A method for identifying fingerprint of soapberry and yunnan soapberry

Abstract

The application discloses a fingerprint spectrum identification method for Gleditsia sinensis and Gleditsia delavayi. The components of Gleditsia sinensis and Gleditsia delavayi are detected by high performance liquid chromatography (HPLC). The HPLC characteristic spectrum has moderate peak time, complete peak separation, good peak shape, no interference, easy positioning, and can accurately detect the quality of Gleditsia sinensis and Gleditsia delavayi. Moreover, the Gleditsia sinensis and Gleditsia delavayi can be distinguished according to the characteristic peaks, and the method provides a reliable method for identifying Gleditsia sinensis and Gleditsia delavayi which lose the appearance identification characteristics or related additional products.

Description

Technical Field

[0001] This invention relates to a method for distinguishing between soapberry rice and Yunnan soapberry rice, and more particularly to a fingerprint spectrum identification method for soapberry rice and Yunnan soapberry rice. Background Technology

[0002] Soapberry rice, derived from the outer endosperm of the seeds of the soapberry (Gleditsia sinensis Lam.), is commonly known as soapberry essence, snow lotus seed, or soapberry kernel. It is the source of the food additive soapberry polysaccharide gum. Soapberry rice mainly contains protein, total sugar, vitamins, amino acids, and trace elements. It has the effects of tonifying the kidneys, moistening the lungs, clearing phlegm and opening the orifices, promoting urination, nourishing the heart and blood vessels, clearing the liver and improving eyesight, and invigorating the spirit. Its total energy is 3.36 kcal / g, total carbohydrates are 61%, and total amino acids are 3.13%. There are eight species of soapberry plants in China, mainly produced in Shandong, Henan, Sichuan, Guizhou, and Yunnan provinces. Research has found that soapberry rice sold in the market also comes from the processed outer endosperm of the seeds of the Yunnan soapberry (Gleditsia japonica var. delavayi), commonly known as Yunnan soapberry rice. It is rich in trace elements and amino acids, and conforms to a high-potassium, low-sodium diet. In the market, both soapberry rice and Yunnan soapberry rice are sold as soapberry rice without distinguishing the varieties. Moreover, the two are quite similar in appearance and are not easy to distinguish. At present, there is a lack of relevant qualitative identification and quality evaluation methods.

[0003] Currently, there are no literature reports on the fingerprint identification of Gleditsia sinensis and Gleditsia yunnanensis. The method of this invention is simple, rapid, requires a small amount of medicinal materials, and yields fingerprint spectral characteristics and differences with high repeatability. It can be used for rapid and accurate identification and quality control of Gleditsia sinensis and Gleditsia yunnanensis. Summary of the Invention

[0004] The purpose of this invention is to provide a fingerprint chromatographic identification method for saponin rice and Yunnan saponin rice. This method uses 50% methanol as the extraction solvent, employs ultrasound-assisted extraction to prepare the test solution, and then uses HPLC to measure the fingerprint chromatogram under certain conditions for the identification of saponin rice and Yunnan saponin rice. This method is simple, rapid, requires a small amount of medicinal material, and yields fingerprint chromatograms with obvious characteristics and differences, exhibiting high repeatability. It can be used for accurate and rapid identification and quality control of saponin rice and Yunnan saponin rice.

[0005] The technical solution of the present invention:

[0006] A fingerprint identification method for saponin rice and Yunnan saponin rice, wherein the fingerprint identification method uses high performance liquid chromatography (HPLC) and includes the following steps:

[0007] (1) Preparation of saponin rice test solution: Take an appropriate amount of saponin rice powder, extract with methanol, filter, take the filtrate to obtain saponin rice test solution;

[0008] (2) Preparation of Yunnan soapberry rice test solution: Take an appropriate amount of Yunnan soapberry rice powder, add methanol to extract, filter the extract, take the filtrate to obtain Yunnan soapberry rice test solution;

[0009] (3) Take samples of the saponin rice test solution and Yunnan saponin rice test solution respectively for high performance liquid chromatography and record the chromatograms;

[0010] (4) Establishment of fingerprint chromatograms: HPLC fingerprint chromatograms of 13 batches of Gleditsia sinensis from different origins and Gleditsia sinensis from Yunnan were determined. The data of the chromatograms were analyzed and compared using the 2004 A edition of the "Similarity Evaluation System for Chromatographic Fingerprint chromatograms of Traditional Chinese Medicine". The common pattern of HPLC fingerprint chromatograms of Gleditsia sinensis and Gleditsia sinensis from Yunnan was calculated and used as the standard fingerprint chromatogram for the identification of Gleditsia sinensis and Gleditsia sinensis from Yunnan. The variety and quality of the medicinal materials can be determined based on the position of the characteristic fingerprint peaks and the size of the main characteristic peak area.

