Identification method for characteristic chromatograms of Bergenia ligulata medicinal materials, cut pieces, extracts and their formula granules

Abstract

The present invention provides a method for identifying HPLC characteristic spectra of bergenia medicinal materials, decoction pieces, extracts and their formula particles, comprising the following steps: A) dissolving a test sample with a solvent, extracting, and obtaining a test solution; B) determining the test solution with high performance liquid chromatography to obtain HPLC characteristic spectra of bergenia medicinal materials, decoction pieces, extracts and their formula particles; the chromatographic conditions of the high performance liquid chromatography are: the chromatographic column is a C18 column; the mobile phase A is methanol, and the mobile phase B is a 0.1% phosphoric acid solution, and the gradient elution. The present invention adopts high performance liquid chromatography, selects methanol-0.1% phosphoric acid solution as the mobile phase for gradient elution, and uses arbutin, gallic acid, catechin, bergenin, and ellagic acid as reference substances to establish HPLC characteristic spectra of bergenia medicinal materials, decoction pieces, extracts and their formula particles, with good repeatability and precision, stable and reliable methods, and can control the quality of bergenia medicinal materials, decoction pieces, extracts and their formula particles.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical detection, and particularly relates to a method for identifying the characteristic chromatograms of Bergenia purpurascens herbs, cut pieces, extracts and their formula granules. Background Art

[0002] Bergenia purpurascens is the dried rhizome of Bergenia purpurascens (Hook.f.et Thoms.) Engl. of the Saxifragaceae family, which is included in the current legal standard of traditional Chinese medicine, "Chinese Pharmacopoeia" 2020 edition. It has the effects of astringing diarrhea, stopping bleeding and relieving cough, and relaxing muscles and activating collaterals. It is used for diarrhea, dysentery, anorexia, bleeding from internal and external injuries, pulmonary tuberculosis cough, tracheitis cough, rheumatic pain, and traumatic injury. Regarding the quality detection of Bergenia purpurascens, it is currently limited to the shape, thin-layer chromatography identification and the determination of the content of individual components by liquid chromatography, and there is no detection method that comprehensively reflects and controls Bergenia purpurascens. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a method for identifying the characteristic chromatograms of Bergenia purpurascens herbs, cut pieces, extracts and their formula granules. The HPLC characteristic chromatogram method for Bergenia purpurascens herbs, cut pieces, extracts and their formula granules constructed by the present invention is stable and reliable, and can control the quality of Bergenia purpurascens herbs, cut pieces, extracts and their formula granules.

[0004] The present invention provides a method for identifying the HPLC characteristic chromatograms of Bergenia purpurascens herbs, cut pieces, extracts and their formula granules, including the following steps:

[0005] A) Dissolve and extract the test sample with a solvent to obtain a test solution;

[0006] B) Determine the test solution by high performance liquid chromatography to obtain the HPLC characteristic chromatograms of Bergenia purpurascens herbs, cut pieces, extracts and their formula granules;

[0007] The chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 column; mobile phase A is methanol, and mobile phase B is 0.1% phosphoric acid solution, with gradient elution.

[0008] In the present invention, both 0.1% phosphoric acid solution and 30% methanol are volume fractions;

[0009] In the present invention, the preparation of a reference substance solution is also included:

[0010] Dissolve arbutin, gallic acid, catechin, bergenin, and ellagic acid with methanol respectively to prepare solutions containing 200 μg, 100 μg, 80 μg, 120 μg, and 40 μg per 1 ml as the reference substance solution of the reference substance;

[0011] The reference substance solution is determined by high performance liquid chromatography to obtain the chromatogram of the reference substance; and the components of the HPLC characteristic chromatograms of Bergenia medicinal materials, decoction pieces, extracts and their formula granules are qualitatively analyzed according to the chromatogram of the reference substance.

[0012] In the present invention, the gradient elution is specifically as follows:

[0013] 0 - 5 min, phase A: 5%, phase B: 95%;

[0014] 5 - 22 min, phase A: 5 - 15%, phase B: 95 - 85%;

[0015] 22 - 32 min, phase A: 15 - 25%, phase B: 85 - 75%;

[0016] 32 - 57 min, phase A: 25 - 35%, phase B: 75 - 45%;

[0017] 57 - 67 min, phase A: 55 - 80%, phase B: 45 - 20%.

[0018] In the present invention, the size of the C18 column is 250×4.6 mm×5 μm; the column temperature is 30°C - 35°C.

[0019] In the present invention, the flow rate of the mobile phase is 0.8 - 1.2 mL / min; the detection wavelength is 240 - 260 nm; the injection volume is 5 - 10 μL, and the number of theoretical plates calculated based on the bergenin peak should be not less than 4500.

[0020] In the present invention, the test sample raw materials are one or more of Bergenia medicinal materials, decoction pieces, Bergenia extracts and Bergenia formula granules;

[0021] If the test sample raw material is Bergenia medicinal material or decoction piece:

[0022] The extraction process includes: mixing the test sample with water, boiling for 25 - 35 min, filtering, evaporating the filtrate to dryness, adding 30% methanol, ultrasonically treating, cooling, shaking well, filtering, and taking the subsequent filtrate to obtain the test solution; the mass ratio of the test sample (g) to the volume of water is 0.5:48 - 52, preferably 0.5:50; the mass ratio of the test sample (g) to the volume of 30% methanol (mL) is 0.5:48 - 52, preferably 0.1:50;

[0023] If the test sample raw material is Bergenia extract or formula granule:

[0024] The extraction process includes:

[0025] Mix the test sample with 30% methanol, ultrasonicate, allow to cool, shake well, filter, and take the subsequent filtrate to obtain the test solution. The mass ratio of the test sample in grams to the volume of 30% methanol in mL is 0.1:48 - 52, preferably 0.1:50.

[0026] In the present invention, the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint is used to evaluate the similarity of the HPLC characteristic fingerprints of Bergenia ligulata medicinal materials and decoction pieces, obtaining the HPLC characteristic fingerprints of Bergenia ligulata medicinal materials and decoction pieces composed of 10 characteristic peaks. Among them, the peak corresponding to the bergenin reference substance is the S peak. In the said standard characteristic fingerprint, calculate the relative retention time of each characteristic peak to the S peak, and its relative retention time should be within ±10% of the specified value; the specified values are: 0.48 (peak 3), 0.75 (peak 4), 0.88 (peak 5), 0.98 (peak 6), 1.42 (peak 8), 1.44 (peak 9), 1.66 (peak 10).

