Thin-layer chromatography rapid identification method for vigor-preserving decoction particles

Abstract

The invention discloses a thin-layer chromatography rapid identification method for vigor-preserving decoction particles, and aims to solve the problems of complicated operation, more interference, low efficiency and the like of the existing method. According to the method, a test solution is prepared through methanol extraction and water-saturated n-butyl alcohol and ammonia test solution extraction, reference medicinal materials and seven reference solutions are matched, a petroleum ether-ethyl acetate-methanol-formic acid mixed solution is adopted as a developing solvent, and inspection is conducted after developing is conducted through a silica gel H thin-layer plate. The method can be used for accurately identifying seven key components in the vitality-preserving decoction granules at one time, is high in specificity, simple and convenient to operate, environment-friendly and high in efficiency, and provides rapid and reliable technical support for quality control of the vitality-preserving decoction granules.

Description

Technical Field

[0001] The invention relates to the technical field of drug identification methods, and specifically proposes a thin-layer chromatography rapid identification method for Baoyuan decoction granules. Background Art

[0002] Classical prescriptions, shining jewels in the treasure trove of Traditional Chinese Medicine, carry thousands of years of medical wisdom and practical experience. Baoyuan Decoction, one of these, boasts a rich history. Its earliest traces can be traced back to the Ming Dynasty physician Wei Zhi's book, "The Mirror of Universal Love." The book details the recipe, which comprises ginseng, astragalus, licorice, cinnamon, and ginger. Designed to invigorate qi and warm yang, it is commonly used to treat pediatric symptoms such as weak vital energy and sunken yang due to yang deficiency. It has long played a vital role in clinical practice and has been highly valued by physicians throughout the ages.

[0003] Thin-layer chromatography (TLC) is a key method for quality control and identification of traditional Chinese medicine (TCM) compounds. Baoyuan Tang granules, a TCM compound, contain many ingredients with similar polarity across different ingredients. Identification using the methods specified in the Chinese Pharmacopoeia for each ingredient is complex and time-consuming, and often results in numerous interfering components with negative samples. This makes it difficult to meet the rapid and accurate identification requirements of modern TCM production. Therefore, a rapid, efficient, and low-interference detection method is needed.

[0004] Regarding the quality control and identification of Baoyuan Decoction, some currently available identification techniques suffer from varying degrees of deficiencies. For example, Zhang Zhuo et al. published a thin-layer chromatography (TLC) method for identifying ginseng, astragalus, and licorice in their "Establishment of a TLC Identification Method for Standardized Decoctions of Baoyuan Decoction." However, this method also has limitations. Under these chromatographic conditions, astragalus' characteristic component, calycosin glucoside, interferes with the method. Furthermore, the single component liquiritin used for identification of licorice fails to fully capture the key components of licorice. Furthermore, in practical applications, the detected spots are unclear and exhibit significant tailing, making it difficult to accurately identify the various medicinal ingredients in Baoyuan Decoction and failing to meet the stringent quality control requirements for Baoyuan Decoction. CN116068075A describes a method for identifying ginseng, astragalus, licorice, cinnamon, and ginger. However, in terms of ingredient identification, there is interference between ginsenoside Rb1 and astragaloside IV spots, resulting in unsatisfactory separation effect, and there is negative interference with the spots of astragaloside I. Only one component in licorice is identified, which makes it difficult to fully and accurately reflect the intrinsic quality of Baoyuan Decoction and provide strong support for the quality control of Baoyuan Decoction. At the same time, the pretreatment process is extremely complicated and requires two independent operations. The detection step also needs to be carried out twice. The entire process is cumbersome, time-consuming, and labor-intensive, which seriously affects the detection efficiency. Macroporous resin waste is generated during the pretreatment process, which places a heavy burden on the environment. Moreover, the developing agent used contains chloroform, which is seriously harmful to the human body and can cause irreversible damage. The detection conditions still need to be improved. CN112697949A describes a method for identifying ginseng, astragalus, and licorice, but the three spots of ginsenoside Rb1, ammonium glycyrrhizate, and astragaloside IV were not completely separated, the spots were unclear, and the tailing phenomenon was serious; and the control medicinal material solutions and negative control solutions of ginseng, astragalus, and licorice on the same silica gel plate were not compared and analyzed with the test sample solutions, which failed to fully and accurately demonstrate that the method did not have negative interference, and the specificity was poor, which reduced the reliability and accuracy of the identification results; at the same time, this method only identified one component, ammonium glycyrrhizate, in licorice, and could not fully cover the key components in licorice, which is not conducive to effective control of the quality of licorice medicinal materials in Baoyuan Decoction.