[0011] (5) Evaluation of similarity: The fingerprint spectra of Gleditsia sinensis and Gleditsia yunnanensis were imported into the 2004 A version of the "Similarity Evaluation System for Chromatographic Fingerprint Spectra of Traditional Chinese Medicine" for similarity analysis to examine the consistency of chromatographic peaks and evaluate the similarity.

[0012] In step (1) above, the preparation of the saponin rice test solution is as follows: take saponin rice powder and pass it through a No. 5 sieve, accurately weigh 0.95-1.05g, place it in a stoppered conical flask, accurately add 10mL of 50% methanol, seal tightly, weigh, sonicate for 30-60min, cool, make up for the weight loss, filter, take the filtrate, and filter it through a 0.45μm microporous membrane to obtain the solution.

[0013] Specifically, in step (1) above, the preparation of the saponin rice test solution is as follows: take saponin rice powder and pass it through a No. 5 sieve, accurately weigh 1.00 g, place it in a stoppered conical flask, accurately add 10 mL of 50% methanol, seal tightly, weigh, sonicate for 30 min, cool, make up for the weight loss, filter, take the filtrate, and filter it through a 0.45 μm microporous membrane to obtain the solution.

[0014] In step (2) above, the preparation of the Yunnan soapberry rice test solution is as follows: Yunnan soapberry rice is pulverized into powder and passed through a No. 5 sieve. 0.95-1.05g is accurately weighed, placed in a stoppered conical flask, 10mL of 50% methanol is accurately added, the flask is sealed tightly, weighed, sonicated for 30-60min, cooled, the weight loss is made up, filtered, and the filtrate is filtered through a 0.45μm microporous membrane to obtain the final solution.

[0015] Specifically, in step (2) above, the preparation of the Yunnan soapberry rice test solution is as follows: take Yunnan soapberry rice powder and pass it through a No. 5 sieve, accurately weigh 1.00 g, place it in a stoppered conical flask, accurately add 10 mL of 50% methanol, seal tightly, weigh, sonicate for 30 min, cool, make up for the weight loss, filter, take the filtrate, and filter it through a 0.45 μm microporous membrane to obtain the solution.

[0016] In step (3) above, the chromatographic conditions are as follows:

[0017] The chromatographic column was Diamnonsil C10. 18 The chromatographic column was 250 mm × 4.6 mm, 5 μm in diameter, with a column temperature of 30 °C, an injection volume of 20 μL, a flow rate of 1 mL / min, and a detection wavelength of 254 nm. Mobile phase A was acetonitrile, and mobile phase B was a 0.01% aqueous solution of phosphoric acid. Gradient elution was used.

[0018] In step (3) above, the gradient elution conditions are as follows: 0-5 min, 5%-13% A; 5-15 min, 13%-14.8% A; 15-17 min, 14.8%-16% A; 17-18 min, 16%-20% A; 18-19 min, 20%-25% A; 19-22 min, 25%-25% A; 22-25 min, 25%-30% A; 25-32 min, 30%-100% A.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] This method is simple to operate, easy to control, has good repeatability and accuracy, and strong characteristic. It can be quickly identified by the number of common peaks and their similarity, and the results are more objective and accurate.

[0021] In the instrument precision study, the RSD values ​​for the relative peak areas of each common peak were 0.18–2.57%, and the RSD values ​​for the relative retention times were 0.05–0.88%. In the method repeatability study, the RSD values ​​for the relative peak areas of each common chromatographic peak were 0.18–2.90%, and the RSD values ​​for the relative retention times were 0.05–0.17%. In the stability study, the RSD values ​​for the relative peak areas of each common chromatographic peak were 0.18–2.57%, and the RSD values ​​for the relative retention times were 0.05–0.88%.