[0027] In the present invention, the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint is used to evaluate the similarity of the HPLC characteristic fingerprint of Bergenia ligulata extract, obtaining the HPLC characteristic fingerprint of Bergenia ligulata extract composed of 10 characteristic peaks. Among them, the peak corresponding to the bergenin reference substance is the S peak. In the said standard characteristic fingerprint, calculate the relative retention time of each characteristic peak to the S peak, and its relative retention time should be within ±10% of the specified value; the specified values are: 0.44 (peak 3), 0.72 (peak 4), 0.86 (peak 5), 0.98 (peak 6), 1.46 (peak 8), 1.49 (peak 9), 1.71 (peak 10).

[0028] In the present invention, the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint is used to evaluate the similarity of the HPLC characteristic fingerprint of Bergenia ligulata formula granules, obtaining the HPLC characteristic fingerprint of Bergenia ligulata formula granules composed of 10 characteristic peaks. Among them, the peak corresponding to the bergenin reference substance is the S peak. In the said standard characteristic fingerprint, calculate the relative retention time of each characteristic peak to the S peak, and its relative retention time should be within ±10% of the specified value; the specified values are: 0.46 (peak 3), 0.74 (peak 4), 0.87 (peak 5), 0.98 (peak 6), 1.43 (peak 8), 1.45 (peak 9), 1.67 (peak 10).

[0029] The present invention provides a method for identifying Bergenia ligulata medicinal materials, decoction pieces, extracts and their formula granules, characterized in that the method described in the above technical solution is used for detection and the detection results are analyzed.

[0030] The newly established characteristic chromatogram method of the present invention has a simple and easy-to-operate preparation method for test samples, can identify more characteristic peaks, and can accurately and reliably detect Bergenia purpurascens and its preparations. It can effectively control the authenticity, quality consistency and stability of Bergenia purpurascens and its preparations, and provide a basis for comprehensively evaluating the quality of Bergenia purpurascens and its preparations. Description of the Drawings

[0031] Figure 1 For the investigation of the extraction method of test samples;

[0032] Figure 2 For the identification of characteristic chromatographic peaks of Bergenia purpurascens extract;

[0033] Figure 3 For the characteristic chromatograms of 16 batches of Bergenia purpurascens medicinal materials;

[0034] Figure 4 For the reference characteristic chromatogram of Bergenia purpurascens medicinal materials;

[0035] Figure 5 For the characteristic chromatograms of 16 batches of Bergenia purpurascens cut pieces;

[0036] Figure 6 For the reference characteristic chromatogram of Bergenia purpurascens cut pieces;

[0037] Figure 7 For the identification of chromatographic peaks of Bergenia purpurascens extract;

[0038] Figure 8 For the characteristic superposition diagram of Bergenia purpurascens extract;

[0039] Figure 9 For the reference characteristic chromatogram of Bergenia purpurascens extract;

[0040] Figure 10 For the chromatograms of Bergenia purpurascens formula granules at different wavelengths;

[0041] Figure 11 For the 3D diagram of Bergenia purpurascens formula granules;

[0042] Figure 12 For the investigation of the flow rate of the characteristic chromatogram of Bergenia purpurascens formula granules;

[0043] Figure 13 For the investigation of the column temperature of the characteristic chromatogram of Bergenia purpurascens formula granules;

[0044] Figure 14 For the chromatograms of Bergenia purpurascens formula granules extracted with different solvents;

[0045] Figure 15 For the chromatograms of Bergenia purpurascens formula granules with different extraction methods;

[0046] Figure 16 For the chromatograms of Bergenia purpurascens formula granules with different extraction times;

[0047] Figure 17 Sampling amount investigation of the characteristic chromatogram of Bergenia ligustifolia formula granules

[0048] Figure 18 Chromatographic peak identification of Bergenia ligustifolia formula granules

[0049] Figure 19 Investigation of different instruments for the characteristic chromatogram of Bergenia ligustifolia formula granules

[0050] Figure 20 Investigation of different chromatographic columns for the characteristic chromatogram of Bergenia ligustifolia formula granules

[0051] Figure 21 Verification charts of the characteristic chromatograms of 3 batches of Bergenia ligustifolia formula granules

[0052] Figure 22 Reference characteristic chromatogram of Bergenia ligustifolia formula granules Specific implementation mode

[0053] The present invention provides a method for identifying the HPLC characteristic chromatograms of Bergenia ligustifolia medicinal materials, cut pieces, extracts and their formula granules, including the following steps:

[0054] A) Dissolve and extract the test sample with a solvent to obtain a test solution;

[0055] B) Determine the test solution by high performance liquid chromatography to obtain the HPLC characteristic chromatograms of Bergenia ligustifolia medicinal materials, cut pieces, extracts and their formula granules;

[0056] The chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 column; mobile phase A is methanol, and mobile phase B is 0.1% phosphoric acid solution, with gradient elution.

[0057] The test samples provided by the present invention include, but are not limited to, one or more of Bergenia ligustifolia medicinal materials, cut pieces, extracts and their formula granules.

[0058] The method for identifying the HPLC characteristic chromatograms of Bergenia ligustifolia medicinal materials, cut pieces, extracts and their formula granules provided by the present invention first takes Bergenia ligustifolia medicinal materials, cut pieces, extracts and their formula granules, dissolves them with a solvent, and extracts them to obtain a test solution.

[0059] The present invention preferably mixes the test sample with water, boils it for 25 - 35 min, filters it, evaporates the filtrate to dryness, then adds methanol with a mass fraction of 30%, ultrasonically treats it, cools it, shakes it well, filters it, and takes the continuous filtrate to obtain a test solution.

[0060] The present invention discovers that when 30% methanol is used as the extraction solvent, both the peak shape and the resolution in the chromatographic peaks are better.

[0061] All the above raw materials of the present invention can be subjected to quality control and qualitative and quantitative detection through the method of the present invention.

[0062] The present invention also includes preparing a reference solution:

[0063] Arbutin, gallic acid, catechin, bergenin, and ellagic acid were dissolved in methanol to prepare solutions containing 200 μg, 100 μg, 80 μg, 120 μg, and 40 μg per ml, respectively, as control solutions;

[0064] The reference substance solution is measured by high performance liquid chromatography to obtain a chromatogram of the reference substance; and the components of the HPLC characteristic spectra of the Bergenia medicinal materials, decoction pieces, extracts and their formula particles are qualitatively identified according to the chromatogram of the reference substance.

[0065] The mobile phase A in the present invention is methanol, the mobile phase B is 0.1% phosphoric acid solution, and the elution is performed by gradient.

[0066] The gradient elution described in the present invention is preferably specifically:

[0067] 0-5min, phase A: 5%, phase B: 95%;

[0068] 5-22min, phase A: 5-15%, phase B 95-85%;

[0069] 22-32min, phase A: 15-25%, phase B: 85-75%;

[0070] 32-57min, phase A: 25-35%, phase B: 75-45%;

[0071] 57~67min, phase A: 55~80%, phase B: 45~20%.