[0005] In summary, existing Baoyuan Decoction identification technologies have shortcomings in terms of process complexity, environmental friendliness, and detection accuracy, making them difficult to meet the requirements for efficient, accurate, and safe quality control of Baoyuan Decoction. Therefore, developing a simple, environmentally friendly, accurate, and efficient method for identifying the main functional ingredients of Baoyuan Decoction, including ginseng, astragalus, and licorice, has become an urgent challenge in this field. Summary of the Invention

[0006] In light of this, the present invention proposes a rapid thin-layer chromatography method for identifying Baoyuan Tang granules. This method can accurately identify ginsenoside Rb1, ginsenoside Rg1, ginsenoside Re, astragaloside IV, liquiritin, apigenin, and isoliquiritin in Baoyuan Tang granules in a single step. The identification method provided by the present invention has high specificity and can significantly improve the efficiency of Baoyuan Tang identification, providing a rapid and accurate technical means for quality control of Baoyuan Tang granules.

[0007] The technical solution of the present invention is achieved as follows: The present invention provides a method for rapid identification of Baoyuan Decoction granules by thin-layer chromatography, characterized in that it comprises the following steps: (1) Preparation of test solution: Take Baoyuantang granules, extract with methanol and filter, evaporate the filtrate and dissolve it in water, extract it with water-saturated n-butanol and ammonia test solution in sequence, evaporate the upper layer and dissolve it in methanol to obtain the test solution; (2) Preparation of control solution: Take ginseng, astragalus, and licorice as control medicinal materials, and prepare control medicinal material solution according to the method of step (1); take ginsenoside Rb1, ginsenoside Rg1, ginsenoside Re, astragaloside IV, liquiritin, apigenin liquiritin, and isoliquiritin as reference substances, and add methanol to prepare a control solution; (3) Thin layer chromatography identification: Spot the test sample solution, control medicinal material solution, and reference substance solution on a silica gel H thin layer plate, develop with a mixed solution of petroleum ether, ethyl acetate, methanol, and formic acid, spray with 10% sulfuric acid ethanol solution, heat until the spots are clear, and inspect under sunlight and 365nm ultraviolet light. Spots of the same color appear in the chromatogram of the test sample and the corresponding positions of the chromatograms of the reference substance and reference medicinal material.

[0008] In some embodiments, in step (1), the ratio of the Baoyuan Decoction granules to methanol is (0.5g-1g): (20-40) mL, and the extraction method is ultrasonic treatment for 20-40 min.

[0009] In some embodiments, in step (1), the number of extractions with water-saturated n-butanol is 1-3 times, and the weight ratio of the volume used in each extraction to the Baoyuan Decoction granules is (20-40) mL: (0.5-1) g.

[0010] In some embodiments, in step (1), the number of extractions with ammonia solution is 1-3 times, and the weight ratio of the volume used for each extraction to the Baoyuan Decoction granules is (20-40) mL: (0.5-1) g.

[0011] In some embodiments, in step (2), the concentration of each reference substance in the reference substance solution is 1 mg per 1 mL.

[0012] In some embodiments, in step (3), the volume ratio of petroleum ether, ethyl acetate, methanol, and formic acid in the developing solvent is 10: (9.5-10.5): (0.5-1.5): (0.5-1.5).

[0013] In some embodiments, in step (3), the volume ratio of petroleum ether, ethyl acetate, methanol, and formic acid in the developing solvent is 10:10:1:1.

[0014] In some embodiments, in step (3), the spotting volume is 2 μL-6 μL.

[0015] In some embodiments, in step (3), the heating temperature is 105°C.

[0016] In some embodiments, the methanol is 80% methanol.