[0022] The chromatograms of Gleditsia sinensis var. sarcodactylis and Gleditsia sinensis var. yunnanensis were imported into the 2004 Edition A of the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" for similarity analysis to examine the consistency of chromatographic peaks and evaluate similarity. The similarity of 10 batches of Gleditsia sinensis var. sarcodactylis was greater than 0.850; the similarity of 3 batches of Gleditsia sinensis var. yunnanensis was greater than 0.940. Compared with Gleditsia sinensis var. yunnanensis, the similarity of Gleditsia sinensis var. sarcodactylis was higher than 0.900, while the similarity of Gleditsia sinensis var. yunnanensis was lower than 0.700. Attached Figure Description

[0023] Figure 1 : It is the fingerprint spectrum of soapberry rice;

[0024] Figure 2: This is a fingerprint spectrum of saponin rice;

[0025] Figure 3 This is the fingerprint spectrum of Yunnan soapberry rice;

[0026] Figure 4 This is a fingerprint spectrum of Yunnan soapberry rice;

[0027] Figure 5 : Fingerprint profiles of Gleditsia sinensis and Gleditsia sinensis from Yunnan;

[0028] Figure 6 : is a comparative fingerprint spectrum of Gleditsia sinensis rice and Gleditsia sinensis rice from Yunnan;

[0029] Figure 7-1 Chromatogram of ethanol extraction solvent;

[0030] Figure 7-2 Chromatogram of water extraction solvent;

[0031] Figure 7-3 Chromatogram of methanol extraction solvent;

[0032] Figure 7-4 Chromatogram of 50% ethanol extraction solvent;

[0033] Figure 7-5 Chromatogram of 50% methanol extraction solvent;

[0034] Figure 8-1 Chromatogram of reflux extraction method;

[0035] Figure 8-2 : Chromatogram of ultrasonic extraction method;

[0036] Figure 9-1 Chromatogram of extraction time at 120 min;

[0037] Figure 9-2 Chromatogram of extraction time (90 min);

[0038] Figure 9-3 Chromatogram of extraction time over 60 min;

[0039] Figure 9-4 Chromatogram of extraction time over 30 min;

[0040] Figure 10-1 Chromatogram of the acetonitrile-water elution system;

[0041] Figure 10-2 Chromatogram of the methanol-water desulfurization system;

[0042] Figure 10-3 Chromatogram of the acetonitrile-0.1% phosphoric acid elution system;

[0043] Figure 10-4Chromatogram of the methanol-0.1% phosphoric acid elution system;

[0044] Figure 11-1 : Conditional chromatogram at 360nm wavelength;

[0045] Figure 11-2 : 330nm wavelength conditional chromatogram;

[0046] Figure 11-3 : Conditional chromatogram at 310nm wavelength;

[0047] Figure 11-4 : 280nm wavelength conditional chromatogram;

[0048] Figure 11-5 : 265nm wavelength conditional chromatogram;

[0049] Figure 11-6 Chromatogram under 254nm wavelength conditions;

[0050] Figure 11-7 : 210nm wavelength conditional chromatogram;

[0051] Figure 12-1 Chromatogram at 25℃ column temperature;

[0052] Figure 12-2 Chromatogram at 30℃ column temperature;

[0053] Figure 12-3 Chromatogram at 35℃ column temperature;

[0054] Figure 13-1 Chromatogram at a flow rate of 0.6 mL / min;

[0055] Figure 13-2 Chromatogram at a flow rate of 0.7 mL / min;

[0056] Figure 13-3 Chromatogram at a flow rate of 0.8 mL / min;

[0057] Figure 13-4 Chromatogram at a flow rate of 1.0 mL / min;

[0058] Figure 14-1 Chromatogram of Agela column;

[0059] Figure 14-2 Symmetry column chromatogram;

[0060] Figure 14-3 Diamnonsil C 18 Chromatogram of a chromatographic column. Detailed Implementation

[0061] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0062] Example 1:

[0063] (1) Preparation of saponin rice test solution: Take saponin rice powder and pass it through a No. 5 sieve. Accurately weigh 1.00 g, place it in a stoppered conical flask, accurately add 10 mL of 50% methanol, seal tightly, weigh, sonicate for 30 min, cool, make up the weight loss, filter, take the filtrate, and filter it through a 0.45 μm microporous membrane to obtain the solution.

