Method for constructing characteristic fingerprint of Swertia chirayita (Roxb.) H. Karst. and method for identifying Swertia chirayita (Roxb.) H. Karst.

Abstract

The present invention provides a method for constructing a characteristic fingerprint of Swertia chirayita and a method for identifying Swertia chirayita. The characteristic fingerprint of Swertia chirayita includes characteristic peaks of the following substances: isomangiferin, swertiamarin, mangiferin, gentiopicroside, sweroside, isoorientin, methylswerinine, oleanolic acid, and ursolic acid. Using the characteristic peaks in the characteristic fingerprint of Swertia chirayita as a measurement index, Swertia chirayita can be accurately identified and the authenticity can be distinguished. In particular, Swertia chirayita with good curative effects on liver and gall diseases can be screened out, which is helpful for the quality control of Swertia chirayita. Moreover, the construction method and the identification method are easy to operate, and have the advantages of good reproducibility, high precision, and strong stability, and are suitable for popularization and application.

Description

Technical Field

[0001] The present invention relates to the field of chromatographic detection, and in particular to a method for constructing a characteristic spectrum of Tibetan Artemisia Capillaris and a method for identifying Tibetan Artemisia Capillaris. Background Art

[0002] Tibetan Artemisia capillaris is one of the eight treasures of Tibetan medicine. It has the effects of clearing heat, detoxifying, clearing the liver and promoting bile secretion. It can be used to treat diseases such as hepatitis, cholecystitis, acute icteric hepatitis and pyelonephritis. The origin of Tibetan Artemisia capillaris is complex, and the ingredients of different types of Tibetan Artemisia capillaris medicinal materials vary greatly, and the content is significantly different. At present, the detection methods for Tibetan Artemisia capillaris include microscopic observation, thin layer chromatography, infrared spectroscopy and high performance liquid chromatography. Among them, the establishment of fingerprints by high performance liquid chromatography conforms to the overall comprehensive characteristics of traditional Chinese medicine and is currently a common method internationally. However, due to the limitation of similarity, fingerprints can only be used to evaluate the quality of medicinal materials of the same kind or within two kinds, and the scope of application is narrow. In addition, the active ingredient evaluation index currently used is single, and the quality of Tibetan Artemisia capillaris cannot be fully controlled, which limits the development and utilization of Tibetan Artemisia capillaris. Summary of the invention

[0003] The present invention aims to solve the technical problems existing in the prior art at least to a certain extent. To this end, the present invention proposes a method for constructing a characteristic spectrum of Tibetan Artemisia Capillaris, a method for identifying Tibetan Artemisia Capillaris, and a method for screening Tibetan Artemisia Capillaris for treating hepatobiliary diseases. The characteristic peaks in the characteristic spectrum of Tibetan Artemisia Capillaris are used as a measurement index to accurately identify Tibetan Artemisia Capillaris, distinguish the authenticity, and especially screen out Tibetan Artemisia Capillaris with good efficacy for hepatobiliary diseases, which is helpful for the quality control of Tibetan Artemisia Capillaris. In addition, the construction method and the identification method are simple to operate, have the advantages of good reproducibility, high precision and strong stability, and are suitable for promotion and application.

[0004] In one aspect of the present invention, the present invention proposes a method for constructing a characteristic spectrum of Tibetan Artemisia capillaris. According to an embodiment of the present invention, the method comprises: (1) preparation of a reference solution: weighing different kinds of reference substances, adding an organic solvent, and preparing a mixed reference solution; (2) preparation of a test solution: taking a Tibetan Artemisia capillaris sample, adding an organic solvent, weighing the weight, ultrasonic treatment, taking it out, cooling it, weighing the weight again, supplementing the reduced weight with the organic solvent, filtering it, and collecting the filtrate; (3) obtaining a characteristic spectrum: using the reference solution as a reference, injecting the reference solution and the test solution into a liquid chromatograph, recording the chromatogram, selecting the chromatograms of multiple batches of Tibetan Artemisia capillaris, determining the common characteristic peaks in the chromatogram, and obtaining a characteristic spectrum of Tibetan Artemisia capillaris. By studying and analyzing the characteristic spectrum of Tibetan Artemisia capillaris, 9 characteristic peaks can be obtained, which is helpful for accurately identifying Tibetan Artemisia capillaris. In addition, the method is simple to operate, has the advantages of good reproducibility, high precision and strong stability, and is suitable for popularization and application.