[0072] The present invention has good baseline separation under the above elution gradient, good separation of each peak and stable baseline.

[0073] The size of the C18 column in the present invention is 250×4.6 mm×5 μm; the present invention adopts the chromatographic column for durability detection, and the durability is good.

[0074] The column temperature in the present invention is 30°C to 35°C. In the present invention, the chromatographic peaks are relatively symmetrical at the above chromatographic column temperature, and the separation is good. 30°C is the optimal solution.

[0075] In the present invention, the flow rate of the mobile phase is 0.8-1.2 mL / min. The present invention found that the chromatographic peaks were better separated and the separation degree was moderate at a flow rate of 1.0 mL / min, which is the most preferred solution.

[0076] The detection wavelength of the present invention is 240-260 nm. The inventors found that at 240 nm, the chromatographic peak has a larger amount of information, the chromatogram baseline is more stable, and the peak area of ​​each peak is larger.

[0077] The theoretical plate number of the present invention calculated based on the bergenin peak should be not less than 4500.

[0078] The sample injection volume of the present invention is 5 - 10 μL, preferably 10 μL.

[0079] The method for detecting the characteristic chromatogram of bergenia and its preparations provided by the present invention can be used for the quality detection of bergenia medicinal materials, cut pieces, extracts and formula granules, and the results are reliable, providing a new method for the quality control of bergenia.

[0080] The similarity of the HPLC characteristic chromatograms of bergenia medicinal materials and cut pieces is evaluated by using the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints. The HPLC characteristic chromatograms of bergenia medicinal materials and cut pieces composed of 10 characteristic peaks are obtained. The peak corresponding to the bergenin reference substance is the S peak. In the standard characteristic chromatogram, the relative retention times of each characteristic peak and the S peak are calculated, and the relative retention times should be within ±10% of the specified values; the specified values are: 0.48 (peak 3), 0.75 (peak 4), 0.88 (peak 5), 0.98 (peak 6), 1.42 (peak 8), 1.44 (peak 9), 1.66 (peak 10).

[0081] The similarity of the HPLC characteristic chromatogram of the bergenia extract is evaluated by using the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints. The HPLC characteristic chromatogram of the bergenia extract composed of 10 characteristic peaks is obtained. The peak corresponding to the bergenin reference substance is the S peak. In the standard characteristic chromatogram, the relative retention times of each characteristic peak and the S peak are calculated, and the relative retention times should be within ±10% of the specified values; the specified values are: 0.44 (peak 3), 0.72 (peak 4), 0.86 (peak 5), 0.98 (peak 6), 1.46 (peak 8), 1.49 (peak 9), 1.71 (peak 10).

[0082] The similarity of the HPLC characteristic chromatogram of the bergenia formula granules is evaluated by using the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints. The HPLC characteristic chromatogram of the bergenia formula granules composed of 10 characteristic peaks is obtained. The peak corresponding to the bergenin reference substance is the S peak. In the standard characteristic chromatogram, the relative retention times of each characteristic peak and the S peak are calculated, and the relative retention times should be within ±10% of the specified values; the specified values are: 0.46 (peak 3), 0.74 (peak 4), 0.87 (peak 5), 0.98 (peak 6), 1.43 (peak 8), 1.45 (peak 9), 1.67 (peak 10).

[0083] Quality judgment criteria: Take the bergenia medicinal materials, decoction pieces, bergenia extract and bergenia formula granules samples, and operate according to the same method as above to obtain the characteristic fingerprints of bergenia medicinal materials, decoction pieces, bergenia extract and bergenia formula granules. Use the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines" (2012 edition) of the Pharmacopoeia Commission of the People's Republic of China to analyze the characteristic fingerprints of bergenia medicinal materials, decoction pieces, bergenia extract and bergenia formula granules and the sample characteristic fingerprints, and the similarity is greater than 0.90.

[0084] The characteristic fingerprints of bergenia medicinal materials, decoction pieces, bergenia extract and bergenia formula granules established by the present invention use arbutin, gallic acid, catechin, bergenin, ellagic acid as reference substances, pay attention to the order of each characteristic peak and the correlation with the medicinal materials and intermediate products, and can comprehensively evaluate the overall quality characteristics of the products. The method is scientific and reliable.

[0085] The present invention provides a method for identifying bergenia medicinal materials, decoction pieces, extracts and their formula granules, which is characterized in that the method described in the above calculation scheme is used for detection and the detection results are analyzed.

[0086] The present invention provides a method for identifying the HPLC characteristic fingerprints of bergenia medicinal materials, decoction pieces, extracts and their formula granules, including the following steps: A) Dissolve and extract the test sample with a solvent to obtain a test solution; B) Determine the test solution by high performance liquid chromatography to obtain the HPLC characteristic fingerprints of bergenia medicinal materials, decoction pieces, extracts and their formula granules; the chromatographic conditions of the high performance liquid chromatography are: the chromatographic column is a C18 column; the mobile phase A is methanol, and the mobile phase B is 0.1% phosphoric acid solution, with gradient elution. The present invention uses high performance liquid chromatography, selects methanol-0.1% phosphoric acid solution as the mobile phase for gradient elution, uses arbutin, gallic acid, catechin, bergenin, ellagic acid as reference substances, and establishes the HPLC characteristic fingerprints of bergenia medicinal materials, decoction pieces, extracts and their formula granules. The method has good repeatability and precision, and is stable and reliable, and can control the quality of bergenia medicinal materials, decoction pieces, extracts and their formula granules.

[0087] In order to further illustrate the present invention, the following examples are used to describe in detail a method for identifying the HPLC characteristic fingerprints of bergenia medicinal materials, decoction pieces, extracts and their formula granules provided by the present invention, but they should not be construed as limiting the protection scope of the present invention.