[0064] (2) Preparation of Yunnan soapberry rice test solution: Take Yunnan soapberry rice powder and pass it through a No. 5 sieve. Accurately weigh 1.00 g, place it in a stoppered conical flask, accurately add 10 mL of 50% methanol, seal tightly, weigh, sonicate for 30 min, cool, make up the weight loss, filter, take the filtrate, and filter it through a 0.45 μm microporous membrane to obtain the solution.

[0065] (3) Inject the saponin rice test solution and the Yunnan saponin rice test solution separately into high performance liquid chromatography for detection, and record the chromatograms; the chromatographic conditions are as follows:

[0066] The chromatographic column was Diamnonsil C10. 18 The chromatographic column was 250 mm × 4.6 mm, 5 μm in diameter, with a column temperature of 30 °C, an injection volume of 20 μL, a flow rate of 1 mL / min, and a detection wavelength of 254 nm. Mobile phase A was acetonitrile, and mobile phase B was a 0.01% aqueous solution of phosphoric acid. Gradient elution was used.

[0067] The aforementioned gradient elution conditions are as follows:

[0068] 0~5min, 5%~13% A; 5~15min, 13%~14.8% A; 15~17min, 14.8%~16% A; 17~18min, 16%~20% A; 18~19min, 20%~25% A; 19~22min, 25%~25% A; 22~25min, 25%~30% A; 25~32min, 30%~100% A.

[0069] (4) Establishment of fingerprint chromatograms: HPLC fingerprint chromatograms of 13 batches of Gleditsia sinensis from different origins and Gleditsia sinensis from Yunnan were determined. The data of the chromatograms were analyzed and compared using the 2004 A edition of the "Similarity Evaluation System for Chromatographic Fingerprint chromatograms of Traditional Chinese Medicine". The common pattern of HPLC fingerprint chromatograms of Gleditsia sinensis and Gleditsia sinensis from Yunnan was calculated and used as the standard fingerprint chromatogram for the identification of Gleditsia sinensis and Gleditsia sinensis from Yunnan. The variety and quality of the medicinal materials can be determined based on the position of the characteristic fingerprint peaks and the size of the main characteristic peak area.

[0070] (5) Evaluation of similarity: The fingerprint spectra of Gleditsia sinensis and Gleditsia yunnanensis were imported into the 2004 A version of the "Similarity Evaluation System for Chromatographic Fingerprint Spectra of Traditional Chinese Medicine" for similarity analysis to examine the consistency of chromatographic peaks and evaluate the similarity.

[0071] Example 2:

[0072] (1) Preparation of saponin rice test solution: Take saponin rice powder and pass it through a No. 5 sieve. Accurately weigh 0.95g and place it in a stoppered conical flask. Accurately add 10mL of 50% methanol, seal tightly, weigh, sonicate for 60min, cool, make up the weight loss, filter, take the filtrate and filter it through a 0.45μm microporous membrane to obtain the solution.

[0073] (2) Preparation of Yunnan soapberry rice test solution: Take Yunnan soapberry rice powder and pass it through a No. 5 sieve. Accurately weigh 0.95g and place it in a stoppered conical flask. Accurately add 10mL of 50% methanol, seal tightly, weigh, sonicate for 60min, cool, make up the weight loss, filter, take the filtrate and filter it through a 0.45μm microporous membrane to obtain the solution.

[0074] (3) Inject the saponin rice test solution and the Yunnan saponin rice test solution separately into high performance liquid chromatography for detection, and record the chromatograms; the chromatographic conditions are as follows:

[0075] The chromatographic column was Diamnonsil C10. 18 The chromatographic column was 250 mm × 4.6 mm, 5 μm in diameter, with a column temperature of 30 °C, an injection volume of 20 μL, a flow rate of 1 mL / min, and a detection wavelength of 254 nm. Mobile phase A was acetonitrile, and mobile phase B was a 0.01% aqueous solution of phosphoric acid. Gradient elution was used.

[0076] The aforementioned gradient elution conditions are as follows:

[0077] 0~5min, 5%~13% A; 5~15min, 13%~14.8% A; 15~17min, 14.8%~16% A; 17~18min, 16%~20% A; 18~19min, 20%~25% A; 19~22min, 25%~25% A; 22~25min, 25%~30% A; 25~32min, 30%~100% A.