[0017] The present invention has the following beneficial effects compared to the prior art: The present invention discloses a thin-layer chromatography (TLC) method for identifying Baoyuan Tang granules. Using TLC, the method can simultaneously detect ginsenoside Rb1, ginsenoside Rg1, ginsenoside Re, astragaloside IV, liquiritin, apigenin, and isoliquiritin in Baoyuan Tang granules. This method exhibits excellent overall separation efficiency, resulting in clearly distinguishable spots. The method possesses strong specificity, good stability, and exceptional durability, providing a precise and reliable basis for quality testing of Baoyuan Tang granules.

[0018] 2. The sample pretreatment process of the present invention is simple to operate, practices the concept of green environmental protection, abandons the use of macroporous resin, avoids the generation of macroporous resin waste, and reduces pollution from the source. The method is also highly stable and feasible, effectively reducing the difficulty and cost of experimental operation.

[0019] 3. The present invention has successfully avoided the risk of irreversible damage to the human body caused by chloroform by carefully screening specific developing agents, and at the same time achieved efficient separation of ginsenoside Rb1, ginsenoside Rg1, ginsenoside Re, astragaloside IV, liquiritin, apigenin, and isoliquiritin in Baoyuan Decoction. In particular, it can achieve multi-component quality control of licorice in the compound, laying a solid foundation for the quality control of Baoyuan Decoction granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1To investigate the specificity of the identification method of the present invention, a heliograph is used; Figure 2 This is a UV inspection diagram to investigate the specificity of the identification method of the present invention; Figure 3 The heliograph of ginseng sample quantity according to the identification method of the present invention; Figure 4 This is a UV inspection diagram of ginseng sample quantity according to the identification method of the present invention; Figure 5 The solar diagram of the astragalus spot sample quantity of the identification method of the present invention; Figure 6 This is an ultraviolet inspection diagram of the astragalus spot sample quantity of the identification method of the present invention; Figure 7 The heliograph of the licorice spot quantity of the identification method of the present invention; Figure 8 This is a UV inspection diagram of the licorice spot sample quantity of the identification method of the present invention; Figure 9 This is the daylight diagram of the identification results of Jiangyou silica gel H plate of the present invention; Figure 10 This is the UV inspection diagram of the identification results of Jiangyou silica gel H plate of the present invention; Figure 11 This is the daylight diagram of the identification results of Qingdao silica gel H plate of the present invention; Figure 12 This is the UV inspection diagram of the identification results of Qingdao silica gel H plate of the present invention; Figure 13 This is a solar diagram showing the high temperature condition identification result of the present invention; Figure 14 This is the UV inspection diagram of the high temperature condition identification result of the present invention; Figure 15 This is a heliograph of the low temperature condition identification result of the present invention; Figure 16 This is the ultraviolet inspection diagram of the low temperature identification results of the present invention; Figure 17 This is a daylight diagram showing the low humidity condition identification result of the present invention; Figure 18 This is the UV inspection diagram of the low humidity condition identification result of the present invention; Figure 19 This is a heliograph showing the high humidity condition identification result of the present invention; Figure 20 This is the UV inspection diagram of the high humidity condition identification result of the present invention; Figure 21 This is the daylight diagram of the identification result under the condition of 110mm stand distance of the present invention; Figure 22 This is the UV inspection diagram of the identification results under the condition of 110mm span distance of the present invention; Figure 23 This is the daylight diagram of the identification result under the condition of 130mm stand distance of the present invention; Figure 24 This is the UV inspection diagram of the identification results of the present invention under the condition of 130mm extension distance; Figure 25 UV inspection images were obtained for Comparative Examples 1-3 of the present invention. DETAILED DESCRIPTION

[0022] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the present invention belong. If the definitions set forth in this section are contrary to or otherwise inconsistent with definitions set forth in the patents, patent applications, published patent applications, and other publications incorporated herein by reference, the definitions listed in this section take precedence over the definitions incorporated herein by reference.

[0024] Unless otherwise specified, the methods used in the following examples are conventional methods. The materials, reagents, and instruments used are conventional materials, reagents, and instruments in the art, unless otherwise specified, and can be obtained commercially by those skilled in the art.