[0088] Example 1

[0089] 1.1 Instruments and materials

[0090] High performance liquid chromatograph;

[0091] Electronic balance: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Co., Ltd.);

[0092] Ultra-pure water machine: Cell type 1810A (Shanghai Moller Scientific Instruments Co., Ltd.);

[0093] Ultrasonic cleaner: Model KQ5200DB (600W, 40KHz; Kunshan Ultrasonic Instruments Co., Ltd.);

[0094] Chromatographic column: C18 250*4.6mm*5μm;

[0095] 1.2 Reagents and test drugs

[0096] Methanol is of chromatographic purity, water is ultra-pure water, and the rest of the reagents are of analytical purity. Bergenin reference crude drug (Sichuan Puxiao Standard Substances Technology Co., Ltd., batch number: PCL-#-Bp126), arbutin (National Institutes for Food and Drug Control, batch number: 111951-201301, content calculated as 99.7%), gallic acid (National Institutes for Food and Drug Control, batch number: 110831-201906, content calculated as 91.5%), catechin (National Institutes for Food and Drug Control, batch number: 110877-202005, content calculated as 95.1%), bergenin (National Institutes for Food and Drug Control, batch number: 111532-202005, content calculated as 94.4%), ellagic acid (National Institutes for Food and Drug Control, batch number: 111959-201903, content calculated as 88.8%),

[0097] Bergenia crude drug (batch numbers: S1~S16);

[0098] The chromatographic conditions and preparation of test solution for the characteristic chromatogram of bergenia crude drug refer to the method of the characteristic chromatogram of bergenia formula granules, as follows:

[0099] 1.3 Chromatographic conditions and system suitability test Octadecylsilane chemically bonded silica gel is used as the filler (column length is 250mm, inner diameter is 4.6mm, particle size is 5μm); methanol is used as mobile phase A, and 0.1% phosphoric acid solution is used as mobile phase B, and gradient elution is carried out according to the regulations in the following table. The detection wavelength is 240nm.

[0100]

[0101] Preparation of test solution Weigh an appropriate amount of the powder of this product, accurately weigh 0.5g, place it in a stoppered conical flask, accurately add 50ml of 30% methanol, stopper it tightly, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, let it cool, shake well, filter, and take the subsequent filtrate, that is obtained.

[0102] 1.4 Investigation on the extraction method of test solution preparation

[0103] Take an appropriate amount of the powder of this product (batch number: S16), about 0.5g, weigh accurately, and place in a stoppered conical flask. When the extraction method of the test sample is reflux and ultrasound (power 600W, frequency 40kHz), add 50ml of 30% methanol for inspection, extract for 30min, cool, shake well, filter, and take the filtrate. When it is water extraction, add 50ml of water, boil for half an hour, filter, evaporate to dryness, and then add 50ml of 30% methanol, ultrasound (power 600W, frequency 40kHz) for 30min, cool, shake well, filter, and take the filtrate. See Figure 1 By Figure 1 It can be seen that when the sample is subjected to ultrasonic extraction, reflux extraction, and water extraction, the water extraction baseline is flatter. In this experiment, the water extraction method is selected as the extraction method of Bergenia medicinal materials.

[0104] The final method for preparing the test sample is: take an appropriate amount of the product powder (passed through No. 3 sieve), about 0.5g, add 50ml of water, boil for half an hour, filter, evaporate the filtrate, then add 50ml of 30% methanol, ultrasonic (power 600W, frequency 40kHz) for 30min, cool, shake well, filter, and take the filtrate.

[0105] 1.5 Methodological Review

[0106] 1.5.1 Chromatographic peak identification

[0107] Preparation of test solution: According to the experimental conditions proposed above, prepare the test solution of Bergenia medicinal material.

[0108] Preparation of test solution: According to the experimental conditions proposed above, prepare the test solution of Bergenia formula granules.

[0109] Preparation of reference solution: Take arbutin, gallic acid, catechin, bergenin, and ellagic acid reference substances, accurately weigh them, and add 30% methanol to make solutions containing 200μg, 100μg, 80μg, 120μg, and 40μg per 1ml, respectively, as reference substance solutions.

[0110] Preparation of control medicinal material solution: Take 0.5 g of Bergenia control medicinal material, add 50 ml of water, boil for 30 minutes, filter, evaporate the filtrate, add 50 ml of 30% methanol to the residue, seal, and ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the control medicinal material reference solution.

[0111] Preparation of negative control solution: According to the experimental conditions proposed above, a negative control solution lacking Bergenia medicinal materials was prepared.

[0112] The characteristic peaks of the bergenia extract were identified. Figure 2 , Figure 2The results showed that the negative solution had no interference and the method had good specificity.

[0113] 1.5.2 Precision test

[0114] Take the Bergenia medicinal material test solution and inject it 6 times continuously according to the proposed experimental method, 10 μl each time, and calculate the retention time of each characteristic peak. See Table 1.

[0115] Table 1 Precision investigation - retention time

[0116]

[0117] The results showed that the RSD of the retention time of each characteristic peak was 0.01%-0.09%. The instrument had good precision.

[0118] 1.5.3 Repeatability study

[0119] Six portions of Bergenia oleifera (batch number: S16) were accurately weighed and prepared and tested according to the proposed experimental method. See Table 2.

[0120] Table 2 Repeatability study - relative retention time ratio

[0121]

[0122]

[0123] The results showed that this method had good repeatability.

[0124] The results showed that the RSD of the relative retention time of each characteristic peak was 0% to 0.12%. This method had good repeatability.

[0125] 1.5.4 Stability test

[0126] Based on the above proposed experimental conditions, take the same test solution and measure it at 0h, 4h, 8h, 12h, 16h and 24h respectively. See Table 3.

[0127] Table 3 Stability Study-Retention Time

[0128]

[0129] The results showed that the RSD of the corresponding characteristic peak retention time was between 0.01% and 0.05%, and the sample solution was relatively stable within 24 hours.

[0130] In summary, the RSD of the relative retention time of each characteristic peak meets the requirements in the above investigations, and this method is good.

[0131] 1.5.5 Determination of characteristic peaks and establishment of reference spectrum

[0132] 1.5.5.1 Establishment of the specified limits for relative retention time

[0133] The summary of the investigation items and verification results of the methodology is shown in Table 4.

[0134] Table 4 Summary of the results of each item of the methodology RSD% Standard - Retention time - Relative retention time

[0135]

[0136] The results showed that the RSD range of peaks 1 to 10 was 0.01% - 0.12%, the elution times of each characteristic peak were stable, and the specified values of the relative retention times of each peak were tentatively set at ±10%.

[0137] Final regulation: The characteristic chromatogram of the test sample should show 10 characteristic peaks, and the peak corresponding to the bergenin reference substance is the S peak.

[0138] 1.5.5.2 Verification results of 16 batches of bergenia decoction pieces

[0139] Using this method, the characteristic chromatograms of 16 batches of samples were analyzed to calculate the relative retention time. See Figure 3 , Table 5. Figure 3 Among them, the batch numbers represented by each chromatogram are S1 - S16.

[0140] Table 5 Relative retention times of 16 batches of bergenia medicinal materials

[0141]

[0142] According to the principle of stable relative retention time and that all batches of samples can be detected with relatively high peaks, a total of 10 peaks with good repeatability were selected as characteristic peaks. It is specified that: the test sample chromatogram should show 10 characteristic peaks, which should correspond to the retention times of 10 characteristic peaks in the chromatogram of the control medicinal material. Among them, peaks 1, 2, and 7 should correspond to the retention times of the corresponding reference substance peaks of the reference substances respectively. The peak corresponding to the bergenin reference substance is the S peak, and the relative retention times of the remaining characteristic peaks and the S peak are calculated. The relative retention times should be within the range of ±10% of the specified values, and the specified values are 0.45 (peak 3), 0.73 (peak 4), 0.87 (peak 5), 0.98 (peak 6), 1.45 (peak 8), 1.47 (peak 9), 1.69 (peak 10).

