A thin layer chromatography identification method for Zexie decoction

Abstract

The present invention discloses a thin layer chromatography identification method of Zexie decoction, belonging to the technical field of traditional Chinese medicine identification methods. Specifically, it comprises: (1) extracting Zexie decoction with n-hexane, drying the extract and dissolving it with a first solvent to obtain a test solution; (2) taking Zexie control medicinal material and Atractylodes macrocephala control medicinal material to prepare a control medicinal material solution; (3) aspirating the test solution and the control medicinal material solution and spotting them on a silica gel G thin layer plate, using a mixed solution of chloroform, acetone and formic acid in a volume ratio of (9-10): (0.1-1): (0.01-0.2) as a developing agent, developing, coloring and inspecting. The identification method of the present invention, based on a specific extraction method and a specific developing agent, can effectively identify Zexie and Atractylodes macrocephala at the same time, reducing the inspection cost and saving the inspection time.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine identification methods, and in particular to a thin layer chromatography identification method for Zexie decoction. Background Art

[0002] The original text of Zexie Decoction is recorded in "Golden Chamber Synopsis·Volume 2·Tanyin Kebing Bingpulse Zheng Bingzhi Chapter 12" 205: "There is a branch of fluid under the heart, and the person suffers from dizziness, and Zexie Decoction is the main treatment. Zexie Decoction Recipe Zexie 5 liang, Baizhu 2 liang, the above two ingredients, add 2 liters of water, boil to 1 liter, divide and take again while warm". The dosage and decoction method have been recorded in the original text, and 1 times the daily prescription amount is used to prepare the extract. According to literature research, the whole prescription is composed of two herbs, Zexie and Baizhu. Zexie Decoction is mainly used to treat water retention under the heart, the clear yang does not rise, and the turbid yin rises, so the head and eyes are dizzy. Zexie in the prescription is sweet and light, and it is diuretic and dehumidifying, so that the water and moisture can be discharged through urination, which is the main medicine. Baizhu is sweet and bitter, strengthens the spleen and replenishes qi, promotes diuresis and reduces swelling, and helps the spleen to transport water and moisture, which is the minister medicine. The two medicines are complementary to each other, focusing on promoting diuresis and strengthening the spleen to control water, which is a good prescription for treating spleen deficiency and water retention. This prescription has been included in the "Catalogue of Ancient Classic Prescriptions (First Batch)" formulated and published by the State Food and Drug Administration. Although it is simple in composition, it has significant efficacy. It is now often used to treat vertigo diseases such as Meniere's disease, benign paroxysmal positional vertigo, and vertebrobasilar artery insufficiency. It has great development value and prospects.

[0003] In recent years, many domestic scholars have conducted research on the pharmacology, pharmacodynamics and pharmacokinetics of the chemical components and effective ingredients in Zexie Decoction, mainly focusing on pharmacology, the material basis of the decoction, extraction technology, multi-index component content determination, fingerprint research, etc., and mainly used instruments such as high-performance liquid chromatography and liquid-mass spectrometry chromatography, which greatly increased the inspection cost. For example, CN202010139733-A method for establishing the fingerprint of Zexie Decoction and its fingerprint, discloses the use of ultra-high performance liquid chromatography, with acetonitrile-phosphoric acid aqueous solution as the mobile phase for gradient elution, which can simultaneously identify 19 common peaks; CN201910065029-A quality detection method and quality evaluation method for the standard decoction of Zexie Decoction, discloses the use of liquid chromatography to detect the content of atractylodes lactone III and 23-acetyl atractylodes alcohol B; CN201611121475-A method for detecting the content of atractylodes lactone III and 23-acetyl atractylodes alcohol B in the standard granules of Zexie Decoction, discloses a method for detecting the content of atractylodes lactone III and 23-acetyl atractylodes alcohol B in the standard granules of Zexie Decoction. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a thin layer chromatography identification method for Zexie decoction, which can simultaneously identify Zexie and Atractylodes macrocephala, reduce the inspection cost, and save the inspection time.

[0005] In order to solve the above technical problems, the present invention provides a thin layer chromatography identification method of Zexie decoction, which comprises:

[0006] (1) extracting Zexie decoction with n-hexane, drying the extract and then dissolving it with a first solvent to obtain a test solution;

[0007] (2) extracting a Rhizoma Alismatis by using n-hexane, drying the extract and dissolving it in a first solvent to obtain a Rhizoma Alismatis control medicinal material solution; extracting a Rhizoma Atractylodis Macrocephala by using n-hexane, drying the extract and dissolving it in a first solvent to obtain a Rhizoma Atractylodis Macrocephala control medicinal material solution;

[0008] (3) Pipette the test sample solution, the control medicinal material solution of Alisma orientalis, and the control medicinal material solution of Atractylodes macrocephala onto a silica gel G thin layer plate, develop the plate with a mixed solution of chloroform, acetone, and formic acid in a volume ratio of (9-10):(0.1-1):(0.01-0.2) as the developing agent, develop the color, and inspect the plate.

[0009] The present invention also discloses a thin layer chromatography identification method of Zexie decoction, which comprises:

[0010] (1) extracting Zexie decoction with n-hexane, drying the extract and dissolving it with methanol to obtain a test solution;

[0011] (2) taking 23-acetyl alismatol C reference substance, atractylodes lactone II reference substance, and atractylodes lactone III reference substance, dissolving them in a second solvent to prepare a reference substance solution;

[0012] (3) Pipette the test solution and the reference solution onto a silica gel G thin layer plate and develop the plate using a mixed solution of chloroform, acetone, and formic acid in a volume ratio of (9-10):(0.1-1):(0.01-0.2) as a developing agent to develop color and examine the plate.