[0078] (4) Establishment of fingerprint chromatograms: HPLC fingerprint chromatograms of 13 batches of Gleditsia sinensis from different origins and Gleditsia sinensis from Yunnan were determined. The data of the chromatograms were analyzed and compared using the 2004 A edition of the "Similarity Evaluation System for Chromatographic Fingerprint chromatograms of Traditional Chinese Medicine". The common pattern of HPLC fingerprint chromatograms of Gleditsia sinensis and Gleditsia sinensis from Yunnan was calculated and used as the standard fingerprint chromatogram for the identification of Gleditsia sinensis and Gleditsia sinensis from Yunnan. The variety and quality of the medicinal materials can be determined based on the position of the characteristic fingerprint peaks and the size of the main characteristic peak area.

[0079] (5) Evaluation of similarity: The fingerprint spectra of Gleditsia sinensis and Gleditsia yunnanensis were imported into the 2004 A version of the "Similarity Evaluation System for Chromatographic Fingerprint Spectra of Traditional Chinese Medicine" for similarity analysis to examine the consistency of chromatographic peaks and evaluate the similarity.

[0080] Example 3:

[0081] (1) Preparation of saponin rice test solution: Take saponin rice powder and pass it through a No. 5 sieve. Accurately weigh 1.05 g, place it in a stoppered conical flask, accurately add 10 mL of 50% methanol, seal tightly, weigh, sonicate for 45 min, cool, make up the weight loss, filter, take the filtrate, and filter it through a 0.45 μm microporous membrane to obtain the solution.

[0082] (2) Preparation of Yunnan soapberry rice test solution: Take Yunnan soapberry rice powder and pass it through a No. 5 sieve. Accurately weigh 1.05 g, place it in a stoppered conical flask, accurately add 10 mL of 50% methanol, seal tightly, weigh, sonicate for 45 min, cool, make up the weight loss, filter, take the filtrate, and filter it through a 0.45 μm microporous membrane to obtain the solution.

[0083] (3) Inject the saponin rice test solution and the Yunnan saponin rice test solution separately into high performance liquid chromatography for detection, and record the chromatograms; the chromatographic conditions are as follows:

[0084] The chromatographic column was Diamnonsil C10. 18 The chromatographic column was 250 mm × 4.6 mm, 5 μm in diameter, with a column temperature of 30 °C, an injection volume of 20 μL, a flow rate of 1 mL / min, and a detection wavelength of 254 nm. Mobile phase A was acetonitrile, and mobile phase B was a 0.01% aqueous solution of phosphoric acid. Gradient elution was used.

[0085] The aforementioned gradient elution conditions are as follows:

[0086] 0~5min, 5%~13% A; 5~15min, 13%~14.8% A; 15~17min, 14.8%~16% A; 17~18min, 16%~20% A; 18~19min, 20%~25% A; 19~22min, 25%~25% A; 22~25min, 25%~30% A; 25~32min, 30%~100% A.

[0087] (4) Establishment of fingerprint chromatograms: HPLC fingerprint chromatograms of 13 batches of Gleditsia sinensis from different origins and Gleditsia sinensis from Yunnan were determined. The data of the chromatograms were analyzed and compared using the 2004 A edition of the "Similarity Evaluation System for Chromatographic Fingerprint chromatograms of Traditional Chinese Medicine". The common pattern of HPLC fingerprint chromatograms of Gleditsia sinensis and Gleditsia sinensis from Yunnan was calculated and used as the standard fingerprint chromatogram for the identification of Gleditsia sinensis and Gleditsia sinensis from Yunnan. The variety and quality of the medicinal materials can be determined based on the position of the characteristic fingerprint peaks and the size of the main characteristic peak area.

[0088] (5) Evaluation of similarity: The fingerprint spectra of Gleditsia sinensis and Gleditsia yunnanensis were imported into the 2004 A version of the "Similarity Evaluation System for Chromatographic Fingerprint Spectra of Traditional Chinese Medicine" for similarity analysis to examine the consistency of chromatographic peaks and evaluate the similarity.

[0089] This invention has undergone extensive experimental research, and the results of this experimental research are as follows:

[0090] I. Fingerprint Identification Methods

[0091] 1. Instruments:

[0092] Waterse-2695 high performance liquid chromatograph (Waters Corporation, USA), PDA detector, column oven, autosampler, HH-6 digital display constant temperature water bath (Changzhou Aohua Instrument Co., Ltd.), powder grinder (Ruian Baixin Pharmaceutical Machinery Co., Ltd., model: LG-01), AL204-IC / 0.001 analytical balance (METTLE RTOLEDO Instrument Co., Ltd.).