[0143] Using the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition) to synthesize the above samples, a reference characteristic chromatogram of the bergenia medicinal material was established. See Figure 4 . Peak 1 is arbutin, peak 2 is gallic acid, peak 6 is catechin, peak 7 (S) is bergenin; peak 10 is ellagic acid.

[0144] Example 2 Detection of the Characteristic Chromatogram of Bergenia Slices

[0145] 1. Experimental Materials

[0146] Bergenia slices (prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: YBC - 01 to YBC - 16).

[0147] 2. The detection method is the same as that of the Bergenia medicinal materials [Characteristic Chromatogram].

[0148] Chromatographic conditions and system suitability test: Use octadecylsilane chemically bonded silica gel as the filler (column length is 250 mm, inner diameter is 4.6 mm, particle size is 5 μm); use methanol as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution according to the regulations in the following table. The theoretical plate number calculated based on the bergenin peak should not be less than 4500.

[0149] Preparation of the reference substance solution: Take about 0.5 g of the reference Bergenia medicinal materials, add 50 ml of water, boil for half an hour, filter, evaporate the filtrate to dryness, then add 50 ml of 30% methanol, sonicate (power 600 W, frequency 40 kHz) for 30 min, cool, shake well, filter, and take the subsequent filtrate as the reference medicinal materials reference substance solution. Take arbutin, gallic acid, and bergenin reference substances, accurately weigh them, and prepare solutions containing 200 μg, 100 μg, and 120 μg per 1 ml with 30% methanol respectively as the reference substance solutions of reference substances.

[0150] Preparation of the test solution: Take an appropriate amount of the powder of this product (passed through No. 3 sieve), about 0.5 g, add 50 ml of water, boil for half an hour, filter, evaporate the filtrate to dryness, then add 50 ml of 30% methanol, sonicate (power 600 W, frequency 40 kHz) for 30 min, cool, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0151] Determination method: Accurately pipette 10 μl of the reference substance solution and the test solution respectively, inject them into the liquid chromatograph, and perform the determination to obtain the results.

[0152] 3. Verification results of 16 batches of Bergenia slices

[0153] Using this method, perform characteristic chromatogram analysis on 16 batches of samples and calculate the relative retention time. See Figure 5 , Table 6; Figure 5 In the figure, the chromatograms from bottom to top represent batch numbers YBC - 01 to YBC - 16.

[0154] Table 6 Relative retention time of 16 batches of Bergenia slices

[0155]

[0156]

[0157] According to the principle of stable relative retention time, detectable in each batch of samples and relatively high peaks, a total of 10 peaks with good repeatability were selected as characteristic peaks. It is stipulated that: 10 characteristic peaks should be presented in the chromatogram of the test sample, and the retention times should correspond to those of the 10 characteristic peaks in the chromatogram of the control crude drug. Among them, the retention times of peak 1, peak 2, and peak 7 should respectively correspond to those of the corresponding reference substance peaks. The peak corresponding to the bergenin reference substance is the S peak, and the relative retention times of the remaining characteristic peaks with respect to the S peak should be within the range of ±10% of the specified values. The specified values are 0.48 (peak 3), 0.75 (peak 4), 0.88 (peak 5), 0.98 (peak 6), 1.42 (peak 8), 1.44 (peak 9), and 1.66 (peak 10).

[0158] Using the Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines (2012 Edition), 16 batches of bergenia decoction pieces were synthesized to establish a reference chromatogram of the characteristic chromatogram of bergenia decoction pieces, as shown in Figure 6 , among which, peak 1: arbutin; peak 2: gallic acid; peak 6: catechin; peak 7 (S): bergenin; peak 10: ellagic acid.

[0159] Example 3 Detection of the Characteristic Chromatogram of Bergenia Extract

[0160] 3.1 Experimental Materials

[0161] Control crude drug of bergenia (Sichuan Puxiao Standard Technolog Co., Ltd., batch number: PCL-#-Bp126),

[0162] Bergenia extract (prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: YBC-BT-01~YBC-BT-16).

[0163] 3.2 Characteristic Method

[0164] The chromatographic conditions for the characteristic chromatogram of bergenia extract and the preparation of the test sample are as follows:

[0165] Chromatographic conditions and system suitability test: The filler is octadecylsilane chemically bonded silica gel (column length is 250 mm, inner diameter is 4.6 mm, particle size is 5 μm); methanol is used as mobile phase A, and 0.1% phosphoric acid solution is used as mobile phase B. Gradient elution is carried out according to the regulations in the following table. The detection wavelength is 240 nm.

[0166]

[0167]

[0168] Preparation of the test solution: Weigh accurately about 0.1 g of the powder of this product, place it in a stoppered conical flask, accurately add 50 ml of 30% methanol, stopper tightly, ultrasonically treat (power 600 W, frequency 40 kHz) for 20 minutes, let it cool, shake well, filter, and take the subsequent filtrate, which is the test solution.

[0169] 3.3 Methodology investigation

[0170] 3.3.1 Chromatographic peak identification

[0171] Preparation of the test solution: Prepare the test solution of the bergenin extract according to the above-mentioned experimental conditions.

[0172] Preparation of the reference solution: Weigh accurately the reference substances of arbutin, gallic acid, catechin, bergenin, and ellagic acid, and separately dissolve them in 30% methanol to prepare solutions containing 200 μg, 100 μg, 80 μg, 120 μg, and 40 μg per 1 ml, respectively, as the reference solution of the reference substances.

[0173] Take 0.5 g of the reference crude drug of bergenin, add 50 ml of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 50 ml of 30% methanol, stopper tightly, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, shake well, filter, and take the subsequent filtrate, as the reference solution of the reference crude drug.

[0174] Preparation of the negative control solution: Prepare the negative control solution without the bergenin extract according to the above-mentioned experimental conditions.

[0175] Identify the characteristic chromatogram peaks of the bergenin extract, as shown in Figure 7 ; Figure 7 It shows that the negative solution has no interference and the method has good specificity.

[0176] 3.3.2 Precision test

[0177] Take the test solution of the bergenin extract, inject 10 μl each time continuously for 6 times according to the proposed experimental method, and calculate the retention time of each characteristic peak. See Table 7:

[0178] Table 7 Precision investigation - Retention time

[0179]

[0180] The results show that the RSD of the retention time of each characteristic peak for precision is 0.01% - 0.04%. The precision of this instrument is good.