[0025] When an amount, concentration or other value or parameter is expressed as a range, a preferred range or a range defined by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any upper range limit or preferred value with any lower range limit or preferred value, regardless of whether the range is disclosed alone. For example, when a range "1 to 5" is disclosed, the described range should be interpreted as including the ranges "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within the range. In the present specification and claims, range definitions may be combined and / or interchanged, and if not otherwise stated, such ranges include all subranges contained therein.

[0026] In the following examples, the ammonia test solution was prepared as follows: 400 mL of concentrated ammonia solution was diluted to 1000 mL with water and prepared according to General Rules 8002 of Part IV of the 2020 edition of the Chinese Pharmacopoeia.

[0027] Exclusive inspection: The methodological investigation of thin layer chromatography identification of ginseng, astragalus and licorice is as follows: Take 0.75 g each of the negative samples of Baoyuan Decoction Granules lacking ginseng, the negative samples of Baoyuan Decoction Granules lacking astragalus, and the negative samples of Baoyuan Decoction Granules lacking licorice, and prepare the Baoyuan Decoction Granules lacking ginseng negative control solution, Baoyuan Decoction Granules lacking astragalus control solution, and Baoyuan Decoction Granules lacking licorice control solution according to the preparation method of the test solution.

[0028] Pipette 4 μL of ginseng reference solution, 4 μL of astragalus reference solution, 4 μL of licorice reference solution and 4 μL of ginseng control medicinal material solution, 4 μL of astragalus control medicinal material solution, 4 μL of licorice control medicinal material solution, 4 μL of test solution, 4 μL of Baoyuan Decoction granules lacking ginseng negative control solution, 4 μL of Baoyuan Decoction granules lacking astragalus reference solution, and 4 μL of Baoyuan Decoction granules lacking licorice reference solution, and spot them on the same silica gel H thin layer plate respectively, and use a solution of petroleum ether, ethyl acetate, methanol, and formic acid with a volume ratio of 10:10:1:1 as the developing agent. Develop, take out, dry, spray with 10% sulfuric acid ethanol, heat at 105°C until the spots are clear, and examine under sunlight and ultraviolet light at a wavelength of 365nm. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatograms of the reference sample and control medicinal material. The picture under sunlight is as follows Figure 1 As shown, the 365nm UV inspection is as follows Figure 2 As shown, from left to right are: 1- ginseng reference solution, 2- astragalus reference solution and licorice reference solution, 3- ginseng control medicinal material solution, 4- astragalus control medicinal material solution, 5- licorice control medicinal material solution, 6- test solution, 7- Baoyuan Decoction granules lacking ginseng negative control solution, 8- Baoyuan Decoction granules lacking astragalus reference solution, 9- Baoyuan Decoction granules lacking licorice reference solution, among which the spots in 1- ginseng reference solution from top to bottom correspond to ginsenoside Rb1 reference, ginsenoside Re reference, and ginsenoside Rg1 reference, and the spots in 2- astragalus reference solution from top to bottom correspond to isoliquiritigenin reference, liquiritin reference, apigenin reference, and astragaloside IV reference.

[0029] Durability inspection: (1) Investigation of different sample quantities Respectively take 2μL, 4μL, 6μL of ginseng reference solution, 2μL, 4μL, 6μL of ginseng control medicinal material solution, 2μL, 4μL, 6μL of test solution, and conduct the test at room temperature (20℃, RH42%). Spot them on different silica gel H thin layer plates, use a solution of petroleum ether, ethyl acetate, methanol, and formic acid with a volume ratio of 10:10:1:1 as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol, heat at 105℃ until the spots are clear, and inspect under sunlight and ultraviolet light with a wavelength of 365nm to compare the development effects of different spot amounts. Figure 3 As shown, the wavelength is 365nm under ultraviolet inspection. Figure 4 As shown, the 3 on the left are reference solutions, the 3 in the middle are ginseng reference medicinal material solutions, and the 3 on the right are test solutions. The substances represented by the spots from top to bottom in the reference solution are ginsenoside Rb1 reference, ginsenoside Re reference, and ginsenoside Rg1 reference, respectively.

[0030] The amount of Astragalus root sample was investigated in the same way as that of Ginseng root sample. Figure 5 As shown, the wavelength is 365nm under ultraviolet inspection. Figure 6 As shown, the 3 on the left are reference solutions, among which the spots are astragaloside reference solutions, the 3 in the middle are ginseng reference medicinal material solutions, and the 3 on the right are test solution.