[0093] 2. Reagents:

[0094] Methanol (Sinopharm Chemical Reagent Co., Ltd., batch number: 20200928), acetonitrile (Sinopharm Chemical Reagent Co., Ltd., batch number: 20201215), phosphoric acid (Chongqing Chuandong Chemical Co., Ltd.), Wahaha purified water (Guizhou Wahaha Group Co., Ltd.), of which methanol and acetonitrile are chromatographic grade, and the remaining chemical reagents are analytical grade.

[0095] 3. Sample:

[0096] Ten batches of Gleditsia sinensis seeds and three batches of Gleditsia japonica seeds were purchased. They were identified by Professor Wang Xiangpei of the School of Ethnic Medicine, Guizhou University for Nationalities, as dried processed products of the outer endosperm of the seeds of Gleditsia sinensis Lam. and Gleditsia japonica var. delavayi.

[0097] Sample information is shown in the table below, along with the HPLC fingerprint chromatograms and sample sources.

[0098]

[0099] 4. Preparation of the test solution:

[0100] Take approximately 1.00g of powdered Gleditsia sinensis and Gleditsia yunnanensis, accurately weigh it, place it in a stoppered conical flask, accurately add 10mL of 50% methanol, seal tightly, weigh, sonicate for 30min, cool, make up the weight loss, filter, take the filtrate, and filter it through a 0.45μm microporous membrane to obtain the final product.

[0101] 5. HPLC chromatographic conditions:

[0102] Diamnonsil C 18 The chromatographic column had dimensions of 250 mm × 4.6 mm and a diameter of 5 μm. The column temperature was 30 °C, the injection volume was 20 μL, the flow rate was 1 mL / min, and the detection wavelength was 254 nm. The mobile phase consisted of acetonitrile (A) and 0.01% aqueous phosphoric acid solution (B). Gradient elution was used with the following conditions: 0–5 min, 5%–13% (A); 5–15 min, 13%–14.8% (A); 15–17 min, 14.8%–16% (A); 17–18 min, 16%–20% (A); 18–19 min, 20%–25% (A); 19–22 min, 25%–25% (A); 22–25 min, 25%–30% (A); 25–32 min, 30%–100% (A).

[0103] 6. Determination methods:

[0104] Take 20 μL of test solution of Gleditsia sinensis and Gleditsia sinensis from Yunnan, inject them into a high performance liquid chromatograph, and determine the chromatograms of Gleditsia sinensis and Gleditsia sinensis from Yunnan to obtain the fingerprint chromatograms for identification of Gleditsia sinensis and Gleditsia sinensis from Yunnan.

[0105] 7. Methodological Investigation and Results of Fingerprinting

[0106] In the instrument precision study, the RSD values ​​for the relative peak areas of each common peak were 0.18–2.57%, and the RSD values ​​for the relative retention times were 0.05–0.88%. In the method repeatability study, the RSD values ​​for the relative peak areas of each common chromatographic peak were 0.18–2.90%, and the RSD values ​​for the relative retention times were 0.05–0.17%. In the stability study, the RSD values ​​for the relative peak areas of each common chromatographic peak were 0.18–2.57%, and the RSD values ​​for the relative retention times were 0.05–0.88%.

[0107] 8. Fingerprint mapping

[0108] Thirteen batches of sample powder were taken and prepared according to the method for test solutions. The fingerprint chromatograms were determined by HPLC. Using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" (2004, Edition A), common peaks were identified. After multi-point correction and full-spectrum peak matching using the median method, sample fingerprint chromatograms were established. Based on these, reference chromatograms for Gleditsia sinensis and Gleditsia japonica were established. Of the relevant parameters given for 10 batches of Gleditsia sinensis test solutions, 17 peaks were common to all batches. These 17 peaks were identified as common peaks. Figure 1 , Figure 2 Comparing the relevant parameters given by three batches of Yunnan soapberry rice test solutions, 16 peaks were found to be common to all batches. These 16 peaks were determined to be their common peaks. (See attached image) Figure 3 , Figure 4 The results of relative peak area (Table 3) and relative retention time (Table 4) show that there are 13 common peaks in both *Gleditsia sinensis* and *Gleditsia yunnanensis*. Figure 5 ,See Figure 6 The similarity is shown in Table 2.