[0181] 3.3.2 Repeatability investigation

[0182] Accurately weigh 6 portions of the bergenin extract (batch number: YBC - BT - 07), prepare and determine them according to the proposed experimental method. See Table 8.

[0183] Table 8 Reproducibility Investigation - Relative Retention Time

[0184]

[0185] The results showed that the relative retention times of the characteristic peaks in the reproducibility were consistent. This method had good reproducibility.

[0186] 3.3.6 Stability Investigation

[0187] Based on the above - determined experimental conditions, the same test solution was taken and determined at 0 h, 4 h, 8 h, 12 h, 16 h, and 24 h respectively. See Table 9.

[0188] Table 9 Stability Investigation - Retention Time

[0189]

[0190] The results showed that the RSD of the retention times of the corresponding characteristic peaks was between 0.01% and 0.05%, and the sample solution was relatively stable within 24 hours.

[0191] 3.3.7 Determination of Characteristic Peaks and Establishment of Reference Chromatogram

[0192] 3.3.7.1 Establishment of the Specified Limit of Relative Retention Time

[0193] The summary of each investigation item and verification result of the methodology is shown in Table 10.

[0194] Table 10 Summary of RSD% of Results of Each Item of Methodology Standard - Retention Time - Relative Retention Time

[0195]

[0196]

[0197] The results showed that the relative retention times of each characteristic peak were relatively stable. The RSD range of peaks 1 to 10 was 0.01% - 0.73%. Combining the data of different instruments and different chromatographic columns, the specified value of the relative retention time of each peak was tentatively set at ±10%.

[0198] Final regulation: 10 characteristic peaks should be presented in the characteristic chromatogram of the test sample, and the peak corresponding to the bergenin reference substance is the S peak.

[0199] 3.3.8 Verification Results of 16 Batches of Bergenia Extracts

[0200] Using the above - mentioned method, the characteristic chromatograms of 16 batches of samples were analyzed, and the relative retention times were calculated. See Figure 8 , Table 11; Figure 8From bottom to top, the batch numbers are successively: YBC - BT - 01 to YBC - BT - 16.

[0201] Table 11 Relative retention times of 16 batches of Bergenia extract

[0202]

[0203] According to the principle of stable relative retention time, all batches of samples can be detected and the peaks are relatively high, the repeatability of the 10 selected characteristic peaks is relatively good. Finally, it is stipulated that: in the chromatogram of the test sample, 10 characteristic peaks should be presented, and the retention times of the 10 characteristic peaks should correspond to those in the chromatogram of the control crude drug. Among them, peak 1, peak 2, and peak 7 should respectively correspond to the retention times of the corresponding reference substance peaks of the reference standards. The peak corresponding to the bergenin reference substance is the S peak, and the relative retention times of the remaining characteristic peaks and the S peak are calculated. The relative retention times should be within the range of ±10% of the specified values. The specified values are 0.44 (peak 3), 0.72 (peak 4), 0.86 (peak 5), 0.98 (peak 6), 1.46 (peak 8), 1.49 (peak 9), and 1.71 (peak 10).

[0204] Using the Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines (2012 Edition) to synthesize 16 batches of Bergenia extract, a reference chromatogram of the characteristic chromatogram of Bergenia extract was established, as shown in Figure 9 , among which, peak 1: arbutin; peak 2: gallic acid; peak 6: catechin; peak 7 (S): bergenin; peak 10: ellagic acid.

[0205] Example 4 Detection of the characteristic chromatogram of Bergenia formula granules

[0206] 4.1 Experimental materials

[0207] Bergenia formula granules S1, S2, S3, S4.

[0208] 4.2 Chromatographic conditions

[0209] Chromatographic conditions and system suitability test: Octadecylsilyl silica gel bonded phase was used as the filler (column length was 250 mm, inner diameter was 4.6 mm, particle size was 5 μm); methanol was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was carried out according to the regulations in the following table.

[0210]

[0211] 4.2.1 Wavelength selection

[0212] On the basis of the above-mentioned experimental conditions, the diode array detector was used to perform full-wavelength scanning on arbutin, gallic acid, catechin, bergenin, ellagic acid, and the test solution, respectively, and the chromatograms of the test solution at wavelengths of 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, and 290 nm were extracted respectively. See Figure 10 , combined with the 3D map of bergenin formula granules (see Figure 11 ). The results showed that when the detection wavelength was 240 - 260 nm, the peak shapes of the chromatographic peaks were good, the proportions of each peak were moderate, and the baseline of the chromatogram was more stable. The most preferred wavelength was 240 nm, so the detection wavelength was determined to be 240 nm.

[0213] 4.2.2 Flow rate investigation

[0214] On the basis of the above-mentioned experimental conditions, investigations were carried out at flow rates of 0.8 ml / min, 1 ml / min, and 1.2 ml / min respectively. See Figure 12 and Table 12.

[0215] Table 12 Flow rate investigation - relative retention time of characteristic peaks

[0216]

[0217] The results showed that when the flow rates were 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min respectively, the peak shapes of the chromatograms of each characteristic peak were good and the resolution was moderate. Considering comprehensively, the most preferred flow rate of 1.0 ml / min was used to carry out the subsequent experiments.

[0218] 4.2.3 Column temperature investigation

[0219] On the basis of the above-mentioned experimental conditions, investigations were carried out at column temperatures of 25 °C, 30 °C, and 35 °C respectively. See Figure 13 , Table 13.

[0220] Table 13 Column temperature investigation - relative retention time of characteristic peaks

[0221]

[0222] The results showed that when the column temperatures were 25 °C, 30 °C, and 35 °C respectively, the relative standard deviation (RSD) of the relative retention time of each characteristic peak was 1.35% - 7.23%. As can be seen from the figure, when the column temperature was 30 °C and 35 °C, the peak shapes of the chromatograms were relatively symmetrical and the resolutions were good. Considering comprehensively, the column temperature was determined to be 30 °C for subsequent investigations.

[0223] In summary, the chromatographic conditions for the characteristic chromatogram of Bergenia formula granules were determined as follows: octadecylsilane chemically bonded silica gel was used as the filler (column length was 250 mm, inner diameter was 4.6 mm, and particle size was 5 μm); methanol was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was carried out according to the regulations in the following table. The detection wavelength was 240 nm.

[0224] 4.4 Preparation of test solution

[0225] 4.4.1 Investigation of extraction solvents: Methanol, ethanol, 70% methanol, 70% ethanol, 50% methanol, 50% ethanol, 30% methanol, 30% ethanol, and water were used as extraction solvents respectively. The results showed that when 30% methanol was used as the extraction solvent, the chromatographic peaks had good peak shapes and good resolution. Therefore, 30% methanol was selected as the extraction solvent. See Figure 14 .