[0031] The amount of licorice was investigated in the same way as that of ginseng. Figure 7 As shown, the wavelength is 365nm under ultraviolet inspection. Figure 8 As shown, label 1-3 are reference solution, and the spots correspond to the substances from top to bottom, namely, isoliquiritin reference, liquiritin reference, and apiosylliquiritin reference, 6 is licorice reference medicinal material solution, and 7-9 are test solution.

[0032] (2) Durability study of thin-layer boards from different manufacturers Aspirate 4 μL of ginseng reference solution, 4 μL of astragalus reference solution, 4 μL of licorice reference solution, 4 μL of ginseng control medicinal material solution, 4 μL of astragalus control medicinal material solution, 4 μL of licorice control medicinal material solution, 4 μL of test solution, 4 μL of Baoyuan Decoction Granules lacking ginseng negative control solution, 4 μL of Baoyuan Decoction Granules lacking astragalus reference solution, and 4 μL of Baoyuan Decoction Granules lacking licorice reference solution. Silica gel H thin layer plates prepared by different manufacturers (Yantai Yinlong silica gel H plate, Qingdao silica gel H plate, Yantai Jiangyou H plate) were tested respectively. A solution of petroleum ether, ethyl acetate, methanol, and formic acid with a volume ratio of 10:10:1:1 was used as the developing agent. The plates were developed, taken out, dried, sprayed with 10% sulfuric acid ethanol, heated at 105°C until the spots were clear, and inspected under sunlight and ultraviolet light at a wavelength of 365nm. The developing effects of thin layer plates from different manufacturers were compared, and it was found that the developing effects were all good. For example, the Yantai Jiangyou H plate Figure 9 and Figure 10 As shown, the spots of 1-ginseng reference solution from top to bottom correspond to ginsenoside Rb1 reference, ginsenoside Re reference, and ginsenoside Rg1 reference, and the spots of 2-astragalus reference solution from top to bottom correspond to isoliquiritin reference, liquiritin reference, apigenin liquiritin reference, and astragaloside IV reference; the sunlight image and UV inspection image of Qingdao silica gel H plate are shown in Figure 2. Figure 11 and Figure 12 .

[0033] (3) Temperature durability test Pipette 4 μL of ginseng reference solution, 4 μL of astragalus reference solution, 4 μL of licorice reference solution, 4 μL of ginseng control medicinal material solution, 4 μL of astragalus control medicinal material solution, 4 μL of licorice control medicinal material solution, 4 μL of test solution, 4 μL of Baoyuan Decoction granules lacking ginseng negative control solution, 4 μL of Baoyuan Decoction granules lacking astragalus reference solution, and 4 μL of Baoyuan Decoction granules lacking licorice reference solution, and conduct tests at low temperature (5°C, RH65%) and high temperature (40°C, RH37%), respectively, and spot them on different silica gel H thin layer plates, use a solution of petroleum ether, ethyl acetate, methanol, and formic acid in a volume ratio of 10:10:1:1 as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol, heat at 105°C until the spots are clear, and inspect under sunlight and ultraviolet light with a wavelength of 365nm to compare the development effects at different temperatures. The sunlight image under high temperature conditions is shown as follows Figure 13 As shown, the UV inspection diagram is as follows Figure 14 As shown, the sunlight diagram under low temperature conditions is as follows Figure 15 As shown, the UV inspection diagram is as follows Figure 16 As shown, it was found that the expansion effects were all good.