[0109] 9. Fingerprint Similarity Evaluation

[0110] The chromatograms of Gleditsia sinensis var. sarcodactylis and Gleditsia sinensis var. yunnanensis were imported into the 2004 Edition A of the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" for similarity analysis to examine the consistency of chromatographic peaks and evaluate similarity. The similarity of 10 batches of Gleditsia sinensis var. sarcodactylis was greater than 0.850; the similarity of 3 batches of Gleditsia sinensis var. yunnanensis was greater than 0.940. Compared with Gleditsia sinensis var. yunnanensis, the similarity of Gleditsia sinensis var. sarcodactylis was higher than 0.900, while the similarity of Gleditsia sinensis var. yunnanensis was lower than 0.700.

[0111] Table 1. Similarity between 10 batches of Gleditsia sinensis seeds and 3 batches of Yunnan Gleditsia sinensis seeds

[0112]

[0113] Table 2. Similarity between Gleditsia sinensis and Gleditsia yunnanensis.

[0114]

[0115] 10. Conditional Screening of the Invention

[0116] (1) Selection of extraction solvent

[0117] Five portions of 1g each of soapberry powder (passed through a No. 5 sieve) were weighed and added to 10mL each of methanol, 50% methanol, ethanol, 50% ethanol, and water, respectively. The mixtures were sonicated for 30 min, filtered through a microporous membrane, and the filtrates were used for analysis. Results showed that the sample extracted with 50% methanol exhibited complete extraction, rich chromatographic information, and good chromatographic resolution and peak shape. (See...) Figure 7-1 , Figure 7-2 , Figure 7-3 , Figure 7-4 , Figure 7-5 .

[0118] (2) Selection of extraction method

[0119] The experiment investigated ultrasonic and reflux extraction methods. Results showed that ultrasonic extraction resulted in better dissolution of sample components, leading to better chromatographic resolution and peak shape. (See...) Figure 8-1 , Figure 8-2 .

[0120] (3) Selection of extraction time

[0121] The experiment examined the chromatograms obtained from different ultrasonic extraction times (30 min, 60 min, 90 min, and 120 min). The results showed little difference in the amount of information contained in the chromatograms obtained from different ultrasonic methods. Therefore, 30 min was selected as the optimal extraction time, as it is fast and resource-efficient. See [link to experiment]. Figure 9-1 , Figure 9-2 , Figure 9-3 , Figure 9-4 .

[0122] (4) Selection of mobile phase

[0123] Using methanol-water, methanol-0.1% phosphoric acid solution, acetonitrile-water, and acetonitrile-0.1% phosphoric acid solution as mobile phases, the gradient elution profiles of each mobile phase were compared. The results showed that the acetonitrile-0.1% phosphoric acid solution system produced the most peaks, with better peak shape and separation performance. (See attached image) Figure 10-1 , Figure 10-4 , Figure 10-3 , Figure 10-4 .

[0124] (5) Selection of detection wavelength

[0125] The experiment examined chromatograms at different wavelengths (210 nm, 254 nm, 265 nm, 280 nm, 310 nm, 330 nm, and 360 nm). The results showed that the chromatogram at 254 nm exhibited the best resolution and peak shape, providing the most complete information. See [link to chromatogram]. Figure 11-1 , Figure 11-2 , Figure 11-3 , Figure 11-4 , Figure 11-5 , Figure 11-6 , Figure 11-7 .

[0126] (6) Selection of different column temperatures

[0127] The experiment investigated column temperatures of 25℃, 30℃, and 35℃. The results showed that the column temperature of 30℃ produced the best chromatographic resolution and peak shape. (See...) Figure 12-1 , Figure 12-2 , Figure 12-3 .

[0128] (7) Selection of different flow velocities

[0129] Flow rates of 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, and 1 mL / min were compared. The results showed that a flow rate of 1 mL / min resulted in better chromatographic resolution and peak shape. (See...) Figure 13-1 , Figure 13-2 , Figure 13-3 , Figure 13-4 .

[0130] (8) Selection of chromatographic column

[0131] This article compares Agela (250mm×4.6mm, 5μm), Symmetry (150mm×3.9mm, 5μm), and Diamnonsil C 18 The separation of the saponin rice sample using three chromatographic columns (250mm × 4.6mm, 5μm) is shown in the figure. Diamnonsil C 18 Good separation. See Figure 14-1 , Figure 14-2 , Figure 14-3 .