[0226] 4.4.2 Investigation of extraction methods: Reflux extraction and ultrasonic extraction were compared. The chromatographic peak information was roughly the same, and finally ultrasonic extraction with simple operation was selected. See Figure 15 .

[0227] 4.4.3 Investigation of extraction time: Ultrasonic extraction for 20 minutes, 30 minutes, and 40 minutes was compared. Finally, ultrasonic extraction for 20 minutes was selected. See Figure 16 .

[0228] 4.4.4 Investigation of sampling amount

[0229] Take this product (batch number 1903074) and take 0.1 g, 0.2 g, and 0.3 g respectively, weigh accurately, place in a stoppered conical flask, accurately add 50 ml of 30% methanol, stopper tightly, ultrasonically treat (power 600 W, frequency 40 kHz) for 20 minutes, let cool, shake well, filter, and take the subsequent filtrate to obtain the test solution. See Figure 17 .

[0230] The results showed that when the sampling amounts were 0.1 g, 0.2 g, and 0.3 g, the chromatographic peak information was consistent. When the sampling amount was 0.1 g, the baseline was more stable. Therefore, the sampling amount of the test solution was determined to be 0.1 g.

[0231] 2.2.4.4.5 Final determination of the preparation method of the test solution:

[0232] Take an appropriate amount of this product, grind it finely, take 0.1 g, weigh accurately, place in a stoppered conical flask, accurately add 50 ml of 30% methanol, stopper tightly, ultrasonically treat (power 600 W, frequency 40 kHz) for 20 minutes, let cool, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0233] 4.5 Methodology investigation

[0234] 4.5.1 Chromatographic peak identification

[0235] Preparation of test solution: Prepare the test solution of Bergenia formula granules according to the above-determined experimental conditions.

[0236] Preparation of reference solution: Take reference substances of arbutin, gallic acid, catechin, bergenin, and ellagic acid, accurately weigh them, and separately prepare solutions containing 200 μg, 100 μg, 80 μg, 120 μg, and 40 μg per 1 ml with 30% methanol as the reference substance reference solutions.

[0237] Preparation of control crude drug solution: Take 0.5 g of Bergenia control crude drug, add 50 ml of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 50 ml of 30% methanol, tightly stopper, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the subsequent filtrate as the control crude drug reference solution.

[0238] Preparation of negative control solution: Prepare the negative control solution of Bergenia formula granules according to the above-determined experimental conditions.

[0239] Locate the characteristic peaks of the Bergenia formula granule fingerprint. See Figure 18 。

[0240] 4.5.2 Precision test

[0241] Take the test solution of Bergenia formula granules (batch number: S1), inject 10 μl each time continuously for 6 times according to the determined experimental method, and calculate the retention time of each characteristic peak. See Table 14.

[0242] Table 14 Precision test - retention time

[0243]

[0244] The results show that the precision of this instrument is good.

[0245] 4.5.3 Repeatability test

[0246] Accurately weigh 6 portions of Bergenia formula granules (batch number: S1), prepare and determine them according to the determined experimental method. See Table 15.

[0247] Table 15 Repeatability test - relative retention time ratio

[0248]

[0249]

[0250] The results show that this method has good repeatability.

[0251] 4.5.4 Intermediate precision test

[0252] 4.5.4.1 Investigation with Different Instruments

[0253] On the basis of the above - determined experimental conditions, take the test solution and determine it on high - performance liquid chromatographs of Instrument 1, Instrument 2, and Instrument 3 respectively. See Figure 19 , Table 16.

[0254] Table 16 Investigation of Instrument Durability - Ratio of Relative Retention Time

[0255]

[0256] The results show that when the test samples are detected with the above - mentioned 3 instruments, the RSD of the relative retention time of each characteristic peak is less than 1%, indicating that the method has good stability.

[0257] 4.5.4.2 Investigation with Different Personnel and at Different Times

[0258] On the basis of the above - determined experimental conditions, accurately weigh Bergenia formula granules (batch number: S1) by different personnel (A, B) at different times (T1, T2), prepare the test solution, and conduct the determination. See Table 17.

[0259] Table 17 Investigation of Personnel and Time - Ratio of Relative Retention Time

[0260]

[0261] The results show that when the same batch of samples is determined by different personnel at different times, the method has good stability.

[0262] 4.5.5 Durability Investigation

[0263] 4.5.5.1 Investigation of Column Durability

[0264] On the basis of the above - determined experimental conditions, investigate C18 columns (4.6 mm×250 mm, 5 μm) with different models but the same specifications respectively. See Figure 20 , Table 18.

[0265] Table 18 Investigation of Column Durability - Ratio of Relative Retention Time

[0266]

[0267] The results show that when the samples are detected with the above - mentioned 3 columns, the RSD of the relative retention time of the characteristic peak is between 0.41% and 4.49%.

[0268] 4.5.5.2 Stability

[0269] On the basis of the above-mentioned experimental conditions, the same test solution was taken and determined at 0h, 3h, 6h, 9h, 12h, and 24h respectively. See Table 19.

[0270] Table 19 Stability study - retention time

[0271]

[0272] The results showed that the RSD of the retention time of the corresponding characteristic peaks was between 0.06% and 0.53%, and the sample solution was relatively stable within 24 hours.

[0273] 4.5.6 Summary of methodology

[0274] When peak 7 was selected as the S peak, the RSD of the relative retention time at each stage is shown in Table 20.

[0275] Table 20 Summary of RSD values when peak 7 is the S peak

[0276]

[0277]

[0278] According to the principle of stable relative retention time, all batches of samples can be detected and the peaks are relatively high. It can be seen from the above table that the flow rate, different instruments, and different chromatographic columns have a greater impact on the relative retention time of each characteristic peak. Therefore, the flow rate was set at 1.0 ml / min, the column temperature was set at 30 °C, and the relative retention times of peak 1 and peak 2 were not specified and were identified by the corresponding reference substances.

[0279] 4.5.7 Determination of characteristic peaks and establishment of reference chromatogram

[0280] 4.5.7.1 Verification results of 3 batches of Bergenia formula granules

[0281] The characteristic chromatograms of 3 batches of samples of this product were determined by the proposed method, and the relative retention times were calculated. See Figure 21 , Table 21.

[0282] Table 21 Relative retention times of 3 batches of Bergenia formula granules

[0283]

[0284] According to the principle of stable relative retention time, all batches of samples can be detected and the peaks are relatively high, a total of 10 peaks with good repeatability were selected as characteristic peaks. The results showed that when peak 7 was used as the S peak, the RSD of the relative retention times of the 10 characteristic peaks of 3 batches of Bergenia formula granules was less than 2%.