[0005] According to an embodiment of the present invention, the Tibetan Artemisia capillaris is selected from: Swertia claspingii, Swertia cerana, Swertia purpurogena, Swertia sichuanensis or Swertia dasyphylla. The inventor collected multiple batches of Swertia genus, Swertia genus, Flat bud genus, and Hairy Flower genus from different regions of Gentianaceae, such as Big Seed Swertia, Swertia cerana, Swertia cerana, Swertia dasyphylla, Swertia dasyphylla indica ... The chemical components of the five species of Tibetan Artemisia capillaris (Swedeia capillaris, Swertia edulis, Swertia purpurogena, Swertia chuanxiensis and Swertia macroseed) are numerous and high in content, all of which contain more than 13 chemical components, of which 10 are shared. The effective ingredients of Tibetan Artemisia capillaris have significant effects on the treatment of acute and chronic hepatitis, chronic cholecystitis, etc. Therefore, it can be considered that the above five species of Tibetan Artemisia capillaris are high-quality medicinal materials for the treatment of liver diseases, and the quality of Tibetan Artemisia capillaris can be evaluated based on the characteristic maps established based on these five species of Tibetan Artemisia capillaris.

[0006] According to an embodiment of the present invention, the different reference substances are selected from at least two of: gentiopicroside, swertiapicroside, danshoin, mangiferin, neomangiferin, isomangiferin, danshool, methyldanshoin, luteolin, danshoflavin, isovitexin, isoorientin, oleanolic acid and ursolic acid. By using the above compounds as reference substances, it is possible to effectively compare which substance the characteristic peaks of the test sample correspond to, thereby facilitating in-depth research.

[0007] According to an embodiment of the present invention, the organic solvent is selected from methanol, ethanol, acetonitrile, isopropanol or acetone. Thus, the components in the Tibetan Artemisia Capillaris can be effectively extracted.

[0008] According to an embodiment of the present invention, the detection conditions of the liquid chromatograph are as follows: the stationary phase of the chromatographic column is octadecyl bonded silica gel; the mobile phases are mobile phase A and mobile phase B, the mobile phase A is acetonitrile, and the mobile phase B is 0.1% phosphoric acid solution; the column temperature is 20-40°C;

[0009] The gradient elution conditions were as follows:

[0010]

[0011] The detection wavelength conditions are as follows:

[0012]

[0013]

[0014] The flow rate conditions are as follows:

[0015] Time (min) Flow rate (mL / min) 0～30 0.7 30～35 Rise from 0.7 to 1 35～135 1 135～138 Drop from 1 to 0.7 138～150 0.7

[0016] Through a large number of experiments, the inventors obtained the above-mentioned preferred detection conditions. Thus, the above 9 characteristic peaks can be separated with high resolution, and then the Herba Swertiae Punicae can be accurately identified to distinguish the genuine from the fake, especially the Herba Swertiae Punicae with good therapeutic effects on hepatobiliary diseases can be obtained. For example, when selecting the detection wavelength, the inventors scanned in the range of 180-600 nm with an ultraviolet spectrophotometer to detect the maximum absorption wavelength, and determined the wavelength according to the ultraviolet absorption spectrum. It was found that the chemical components in the Herba Swertiae Punicae are complex, and all substances cannot be detected by using a constant wavelength, which is not conducive to the quality control of the Herba Swertiae Punicae. Moreover, the inventors studied the retention times of various substances in the Herba Swertiae Punicae and found that when the chromatographic analysis time is greater than 60 minutes, especially up to 150 minutes, the characteristic peaks can be effectively separated, which helps to identify the Herba Swertiae Punicae, distinguish the genuine from the fake, and especially the Herba Swertiae Punicae with good therapeutic effects on hepatobiliary diseases can be obtained.