[0132] Table 3. Relative peak areas of Gleditsia sinensis (A) and Gleditsia sinensis (B)

[0133]

[0134]

[0135] Table 4. Relative retention time of Gleditsia sinensis (A) and Gleditsia sinensis (B) from Yunnan.

[0136]

[0137] .

Claims

1. A fingerprint identification method for Gleditsia sinensis and Gleditsia delavayi, characterized in that: The fingerprint identification method is detected by high performance liquid chromatography, comprising the following steps: (1) Gleditsia sinensis L. test solution preparation: take Gleditsia sinensis L. powder through No. 5 sieve, accurately weigh 0.95-1.05 g, put into a conical flask with a plug, accurately add 50% methanol 10 mL, tightly plug, weigh, ultrasonic 30-60 min, cool, make up the weight loss, filter, take the filtrate, pass through 0.45 μm microporous filter membrane, and then obtain; (2) Yunnan Gleditsia sinensis L. test solution preparation: take Yunnan Gleditsia sinensis L. powder through No. 5 sieve, accurately weigh 0.95-1.05 g, put into a conical flask with a plug, accurately add 50% methanol 10 mL, tightly plug, weigh, ultrasonic 30-60 min, cool, make up the weight loss, filter, take the filtrate, pass through 0.45 μm microporous filter membrane, and then obtain; (3) High performance liquid chromatography sample detection is carried out by sucking Gleditsia sinensis L. test solution and Yunnan Gleditsia sinensis L. test solution respectively, and recording the chromatogram; (4) Fingerprint establishment: the HPLC fingerprints of 10 batches of Gleditsia sinensis L. from different producing areas and 3 batches of Yunnan Gleditsia sinensis L. from different producing areas are determined, the data of the fingerprints are analyzed and compared by using the 2004 A version of "Chinese medicine chromatographic fingerprint similarity evaluation system", the common mode of the HPLC fingerprints of Gleditsia sinensis L. and Yunnan Gleditsia sinensis L. is calculated, and the standard fingerprint for identifying Gleditsia sinensis L. and Yunnan Gleditsia sinensis L. is obtained; the variety and quality of the medicinal material can be determined according to the characteristic fingerprint peak position and the size of the main characteristic peak area; The chromatographic conditions are as follows: The chromatographic column was Diamnonsil C 18 The chromatographic column was 250 mm x 4.6 mm, 5 μm, the column temperature was 30℃, the injection volume was 20 μL, the flow rate was 1 mL / min, the detection wavelength was 254 nm, the mobile phase A was acetonitrile, the mobile phase B was 0.01% phosphoric acid aqueous solution, and the elution mode was gradient elution. The gradient elution conditions are as follows: 0-5 min, 5%-13% A; 5-15 min, 13%-14.8% A; 15-17 min, 14.8%-16% A; 17-18 min, 16%-20% A; 18-19 min, 20%-25% A; 19-22 min, 25%-25% A; 22-25 min, 25%-30% A; 25-32 min, 30%-100% A; (5) Similarity evaluation: the fingerprints of Gleditsia sinensis L. and Yunnan Gleditsia sinensis L. are introduced into the 2004 A version of "Chinese medicine chromatographic fingerprint similarity evaluation system" for similarity analysis, the consistency of the chromatographic peaks is investigated, and the similarity evaluation is carried out.

2. The method according to claim 1, wherein the method is characterized by: In step (1), the Gleditsia sinensis L. test solution preparation: take Gleditsia sinensis L. powder through No. 5 sieve, accurately weigh 1.00 g, put into a conical flask with a plug, accurately add 50% methanol 10 mL, tightly plug, weigh, ultrasonic 30 min, cool, make up the weight loss, filter, take the filtrate, pass through 0.45 μm microporous filter membrane, and then obtain.

3. The method according to claim 1, wherein the method is characterized by the following steps: In step (2), the Yunnan Gleditsia sinensis L. test solution preparation: take Yunnan Gleditsia sinensis L. powder through No. 5 sieve, accurately weigh 1.00 g, put into a conical flask with a plug, accurately add 50% methanol 10 mL, tightly plug, weigh, ultrasonic 30 min, cool, make up the weight loss, filter, take the filtrate, pass through 0.45 μm microporous filter membrane, and then obtain.

Images