the separation effect of the chromatographic peaks of each component is good.

[0104] Experimental Example 5

[0105] This experimental example investigates the centrifugation conditions in the preparation method of the test solution. The preparation method of the test solution is basically the same as the preparation method in Example 1, except that the centrifugation conditions are different. The centrifugation conditions in this experimental example are 10000rpm centrifugation for 15min. The prepared test solution is tested according to the liquid phase conditions in Example 1 and the ratio of the peak area of ​​each peak to the sample weight (A / g) is calculated. The calculation results are shown in Table 8 below.

[0106] Experimental Example 6

[0107] This experimental example investigates the solid-liquid separation method in the preparation method of the test solution. Specifically, the "15000rpm centrifugation for 5min, taking the supernatant" in Example 1 is replaced by "using a 0.22μm microporous membrane to filter, taking the filtrate", and the rest of the preparation method is consistent with the preparation method of the test solution in Example 1. The prepared test solution is tested according to the liquid phase conditions in Example 1 and the ratio of the peak area of ​​each peak to the sample weight (A / g) is calculated. The calculation results are shown in Table 8 below.

[0108] Experimental Example 7

[0109] This experimental example investigates the solid-liquid separation method in the preparation method of the test solution. Specifically, the "15000rpm centrifugation for 5min, taking the supernatant" in Example 1 is replaced by "using a 0.80μm microporous membrane to filter, taking the filtrate", and the rest of the preparation method is consistent with the preparation method of the test solution in Example 1. The prepared test solution is tested according to the liquid phase conditions in Example 1 and the ratio of the peak area of ​​each peak to the sample weight (A / g) is calculated. The calculation results are shown in Table 8 below.

[0110] Table 8 Calculation results - A / g

[0111]

[0112] It can be seen from Table 8 that the results of the peak areas of each peak under different conditions are slightly different, indicating that the solid-liquid separation method in the preparation process of the test solution has little effect on the test results, and Example 6 is the best.

[0113] Experimental Example 8

[0114] This experimental example investigates the detection wavelength. The liquid chromatography conditions of this experimental example are basically the same as those of Example 1, except that the detection wavelengths are different. The detection wavelengths of this experimental example are 270 nm, 280 nm, 296 nm, and 310 nm, respectively. The test solution prepared according to the preparation method of Example 1 is tested according to the liquid chromatography conditions of this experimental example.

[0115] Test results see Figure 14-17 , Table 9-12. From the results, it can be seen that the peak area is the largest at the detection wavelength of 296nm.

[0116] Table 9 Test results - 270nm

[0117] Serial number Retention time / min Peak area Separation Theoretical plates 1 13.279 48415 - 8674.69 2 21.474 81455 13.93 17126.63 3 24.140 53706 4.83 39711.81 4 25.236 46551 2.40 47511.13 5 32.287 53165 23.31 433020.48 6 33.029 43049 3.98 464580.95 7 34.309 39716 6.89 474713.73

[0118] Table 10 Test results - 280nm

[0119] Serial number Retention time / min Peak area Separation Theoretical plates 1 13.279 75094 - 8124.48 2 21.474 129491 13.81 17284.41 3 24.137 91805 4.83 36322.76 4 25.251 71258 2.44 50042.82 5 32.287 82870 23.18 456020.97 6 33.029 59757 4.02 483631.73 7 34.310 60483 6.99 486683.22

[0120] Table 11 Test results - 296nm

[0121] Serial number Retention time / min Peak area Separation Theoretical plates 1 13.279 125620 - 7087.29 2 21.474 185219 13.50 17465.16 3 24.145 134129 4.83 37424.02 4 25.236 96124 2.37 50967.43 5 32.288 116787 22.98 474557.87 6 33.030 67919 4.06 513288.19 7 34.310 74992 7.06 501305.79

[0122] Table 12 Test results - 310nm

[0123] Serial number Retention time / min Peak area Separation Theoretical plates 1 13.279 112655 - 6775.80 2 21.475 157480 13.47 17013.62 3 24.140 119741 4.82 37472.62 4 25.250 73542 2.43 52241.49 5 32.288 110409 23.19 461117.80 6 33.029 55990 4.04 488030.42 7 34.310 56569 6.99 478006.72

[0124] Experimental Example 9

[0125] This experimental example investigates the injection volume. The liquid chromatography conditions of this experimental example are basically the same as those of Example 1, except that the injection volume is different, and the injection volumes of this experimental example are 20 μL, 25 μL, and 35 μL, respectively. The test solution prepared according to the preparation method of Example 1 is tested according to the liquid chromatography conditions of this experimental example.

[0126] Test results see Figure 18-20 , Table 13-15. From the results, it can be seen that when the injection volume is 25μL, the peak shape and peak area are better.