[0017] According to the embodiments of the present invention, the inventors analyzed the constructed characteristic spectrum and learned that there are 9 common characteristic peaks in the characteristic spectrum. Taking sweroside as the standard peak among the characteristic peaks, the relative elution times of the remaining characteristic peaks are 0.349-0.364, 0.633-0.650, 0.848-0.857, 0.898-0.925, 1.000, 1.605-1.838, 4.176-4.667, 4.503-4.927, and 5.429-5.475 in sequence from front to back. At the above elution times, peak 1 comes from isomangiferin; peak 2 comes from swertiamarin; peak 3 comes from mangiferin; peak 4 comes from gentiopicroside; peak 5 comes from sweroside; peak 6 comes from isoorientin; peak 7 comes from methylswerin; peak 8 comes from an unknown substance; peak 9 comes from oleanolic acid and ursolic acid. The characteristic peak of the unknown substance exists in various Herba Swertiae Punicae, but the substance corresponding to this peak has not been identified yet. Therefore, taking the peak of the unknown substance as a characteristic peak can further improve the accuracy of the identification of the Herba Swertiae Punicae; oleanolic acid and ursolic acid are isomers and cannot be completely separated under this condition, and they are both on peak 9. Using the above 9 characteristic peaks as measurement indicators can accurately and effectively identify the Herba Swertiae Punicae, not only identify its authenticity, but more importantly, evaluate its quality, that is, whether high-quality Herba Swertiae Punicae with the efficacy of treating hepatobiliary diseases is obtained. Thus, it helps to control the quality of the Herba Swertiae Punicae and is suitable for popularization and application.

[0018] In another aspect of the present invention, the present invention provides a method for identifying Swertia punicea Hemsl. According to an embodiment of the present invention, the method includes: pretreating a sample to be tested to obtain a sample solution for injection; performing liquid chromatography detection on the sample solution for injection to obtain a chromatogram; determining whether the chromatogram contains characteristic peaks in the characteristic chromatogram of Swertia punicea Hemsl. obtained by the method for constructing the characteristic chromatogram of Swertia punicea Hemsl. described above; and when the chromatogram contains the characteristic peaks, it is an indication that the sample to be tested is Swertia punicea Hemsl. Thus, according to the method of the embodiment of the present invention, it can be accurately determined whether the sample to be tested is Swertia punicea Hemsl., with good reproducibility, high precision, strong stability, and is suitable for popularization and application.

[0019] According to an embodiment of the present invention, the detection conditions of the liquid chromatography are as defined in the method for constructing the characteristic chromatogram of Swertia punicea Hemsl. described above. Thus, the above 9 characteristic peaks can be effectively separated with high resolution.

[0020] According to an embodiment of the present invention, Swertia punicea Hemsl. has the efficacy of treating hepatobiliary diseases. Thus, by using the method of the present invention, high-quality Swertia punicea Hemsl. with good therapeutic effects on hepatobiliary diseases can be screened out.

[0021] Advantageous effects:

[0022] The present invention breaks through the limitation of fingerprint similarity, establishes a characteristic chromatogram of Swertia punicea Hemsl. (only measuring relative retention time), which has a wide range of applications. Through the study of five types of Swertia punicea Hemsl. (Swertia franchetiana H. Smith, Swertia diluta (Turcz.) Benth., Swertia punicea Hemsl., Swertia mussotii Franch., and Swertia macrosperma C. B. Clarke), 9 common peaks are obtained, and 8 of them are identified. It overcomes the defect of only using common components such as swertiamarin and mangiferin to identify Swertia punicea Hemsl., and can comprehensively identify the quality of Swertia punicea Hemsl.