[0034] (4) Humidity durability test Absorb 4 μL of ginseng reference solution, 4 μL of astragalus reference solution, 4 μL of licorice reference solution, 4 μL of ginseng control medicinal material solution, 4 μL of astragalus control medicinal material solution, 4 μL of licorice control medicinal material solution, 4 μL of test solution, 4 μL of Baoyuan Decoction granules lacking ginseng negative control solution, 4 μL of Baoyuan Decoction granules lacking astragalus reference solution, and 4 μL of Baoyuan Decoction granules lacking licorice reference solution. According to the thin layer conditions of Example 1, the test was carried out under low humidity (25°C, RH32%) and high humidity (25°C, RH75%), and they were spotted on different silica gel H thin layer plates, respectively, with a solution of petroleum ether, ethyl acetate, methanol, and formic acid in a volume ratio of 10:10:1:1 as the developing agent, developed, taken out, dried, sprayed with 10% sulfuric acid ethanol, heated at 105°C until the spots were clear, and inspected under sunlight and at a wavelength of 365nm ultraviolet light to compare the development effects under different humidity conditions. The sunlight image under low humidity conditions is as shown in the figure below. Figure 17 As shown, the UV inspection diagram is as follows Figure 18 As shown, the sunlight diagram under high humidity conditions is as follows Figure 19 As shown, the UV inspection diagram is as follows Figure 20 As shown, it was found that the expansion effects were all good.

[0035] (5) Exhibition distance inspection Aspirate 4 μL of ginseng reference solution, 4 μL of astragalus reference solution, 4 μL of licorice reference solution, 4 μL of ginseng control medicinal material solution, 4 μL of astragalus control medicinal material solution, 4 μL of licorice control medicinal material solution, 4 μL of test solution, 4 μL of Baoyuan Decoction granules lacking ginseng negative control solution, 4 μL of Baoyuan Decoction granules lacking astragalus reference solution, and 4 μL of Baoyuan Decoction granules lacking licorice reference solution, with development distances of 110 mm and 130 mm, respectively, and spot them on different silica gel H thin layer plates, using a solution of petroleum ether, ethyl acetate, methanol, and formic acid in a volume ratio of 10:10:1:1 as the developing agent. Develop, take out, dry, spray with 10% sulfuric acid ethanol, heat at 105°C until the spots are clear, and examine under sunlight and ultraviolet light with a wavelength of 365 nm. Compare the development effects under different development distances and find that the development effects are all good. Among them, the sunlight image and ultraviolet image with a development distance of 110 mm are as shown in the figure below. Figure 21 and Figure 22 As shown, the daylight and ultraviolet images of the display with a 130mm stand distance and 110mm are as follows Figure 23 and Figure 24 shown.

[0036] (6) Sampling method of investigation Aspirate 4μL of ginseng reference solution, 4μL of astragalus reference solution, 4μL of licorice reference solution, 4μL of ginseng control medicinal material solution, 4μL of astragalus control medicinal material solution, 4μL of licorice control medicinal material solution, 4μL of test solution, 4μL of Baoyuan Decoction Granules lacking ginseng negative control solution, 4μL of Baoyuan Decoction Granules lacking astragalus reference solution, and 4μL of Baoyuan Decoction Granules lacking licorice reference solution, and examine the strip spotting and dot spotting respectively. Spot them on different silica gel H thin layer plates, and use a solution of petroleum ether, ethyl acetate, methanol, and formic acid in a volume ratio of 10:10:1:1 as the developing agent. Develop, take out, dry, spray with 10% sulfuric acid ethanol, heat at 105℃ until the spots are clear, and inspect under sunlight and ultraviolet light with a wavelength of 365nm. Compare the development effects under different spotting methods and find that the development effects are all good.

[0037] Example 1 1. Preparation of test solution: Take 0.75 g of Baoyuan Decoction granules, add 30 mL of 80% methanol, ultrasonically treat (power 500 W, frequency 53 kHz) for 30 min, filter, evaporate to dryness, and dissolve in 30 mL of water; the obtained solution is extracted twice with water-saturated n-butanol, and twice with ammonia test solution, each time with 30 mL. After evaporating the upper layer, dissolve it in 1 mL of 80% methanol to obtain test solution I.

[0038] 2. Preparation of reference substances and reference medicinal material solutions Take 0.75 g each of ginseng, astragalus, and licorice control medicinal materials, add 30 mL of 80% methanol, ultrasonically treat (power 500 W, frequency 53 kHz) for 30 min, filter, evaporate to dryness, and add 20 mL of water to dissolve; the resulting solution is extracted twice with water-saturated n-butanol, each time with 30 mL, and the obtained upper layer liquid is extracted twice with ammonia test solution, each time with 30 mL. After the upper layer liquid is evaporated to dryness, it is dissolved in 1 mL of 80% methanol to serve as the ginseng control medicinal material solution, the astragalus control medicinal material solution, and the licorice control medicinal material solution.