[0127] Table 13 Test results - 20 μL

[0128] Serial number Retention time / min Peak area Separation Theoretical plates 1 13.349 95802 - 13600.37 2 21.602 157312 16.51 25376.31 3 24.262 103592 5.59 55124.56 4 25.355 85316 2.81 75172.77 5 32.290 98088 25.05 488886.19 6 33.030 56216 4.21 573133.90 7 34.302 52395 7.42 601804.01

[0129] Table 14 Test results - 25 μL

[0130] Serial number Retention time / min Peak area Separation Theoretical plates 1 13.302 128953 - 7791.26 2 21.487 185369 13.41 16800.46 3 24.159 131486 4.86 38187.96 4 25.252 96528 2.37 56573.17 5 32.278 117541 22.69 481848.84 6 33.019 68583 4.06 497919.01 7 34.300 67784 7.15 596244.15

[0131] Table 15 Test results - 35 μL

[0132] Serial number Retention time / min Peak area Separation Theoretical plates 1 13.380 168314 - 8348.33 3 21.615 271035 13.53 14568.81 4 24.270 186641 4.33 27514.97 5 25.346 150192 2.09 37369.25 6 32.278 168514 20.34 408460.95 7 33.018 95437 3.73 420623.84 8 34.290 91426 6.53 465655.46

[0133] Experimental Example 10

[0134] 1. Precision test

[0135] The test solution was prepared by the preparation method of the test solution in Example 1, and the same test solution was accurately aspirated. After repeated injection 6 times according to the method in Example 1, the peak area and retention time of each common peak were determined. Peak No. 2 was used as the reference peak, and the relative retention time and relative peak area of ​​each common peak and their RSD values ​​were calculated. The results are shown in Tables 16 and 17. As can be seen from the table, the relative retention time and relative peak area RSD values ​​of each common peak are less than or equal to 2.06%, indicating that the method has good precision.

[0136] Table 16 Precision test results - relative retention time

[0137]

[0138]

[0139] Table 17 Precision test results - relative peak area

[0140]

[0141] 2. Repeatability test

[0142] The preparation method of the test solution in Example 1 was adopted, and 6 test solutions were prepared in parallel by the same experimenter. The peak area and retention time of each common peak were determined according to the method in Example 1. Peak No. 2 was used as the reference peak, and the relative retention time and relative peak area of ​​each common peak and their RSD values ​​were calculated. The results are shown in Tables 18 and 19. As can be seen from the table, the relative retention time and relative peak area RSD values ​​of each common peak are less than or equal to 2.36%, indicating that the method has good repeatability.

[0143] Table 18 Repeatability test results - relative retention time

[0144]

[0145] Table 19 Repeatability test results - relative peak area

[0146]

[0147]

[0148] 3. Reproducibility test

[0149] The preparation method of the test solution in Example 1 was adopted, and 3 experimenters prepared 1 test solution respectively. The peak area and retention time of each common peak were determined according to the method in Example 1. Peak No. 2 was used as the reference peak, and the relative retention time and relative peak area of ​​each common peak and their RSD values ​​were calculated. The results are shown in Tables 20 and 21. As can be seen from the table, the relative retention time and relative peak area RSD values ​​of each common peak are less than or equal to 2.87%, indicating that the method has good reproducibility.

[0150] Table 20 Reproducibility test results - relative retention time

[0151]

[0152] Table 21 Reproducibility test results - relative peak area

[0153]

[0154] 4. Stability test

[0155] One sample solution was prepared by the preparation method of the sample solution of Example 1, placed at room temperature, and samples were taken at different time points, and the peak area and retention time of each common peak were determined according to the method of Example 1 to investigate the stability of the sample solution at room temperature. Peak No. 2 was used as the reference peak, and the relative retention time and relative peak area of ​​each common peak and their RSD values ​​were calculated. The results are shown in Tables 22 and 23. As can be seen from the table, the relative retention time and relative peak area RSD values ​​of each common peak were less than or equal to 2.71%, indicating that the sample was basically stable when placed at room temperature for 24 hours.

[0156] Table 22 Stability test results - relative retention time

[0157]

[0158]

[0159] Table 23 Stability test results - relative peak area

[0160]

[0161] Through systematic methodological investigation, it can be seen from the table data that the RSD values ​​of relative retention time and relative peak area of ​​precision, stability, repeatability and reproducibility are all less than 2.87%, indicating that the construction method of the fingerprint of Huachansu tablets meets the research requirements of fingerprint.

[0162] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for separating the chemical components of a toad skin extract and its preparation or a method for constructing a fingerprint, characterized in that: The following steps are involved: (1) Preparation of test solution; (2) The sample solution was tested by high performance liquid chromatography. The filler was octadecylsilane bonded silica gel. The mobile phase included acetonitrile and water. The gradient elution program included: 0→2 min→15 min→25 min→35 min→40 min→45 min. The volume percentage of acetonitrile in the mobile phase was 22%→22%→24%→30%→60%→22%→22%.