[0023] The additional aspects and advantages of the present invention will be partly given in the following description, partly will become obvious from the following description, or will be understood through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The above and / or additional aspects and advantages of the present invention will become obvious and easy to understand from the description of the embodiments in conjunction with the following drawings, wherein:

[0025] Figure 1 Shows the HPLC characteristic chromatogram of Swertia punicea Hemsl. according to Embodiment 1 of the present invention;

[0026] Figure 2 Shows the HPLC characteristic chromatogram of Swertia punicea Hemsl. according to Comparative Example 7 of the present invention;

[0027] Figure 3 Shows the HPLC characteristic chromatogram of Swertia punicea Hemsl. according to Comparative Example 8 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] The solution of the present invention will be explained below in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. For those without specific technical or conditions noted in the embodiments, the techniques or conditions described in the literature in the art or according to the product specifications are followed. For reagents or instruments without the manufacturer noted, they are all conventional products that can be obtained through commercial purchase.

[0029] Example 1: Construction of HPLC characteristic chromatogram

[0030] 1.1 Experimental instruments and reagents: Agilent 1260 high performance liquid chromatograph, Yuexu Ultimate XB-C18 chromatographic column, KQ-700VDB ultrasonic instrument, chromatographically pure acetonitrile, analytical pure phosphoric acid, analytical pure methanol and ultrapure water.

[0031] 1.2 Reference substances: Gentian bitter glycoside, Swertiamarin, Sweroside, Mangiferin, Neomangiferin, Isomangiferin, Sweroside alcohol glycoside, Methylswerinine, Luteolin, Swertisin, Isovitexin, Isorientin, Oleanolic acid and Ursolic acid, purchased from the National Institutes for Food and Drug Control.

[0032] 1.3 Test samples: A total of 23 batches of samples were selected, involving five types of Swertia mussotii Franch. Specifically, there were 6 batches of Swertia franchetiana H. Smith, 1 batch of Swertia diluta (Turcz.) Benth., 2 batches of Swertia punicea Hemsl., 9 batches of Swertia mussotii Franch. and 5 batches of Swertia macrosperma C. B. Clarke.

[0033]

[0034]

[0035] 1.4 Steps

[0036] (1) Chromatographic detection conditions: Using octadecylsilane chemically bonded silica as the stationary phase, acetonitrile as mobile phase A, 0.1% phosphoric acid as mobile phase B, column temperature 30 °C, flow rate 0.7 - 1 ml / min, detection wavelength 205 - 240 nm. Gradient elution was carried out according to the following table, and the detection wavelength and flow rate were changed according to the following table. The theoretical plate number should not be less than 10,000 calculated based on the Sweroside peak, and the analysis time was 150 minutes.

[0037]

[0038] (2) Preparation of reference substance solution: Take appropriate amounts of reference substances such as Gentian bitter glycoside, Swertiamarin, Sweroside, Mangiferin, Neomangiferin, Isomangiferin, Sweroside alcohol glycoside, Methylswerinine, Luteolin, Swertisin, Isovitexin, Isorientin, Oleanolic acid and Ursolic acid, accurately weigh them, and make a solution containing 0.5 mg per 1 ml with methanol to obtain the reference substance solution;

[0039] (3) Preparation of test solution: Weigh accurately 0.2 g of the Swertia mussotii Franch. sample, place it in a 100-ml stoppered Erlenmeyer flask, add 25 ml of 80% methanol, weigh again, ultrasonically treat (power 270 W, frequency 40 kHz) for 40 minutes, take out, let it cool, weigh again, make up the lost weight with 80% methanol, filter through a dry filter paper, discard the initial filtrate, and filter the subsequent filtrate through a 0.45-μm microporous filter membrane to obtain the test solution;

[0040] (4) Obtaining the characteristic chromatogram: Pipette 10 μl each of the test solution and the reference solution, inject them into the liquid chromatograph, record the chromatogram, select the chromatograms of qualified batches of Swertia mussotii Franch., determine the common characteristic peaks in the chromatogram, and thus obtain the characteristic chromatogram of Swertia mussotii Franch.