[0039] Take ginsenoside Rb1 reference substance, ginsenoside Rg1 reference substance, and ginsenoside Re reference substance, add 80% methanol to make a solution containing 1 mg of each per 1 mL, which is used as the ginseng reference solution.

[0040] Take the astragaloside IV reference substance and add a solution containing 1 mg of astragaloside IV per 1 mL of 80% methanol to obtain the astragaloside IV reference substance solution.

[0041] Take liquiritin reference substance, apigenin liquiritin, and isoliquiritin, add 80% methanol to make a solution containing 1 mg of each per 1 mL, which is used as the licorice reference substance solution.

[0042] 3. Identification method: Pipette 4μL of ginseng reference solution, 4μL of astragalus reference solution, 4μL of licorice reference solution, 4μL of ginseng control medicinal material solution, 4μL of astragalus control medicinal material solution, 4μL of licorice control medicinal material solution, and 4μL of test sample solution, and spot them on the same silica gel H thin layer plate respectively. Use a solution of petroleum ether, ethyl acetate, methanol, and formic acid in a volume ratio of 10:10:1:1 as the developing agent. Develop the plate, take it out, dry it, spray it with 10% sulfuric acid ethanol, heat it at 105℃ until the spots are clear, and examine it under sunlight and ultraviolet light at a wavelength of 365nm. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatograms of the reference sample and control medicinal material.

[0043] Comparative Example 1 This comparative example provides a method for identifying Baoyuan Decoction, which differs from Example 1 in that: In the method for identifying ginseng, astragalus, and licorice, the test solution is prepared as follows: 1.5g of Baoyuan Decoction freeze-dried powder is added to 30mL of water. Ultrasonic treatment (power 500W, frequency 40kHz) is performed for 30 minutes, and the solution is extracted three times with 15mL of ether. The lower layer is removed and extracted three times with water-saturated n-butanol (15mL each). The combined water-saturated n-butanol solutions are evaporated to dryness. The residue is dissolved in 5mL of water and applied to a D101 macroporous resin (1.5cm inner diameter, 15cm height). The solution is eluted with 20mL of water, the eluate is discarded, and the solution is eluted with 20mL of 40% ethanol solution. The eluate is discarded and the solution is eluted with 30mL of 70% ethanol solution. The eluate is collected and evaporated to dryness. The residue is dissolved in 2mL of 80% methanol to serve as test solution B. All other procedures are the same as in Example 1.

[0044] In the atlas of this embodiment, spots of the same color appear at the corresponding positions of the chromatogram of the test solution B and the chromatogram of the licorice reference substance, but spots of the same color do not appear at the positions of the ginseng reference substance and the astragalus reference substance.

[0045] Comparative Example 2 This comparative example provides a method for identifying Baoyuan Decoction, which differs from Example 1 in that: In the method for identifying ginseng, astragalus, and licorice, the test solution is prepared as follows: 1.5 g of Baoyuan Decoction granules is added to 30 mL of water, ultrasonically treated (power 500 W, frequency 40 kHz) for 30 minutes, extracted three times with 15 mL of ether, the lower layer is removed, and then extracted three times with water-saturated n-butanol (15 mL each). The water-saturated n-butanol solutions are combined and evaporated to dryness. The residue is diluted to volume in a 5 mL volumetric flask with methanol, and filtered through a 0.22 μm microporous membrane to obtain test solution C. All other procedures are the same as in Example 1.

[0046] In the atlas of this embodiment, the chromatogram of the test solution C shows spots of the same color at the corresponding positions of the chromatogram of the licorice reference substance, but no spots of the same color appear at the positions of the ginseng reference substance and the astragalus reference substance.

[0047] Comparative Example 3 This comparative example provides a method for identifying Baoyuan Decoction, which differs from Example 1 in that: In the method for identifying ginseng, astragalus, and licorice, the test solution is prepared by aspirating 5 mL of the standard decoction, passing it through a solid-phase extraction column, first washing with 10 mL of water to remove impurities, then eluting the sample with 10 mL of methanol. The eluate is collected and evaporated to dryness in a 60°C water bath. The volume is then diluted with methanol into a 5 mL volumetric flask, and filtered through a 0.22 μm microporous membrane to obtain the test solution D. All other procedures are the same as in Example 1.