[0285] 4.5.7.2 Establishment of the specified limit of relative retention time

[0286] The test solution chromatogram should show 10 characteristic peaks, which should correspond to the retention times of the 10 characteristic peaks in the chromatogram of the control crude drug. Among them, Peak 1, Peak 2, and Peak 7 should respectively correspond to the retention times of the corresponding reference substance peaks of the reference standards. The peak corresponding to the bergenin reference substance is the S peak. Calculate the relative retention times of the remaining characteristic peaks with respect to the S peak, and the relative retention times should be within the range of ±10% of the specified values. The specified values are 0.46 (Peak 3), 0.74 (Peak 4), 0.87 (Peak 5), 0.98 (Peak 6), 1.43 (Peak 8), 1.45 (Peak 9), and 1.67 (Peak 10).

[0287] Using the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition), the fingerprints of 3 batches of bergenia formula granules were synthesized, and a reference fingerprint of the bergenia formula granules was established, as shown in Figure 22 ; among them, Peak 1: arbutin; Peak 2: gallic acid; Peak 6: catechin; Peak 7 (S): bergenin; Peak 10: ellagic acid.

[0288] As can be seen from the above examples, the present invention provides a method for identifying the HPLC fingerprints of bergenia crude drug, cut crude drug, extract, and its formula granules, including the following steps: A) Dissolve and extract the test sample with a solvent to obtain a test solution; B) Determine the test solution by high performance liquid chromatography to obtain the HPLC fingerprints of bergenia crude drug, cut crude drug, extract, and its formula granules; the chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 column; mobile phase A is methanol, and mobile phase B is 0.1% phosphoric acid solution, with gradient elution. The present invention uses high performance liquid chromatography, selects methanol - 0.1% phosphoric acid solution as the mobile phase for gradient elution, and uses arbutin, gallic acid, catechin, bergenin, and ellagic acid as reference substances to establish the HPLC fingerprints of bergenia crude drug, cut crude drug, extract, and its formula granules. The method has good repeatability and precision, and is stable and reliable, and can control the quality of bergenia crude drug, cut crude drug, extract, and its formula granules.

[0289] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for identifying the HPLC characteristic fingerprints of Bergenia ligulata medicinal materials, cut pieces, extracts and their formula granules, comprising the following steps: A) Dissolve and extract the test sample with a solvent to obtain a test solution; The test sample is one or more of Bergenia ligulata medicinal materials, cut pieces, Bergenia ligulata extracts and Bergenia ligulata formula granules; The extraction process includes: mixing the test sample with water, boiling for 25 - 35 min, filtering, evaporating the filtrate to dryness, adding methanol with a mass fraction of 30%, ultrasonically treating, cooling, shaking well, filtering, and taking the subsequent filtrate to obtain the test solution; B) Determine the test solution by high performance liquid chromatography to obtain the HPLC characteristic fingerprints of Bergenia ligulata medicinal materials, cut pieces, extracts and their formula granules; The chromatographic conditions for the high performance liquid chromatography are: the chromatographic column is a C18 column; mobile phase A is methanol, mobile phase B is 0.1% phosphoric acid solution, with gradient elution; the column temperature is 30°C - 35°C; It also includes preparing a reference substance solution: Dissolve arbutin, gallic acid, catechin, bergenin, and ellagic acid with methanol respectively to prepare solutions containing 200 μg, 100 μg, 80 μg, 120 μg, and 40 μg per 1 ml as the reference substance solution for the reference substance; Determine the reference substance solution by high performance liquid chromatography to obtain the chromatogram of the reference substance; and qualitatively analyze the components of the HPLC characteristic fingerprints of Bergenia ligulata medicinal materials, cut pieces, extracts and their formula granules according to the chromatogram of the reference substance; The specific gradient elution is as follows: 0 - 5 min, mobile phase A: 5%, mobile phase B: 95%; 5 - 22 min, mobile phase A: 5 - 15%, mobile phase B: 95 - 85%; 22 - 32 min, mobile phase A: 15 - 25%, mobile phase B: 85 - 75%; 32 - 57 min, mobile phase A: 25 - 55%, mobile phase B: 75 - 45%; 57 - 67 min, mobile phase A: 55 - 80%, mobile phase B: 45 - 20%; The flow rate of the mobile phase is 0.8 - 1.2 mL / min; the detection wavelength is 240 - 260 nm; the injection volume is 5 - 10 μL, and the theoretical plate number calculated based on the bergenin peak should be not less than 4500.

2. The identification method according to claim 1, wherein The size of the C18 is 250×4.6 mm×5 μm.

3. The identification method according to claim 1, characterized in that, Use the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints to evaluate the similarity of the HPLC characteristic fingerprints of Bergenia ligulata medicinal materials and cut pieces, and obtain the HPLC characteristic fingerprints of Bergenia ligulata medicinal materials and cut pieces composed of 10 characteristic peaks. Among them, the peak corresponding to the bergenin reference substance is the S peak. In the said characteristic fingerprints, calculate the relative retention time of each characteristic peak and the S peak, and its relative retention time should be within ±10% of the specified value; the specified values are: 0.48 (peak 3), 0.75 (peak 4), 0.88 (peak 5), 0.98 (peak 6), 1.42 (peak 8), 1.44 (peak 9), 1.66 (peak 10).

4. The identification method according to claim 1, characterized in that The similarity of the HPLC characteristic fingerprint of Bergenia extract was evaluated using the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints, and the HPLC characteristic fingerprint of Bergenia extract consisting of 10 characteristic peaks was obtained. Among them, the peak corresponding to the bergenin reference substance was the S peak. In the said characteristic fingerprint, the relative retention times of each characteristic peak and the S peak were calculated, and the relative retention times should be within ±10% of the specified values; the specified values were: 0.44 (peak 3), 0.72 (peak 4), 0.86 (peak 5), 0.98 (peak 6), 1.46 (peak 8), 1.49 (peak 9), 1.71 (peak 10).

5. The authentication method according to claim 1, characterized in that The similarity of the HPLC characteristic fingerprint of Bergenia formula granules was evaluated using the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints, and the HPLC characteristic fingerprint of Bergenia formula granules consisting of 10 characteristic peaks was obtained. Among them, the peak corresponding to the bergenin reference substance was the S peak. In the said characteristic fingerprint, the relative retention times of each characteristic peak and the S peak were calculated, and the relative retention times should be within ±10% of the specified values; the specified values were: 0.46 (peak 3), 0.74 (peak 4), 0.87 (peak 5), 0.98 (peak 6), 1.43 (peak 8), 1.45 (peak 9), 1.67 (peak 10).

6. A method for identifying bergenia medicinal materials, cut pieces, extracts and their formula granules, characterized in that, The method according to any one of claims 1 to 5 was used for detection, and the detection results were analyzed.

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