[0041] According to the above detection method, the characteristic chromatogram of Swertia mussotii Franch. obtained is shown in Figure 1 , there are 9 characteristic peaks, among which the 5th peak is the standard peak. Compared with the standard peak, the relative retention times of the 9 peaks are respectively: 0.349, 0.633, 0.848, 0.898, 1.000, 1.838, 4.667, 4.927, 5.475. The substance corresponding to the 1st peak is isomangiferin; the substance corresponding to the 2nd peak is swertiamarin; the substance corresponding to the 3rd peak is mangiferin; the substance corresponding to the 4th peak is gentiopicroside; the substance corresponding to the 5th peak is sweroside; the substance corresponding to the 6th peak is isoorientin; the substance corresponding to the 7th peak is methylswerinine; the substance corresponding to the 9th peak is oleanolic acid and ursolic acid. (The substance of the 8th peak is not determined)

[0042] Example 2: Methodology investigation of the characteristic chromatogram detection method

[0043] 2.1 Repeatability test

[0044] Take 6 portions of the same sample, prepare the test solution, record the chromatogram, and calculate the RSD of the relative retention times of each common peak < 0.43%, meeting the requirement of RSD < 5%, indicating good reproducibility of the method.

[0045]

[0046] 2.2 Precision test

[0047] Take the same test solution, inject it continuously for 6 times, record the chromatogram, and calculate the RSD of the relative retention times of each common peak < 0.43%, meeting the requirement of RSD < 5%, indicating good precision of the instrument.

[0048]

[0049]

[0050] 2.3 Stability test

[0051] Take the same test sample solution, record the chromatograms at 0 h, 3 h, 5 h, 10 h, 15 h, 20 h, and 24 h. The RSD of the relative retention times of each chromatographic peak is < 1.02%, meeting the requirement of RSD < 5%, indicating that the sample is stable within 24 h.

[0052]

[0053] Comparative Example 1

[0054] In this comparative example, the chromatographic detection wavelength in Example 1 was replaced with a constant wavelength of 240 nm, and the remaining chromatographic detection methods were the same as those in Example 1.

[0055] The results showed that the number of characteristic peaks in the chromatogram obtained with a constant wavelength decreased, and the characteristic peaks of oleanolic acid and ursolic acid did not appear, which was not conducive to the quality control of Swertia mussotii Franch. In contrast, the present invention adjusted the wavelength within 115 - 135 minutes, which not only ensured the smooth elution of the first 8 substances in the chromatogram but also detected oleanolic acid and ursolic acid, effectively controlling the quality of Swertia mussotii Franch.

[0056] Comparative Example 2

[0057] In this comparative example, the flow rate in the chromatography of Example 1 was replaced with a constant flow rate of 1 ml / min, and the remaining chromatographic detection methods were the same as those in Example 1.

[0058] The results showed that the first 5 characteristic peaks in the chromatogram obtained with a constant flow rate of 1 ml / min appeared earlier and overlapped, with poor resolution and unable to meet the detection requirements. In contrast, the present invention set the flow rate at 0.7 ml / min within 0 - 30 minutes, avoiding the overlap phenomenon due to the close distance of the first 5 characteristic peaks, with good separation effect and able to meet the detection requirements.

[0059] Comparative Example 3

[0060] In this comparative example, the mobile phase in Example 1 was replaced with methanol - 0.1% phosphoric acid solution, and the remaining chromatographic detection methods were the same as those in Example 1.

[0061] The results showed that the substances in the obtained chromatogram could not be completely separated, with chromatographic peak overlap, and still could not meet the detection requirements within the elution time after adjusting the elution conditions. In contrast, with the mobile phase of the present invention, the substances could be effectively separated, and the obtained chromatographic peaks were sharp without tailing, with good detection effect.

[0062] Comparative Example 4

[0063] In this comparative example, the mobile phase in Example 1 was replaced with acetonitrile - methanol, and the remaining chromatographic detection methods were the same as those in Example 1.