[0048] In the atlas of this embodiment, the chromatogram of the test solution D shows spots of the same color at the corresponding positions of the chromatograms of the ginseng reference substance and the licorice reference substance, but does not show spots of the same color at the position of the astragalus reference substance.

[0049] The spectrum of comparative example 1-3 is as follows Figure 25 , from left to right: 1: from top to bottom: ginsenoside Rb1 reference substance, ginsenoside Re reference substance, ginsenoside Rg1 reference substance, 2: from top to bottom: isoliquiritigenin reference substance, liquiritin reference substance, apiosylliquiritin reference substance, 3: astragaloside IV reference substance, 4: test solution B, 5: test solution C, 6: test solution D.

[0050] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for rapid identification of Baoyuantang granules by thin layer chromatography, characterized in that: The steps include: (1) Preparation of test solution: Take Baoyuantang granules, extract with methanol and filter, evaporate the filtrate and dissolve it in water, extract it with water-saturated n-butanol and ammonia test solution in sequence, evaporate the upper layer and dissolve it in methanol to obtain the test solution; (2) Preparation of control solution: Take ginseng, astragalus, and licorice as control medicinal materials, and prepare control medicinal material solution according to the method of step (1); take ginsenoside Rb1, ginsenoside Rg1, ginsenoside Re, astragaloside IV, liquiritin, apigenin liquiritin, and isoliquiritin as reference substances, and add methanol to prepare a control solution; (3) Thin layer chromatography identification: Spot the test sample solution, control medicinal material solution, and reference substance solution on a silica gel H thin layer plate, develop with a mixed solution of petroleum ether, ethyl acetate, methanol, and formic acid, spray with 10% sulfuric acid ethanol solution, heat until the spots are clear, and inspect under sunlight and 365nm ultraviolet light. Spots of the same color appear in the chromatogram of the test sample and the corresponding positions of the chromatograms of the reference substance and reference medicinal material.

2. The thin layer chromatography rapid identification method of Baoyuantang granules according to claim 1, characterized in that: In step (1), the dosage ratio of the Baoyuan Decoction granules to methanol is (0.5 g-1 g): (20-40) mL, and the extraction method is ultrasonic treatment for 20-40 min.

3. The thin layer chromatography rapid identification method of Baoyuantang granules according to claim 1, characterized in that: In step (1), the number of extractions with water-saturated n-butanol is 1-3 times, and the weight ratio of the volume used in each extraction to the Baoyuan Decoction granules is (20-40) mL: (0.5-1) g.

4. The thin layer chromatography rapid identification method of Baoyuantang granules according to claim 1, characterized in that: In step (1), the number of extractions with ammonia solution is 1-3 times, and the weight ratio of the volume used for each extraction to the Baoyuan Decoction granules is (20-40) mL: (0.5-1) g.

5. The thin layer chromatography rapid identification method of Baoyuantang granules according to claim 1, characterized in that: In step (2), the concentration of each reference substance in the reference substance solution is 1 mg per 1 mL.

6. The thin layer chromatography rapid identification method of Baoyuantang granules according to claim 1, characterized in that: In step (3), the volume ratio of petroleum ether, ethyl acetate, methanol and formic acid in the developing solvent is 10: (9.5-10.5): (0.5-1.5): (0.5-1.5).

7. The thin layer chromatography rapid identification method of Baoyuantang granules according to claim 1, characterized in that: In step (3), the volume ratio of petroleum ether, ethyl acetate, methanol and formic acid in the developing solvent is 10:10:1:

1.

8. The thin layer chromatography rapid identification method of Baoyuantang granules according to claim 1, characterized in that: In step (3), the sample volume is 2 μL-6 μL.

9. The thin layer chromatography rapid identification method of Baoyuantang granules according to claim 1, characterized in that: In step (3), the heating temperature is 105°C.

10. The thin layer chromatography rapid identification method of Baoyuantang granules according to claim 1, characterized in that: The methanol is 80% methanol.

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