2. The method for separating the chemical components of the toad skin extract and its preparation or the method for constructing the fingerprint of the toad skin extract or its preparation according to claim 1, characterized in that: Step (1) comprises: 1) Take the object to be tested, add a solvent to extract it, and obtain an extract; 2) Separate the solid and liquid of the extract and take the liquid, which is the test sample solution.

3. The method for separating the chemical components of the toad skin extract and its preparation or the method for constructing the fingerprint spectrum according to claim 2, characterized in that: Step (1) also satisfies any one or more of the following AE: A. In step 1), the solvent is selected from methanol and a methanol aqueous solution having a volume percentage concentration greater than or equal to 50%; B. In step 1), the extraction is performed by heating and reflux extraction, and the extraction time is 90-150 min; more preferably, the extraction time is 120 min; C. In step 1), the ratio of the mass of the analyte to the volume of the solvent is 1 g: 8-50 mL; D. In step 2), the solid-liquid separation is centrifugation or filtration; more preferably, the centrifugal speed is 10000-15000 rpm, and the time is 5-15 min; more preferably, the specification of the filter membrane for filtration is 0.22-0.80 μm; E. In step 1), the analyte is a Bufo bufo slice.

4. The method for separating the chemical components of the toad skin extract and its preparation or the method for constructing the fingerprint spectrum according to claim 2, characterized in that: The ratio of the mass of the analyte to the volume of the solvent is 1 g:20-30 mL.

5. The method for separating the chemical components of the toad skin extract and its preparation or the method for constructing the fingerprint spectrum according to any one of claims 1 to 4, characterized in that: The chromatographic conditions of the high performance liquid chromatography in step (2) also include: a detection wavelength of 190-400 nm, a flow rate of 1.0-2.0 ml / min, a column temperature of 30-35° C., and an injection volume of 20-50 μl; Preferably, the specifications of the chromatographic column are 4.6×250 mm, 5 μm.

6. The method for separating the chemical components of the toad skin extract and its preparation or the method for constructing the fingerprint spectrum according to claim 5, characterized in that: The detection wavelength is 296 nm.

7. The method for separating the chemical components of the toad skin extract and its preparation or the method for constructing the fingerprint spectrum according to any one of claims 1 to 6, characterized in that: The construction method also includes the step of preparing a reference substance solution using at least one of the reference substances selected from the group consisting of bufotoxin, bufotoxin, bufotoxin, deacetylcintoxin, telocinobufacine, bufotoxin, and cinobufacine, and the step of detecting the reference substance solution by high performance liquid chromatography according to the construction method described in any one of claims 1 to 6 to obtain a reference spectrum of the reference substance.

8. The method for separating the chemical components of the toad skin extract and its preparation or the method for constructing the fingerprint spectrum according to any one of claims 1 to 7, characterized in that: The fingerprint of the toad skin extract and its preparation is selected from any one of the following (1)-(3): (1) It has 7 characteristic peaks, with peak No. 2 as the reference peak, and the relative retention time of each characteristic peak and the reference peak is within the range of ±5% of the specified value; the specified value is: 0.62 (peak 1), 1.00 (peak 2), 1.12 (peak 3), 1.17 (peak 4), 1.50 (peak 5), 1.53 (peak 6), 1.59 (peak 7); (2) It has 7 characteristic peaks, with the peak corresponding to the peak of the reference substance of the ferret toxin as the reference peak, and the relative retention time of each characteristic peak and the reference peak is within the range of ±5% of the specified value, and the specified value is: 0.62 (peak 1), 1.00 (peak 2), 1.12 (peak 3), 1.17 (peak 4), 1.50 (peak 5), 1.53 (peak 6), 1.59 (peak 7); (3) A fingerprint constructed by the construction method described in any one of claims 1 to 7.

9. Application of the method for separating the chemical components of a toad skin extract and a preparation thereof or the method for constructing a fingerprint spectrum thereof as described in any one of claims 1 to 8 in the quality control of a toad skin extract and a preparation thereof.

10. A method for quality control of a toad skin extract and its preparation, characterized in that: The method comprises the steps of comparing the fingerprint of the toad skin extract product to be tested with the fingerprint of the toad skin extract and its preparation; the fingerprint of the toad skin extract product to be tested is constructed using the toad skin extract product to be tested according to the chemical component separation method of the toad skin extract and its preparation or the fingerprint construction method of any one of claims 1 to 8, and the fingerprint of the toad skin extract and its preparation is the fingerprint of the toad skin extract and its preparation according to claim 8.

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