[0064] The results show that the substances in the obtained chromatogram cannot be completely separated, and the chromatographic peaks overlap. After adjusting the elution conditions, the detection requirements cannot be met within the elution time. However, by using the mobile phase of the present invention, the substances can be effectively separated, and the chromatographic peaks are sharp without tailing, and the detection effect is good.

[0065] Comparative Example 5

[0066] In this comparative example, the mobile phase in Example 1 was replaced with acetonitrile-0.02 phosphoric acid solution, and the rest of the chromatographic detection method was the same as in Example 1.

[0067] The results show that the separation of the obtained chromatogram is improved, but the peak shape and theoretical plate number are not ideal, and the detection effect is still not ideal after adjusting the elution conditions. However, by using the mobile phase of the present invention, the characteristics of the obtained chromatogram are obvious, the peak shape is sharp, there is no tailing, and the detection effect is good.

[0068] Comparative Example 6

[0069] In this comparative example, the mobile phase elution condition in Example 1 was replaced with an acetonitrile-0.1 phosphoric acid solution with a fixed volume ratio of 12:88, and the rest of the chromatographic detection method was the same as in Example 1.

[0070] The results show that the separation of the last four characteristic peaks in the obtained chromatogram is poor, and the chromatographic peaks overlap, which cannot meet the detection requirements. However, by using the mobile phase elution conditions of the present invention, the characteristic peaks in the obtained chromatogram are obvious, the peak shape is sharp, there is no tailing, and the detection effect is good.

[0071] Comparative Example 7

[0072] In this comparative example, 11 species of Tibetan Artemisia capillaris (Swettia claspingii, Swertia cerasifera in the north, Swertia purpurogena, Swertia chuanxiensis, Swertia dasyphylla in the north China, Swertia pratensis, Swertia dasyphylla in the south China, Swertia dasyphylla in the south China, Swertia dasyphylla in the south China, Swertia dasyphylla in the south China, Swertia dasyphylla in the south China, Swertia dasyphylla in the south China, Swertia dasyphylla in the south China, and Swertia dasyphylla in the south China) were selected for research, of which 23 batches of samples (Swettia claspingii, Swertia cerasifera in the north, Swertia purpurogena, Swertia chuanxiensis, and Swertia dasyphylla in the south China) were derived from the same sources as in Example 1, and the sources of the other 6 species of Tibetan Artemisia capillaris were shown in the table below. The above Tibetan Artemisia capillaris was subjected to chromatographic detection and analysis using the method in Example 1.

[0073] Results Figure 2 Increasing the number of Tibetan Artemisia capillaris species resulted in a decrease in common characteristic peaks. There were only three common characteristic peaks among the 11 Tibetan Artemisia capillaris species, namely, scutellaria baicalensis, arachidinoside and oleanolic acid. These three components are relatively common in Tibetan Artemisia capillaris and cannot fully evaluate the quality of Tibetan Artemisia capillaris.

[0074]

[0075] Comparative Example 8

[0076] In this comparative example, three kinds of Swertia chirayita (excluding Swertia mussotii and Swertia macrosperma) in Example 1 were selected for research, and the chromatographic detection method was the same as that in Example 1.

[0077] The results are shown in Figure 3 , reducing the types of Swertia chirayita led to an increase in the total number of characteristic peaks to 12, and an increase in the characteristic peaks of substances such as isovitexin and sweroside. Although the measurement indicators increased, the screening conditions were too harsh, resulting in a narrower applicable range of the characteristic chromatogram, which was not conducive to the development and utilization of Swertia chirayita.

[0078] Comparative Example 9

[0079] In this comparative example, Swertia mussotii in Example 1 was replaced with Swertia tetraptera for research, and the chromatographic detection method was the same as that in Example 1.

[0080] The results showed that replacing Swertia mussotii with Swertia tetraptera would cause a decrease in the total number of characteristic peaks. There were only 4 characteristic peaks in common, which were the characteristic peaks of swertiamarin, gentiopicroside, swertisin, oleanolic acid, and ursolic acid. These five components were relatively common in Swertia chirayita and could not comprehensively evaluate the quality of Swertia chirayita.

[0081] In the description of this specification, the descriptions with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0082] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for constructing a characteristic fingerprint of Swertia chirayita, characterized in that Comprising: (1) Preparation of reference substance solution: Weigh different reference substances, add organic solvent, and prepare a mixed reference substance solution; (2) Preparation of test solution: Take samples of Swertia franchetiana, Swertia diluta, Swertia punicea, Swertia mussotii, and Swertia macrosperma, and perform the following steps respectively: Add organic solvent, weigh, ultrasonically treat, take out, cool, weigh again, make up the reduced weight with the organic solvent, filter, and collect the filtrate; (3) Obtaining the characteristic chromatogram: Using the reference substance solution as a reference, inject the reference substance solution, the test solutions of Swertia franchetiana, Swertia diluta, Swertia punicea, Swertia mussotii, and Swertia macrosperma into the liquid chromatograph, record the chromatogram, select the chromatograms of multiple batches of Swertia franchetiana, Swertia diluta, Swertia punicea, Swertia mussotii, and Swertia macrosperma, determine the common characteristic peaks in the chromatogram, and obtain the characteristic chromatogram of Herba Swertiae Punicae. The detection conditions of the liquid chromatograph are as follows: The mobile phase comprises: mobile phase A and mobile phase B. Mobile phase A is acetonitrile, and mobile phase B is a phosphoric acid solution with a concentration of 0.05 - 0.2%; Gradient elution is adopted, and the flow rate is 0.7 - 1 ml / min; The detection wavelength is 205 - 240 nm, and the column temperature is 20 - 40 °C; The gradient elution conditions are as follows: The detection wavelength conditions are as follows: ； The flow rate conditions are as follows: ； The different reference substances are selected from gentiopicroside, swertiamarin, sweroside, mangiferin, neomangiferin, isomangiferin, swertianolin, methylswerin, luteolin, swertisin, isovitexin, isoorientin, oleanolic acid, and ursolic acid.

2. The method according to claim 1, wherein The organic solvent is selected from methanol, ethanol, acetonitrile, isopropanol, or acetone.

3. The method according to claim 1, wherein There are 9 common characteristic peaks in the characteristic chromatogram. Taking sweroside as the standard peak, the relative elution times of the remaining characteristic peaks are 0.349 - 0.364, 0.633 - 0.650, 0.848 - 0.857, 0.898 - 0.925, 1.000, 1.605 - 1.838, 4.176 - 4.667, 4.503 - 4.927, and 5.429 - 5.475 in sequence from front to back.

4. The method according to claim 1, wherein The characteristic chromatogram includes the characteristic peaks of the following substances: isomangiferin, swertiamarin, mangiferin, gentiopicroside, sweroside, isoorientin, methylswerin, oleanolic acid, and ursolic acid.

5. The method according to claim 4, wherein The characteristic chromatogram further includes the characteristic peaks of unknown substances, and the relative retention time of the characteristic peaks of unknown substances is 4.503 - 4.

927.

6. A method for identifying Swertia chirayita, characterized in that, Comprising: Pretreat the sample to be tested to obtain the sample loading solution; Perform liquid chromatography detection on the sample loading solution to obtain a chromatogram; Determine whether the chromatogram contains the characteristic peaks in the characteristic chromatogram of Herba Swertiae Punicae obtained by the method for constructing the characteristic chromatogram of Herba Swertiae Punicae according to any one of claims 1 - 5; When the chromatogram contains the characteristic peaks, it is an indication that the sample to be tested is Herba Swertiae Punicae; The detection conditions of the liquid chromatography are as defined in the method of claim 1.

7. The method according to claim 6, characterized in that, The Herba Swertiae Punicae has the efficacy of treating liver and gallbladder diseases.

Images