[0013] The present invention also discloses a thin layer chromatography identification method of Zexie decoction, which comprises:

[0014] (1) extracting Zexie decoction with n-hexane, drying the extract and dissolving it with methanol to obtain a test solution;

[0015] (2) extracting a Rhizoma Alismatis with n-hexane, drying the extract and dissolving it with a first solvent to obtain a Rhizoma Alismatis reference medicinal material solution; extracting a Rhizoma Atractylodis Macrocephala with n-hexane, drying the extract and dissolving it with a first solvent to obtain a Rhizoma Atractylodis Macrocephala reference medicinal material solution; and dissolving 23-acetyl alismatol C reference substance, atractylodes lactone II reference substance, and atractylodes lactone III reference substance with a second solvent to prepare reference substance solutions;

[0016] (3) Pipette the test sample solution, the control medicinal material solution of Alisma orientalis, the control medicinal material solution of Atractylodes macrocephala, and the control sample solution onto a silica gel G thin layer plate, develop the plate with a mixed solution of chloroform, acetone, and formic acid in a volume ratio of (9-10):(0.1-1):(0.01-0.2) as the developing agent, develop the color, and inspect the plate.

[0017] As an improvement of the above technical solution, in step (3), the volume ratio of chloroform, acetone and formic acid is 9.5:0.5:0.06.

[0018] As an improvement of the above technical solution, step (3) includes: taking out after unfolding, drying, spraying with a color developer solution, heating at 100-110° C. until the color is clear, and then inspecting under ultraviolet light.

[0019] As an improvement of the above technical solution, the developer solution is a sulfuric acid ethanol solution, the volume concentration of sulfuric acid is 5-20%, and the wavelength of the ultraviolet light is 360-370nm.

[0020] As an improvement of the above technical solution, the spreading distance of the silica gel G thin layer plate is 8-12 cm.

[0021] As an improvement of the above technical solution, during heating, the heating time of the spots at a spread distance of 0 to 4 cm is ≤ 1 min, and the heating time of the spots at a spread distance of 4 to 8 cm is > 1 min.

[0022] As an improvement of the above technical solution, in step (3), the spotting volume of the test solution is 5-15 μL, the spotting volume of the Rhizoma Alismatis control medicinal material solution is 5-20 μL, the spotting volume of the Rhizoma Atractylodis Macrocephalae control medicinal material solution is 5-15 μL, and the spotting volume of the reference solution is 1-3 μL.

[0023] As an improvement of the above technical solution, step (1) includes:

[0024] (1.1) Take a sample of Zexie decoction and add water to dissolve it; wherein the ratio of the weight of Zexie decoction to the volume of water added is (1-3) g: (20-50) mL;

[0025] (1.2) The solution obtained by dissolving in step (1.1) is extracted 1 to 4 times with n-hexane, the extracts are combined, and evaporated to dryness in a water bath at 50 to 80°C. Methanol is added to the residue to dissolve it and the solution is used as the test solution; wherein the ratio of the volume of n-hexane added for each extraction to the weight of the Zexie decoction is (15 to 30) mL: (1 to 3) g; and the ratio of the volume of methanol used for dissolution to the weight of the Zexie decoction is (0.5 to 1.5) mL: (1 to 3) g.

[0026] As an improvement of the above technical solution, in step (2):

[0027] Take 1-5g of Rhizoma Zedoariae reference medicinal material, add 30-80mL of water, boil for 20-60min, centrifuge and take the supernatant, extract the supernatant with n-hexane 1-4 times, combine the extracts, evaporate to dryness in a water bath at 50-80°C, add methanol to the residue to dissolve it, and use it as the Rhizoma Zedoariae reference medicinal material solution;

[0028] Take 0.5-2 g of Atractylodes macrocephala reference medicinal material, add 30-80 mL of water, boil for 20-60 min, centrifuge and take the supernatant, extract the supernatant with n-hexane 1-4 times, combine the extracts, evaporate to dryness in a water bath at 50-80°C, add methanol to the residue to dissolve it, and use it as the Atractylodes macrocephala reference medicinal material solution.

[0029] As an improvement of the above technical solution, in step (2):

[0030] The reference solution includes 23-acetyl alismatol C reference solution, atractylodes lactone II reference solution, and atractylodes lactone III reference solution; and the preparation methods thereof are respectively:

[0031] Take 23-acetyl alismatol C reference substance, add methanol to make a solution with a concentration of 0.5-1 mg / mL, and obtain acetyl alismatol reference substance solution;

[0032] Take the Atractylodes lactone II reference substance, add methanol to make a solution with a concentration of 0.5-1 mg / mL, and obtain the Atractylodes lactone II reference substance solution;

[0033] Take atractylodes lactone III reference substance, add methanol to make a solution with a concentration of 0.5-1 mg / mL, and obtain an atractylodes lactone III reference substance solution;

[0034] In step (3), the spotting volume of 23-acetyl alismatol C reference solution, atractylodes lactone II reference solution, and atractylodes lactone III reference solution is 1 to 3 μL.

[0035] As an improvement of the above technical solution, the Zexie decoction is composed of the following raw materials in parts by weight:

[0036] There are 18.65 parts of Alisma orientalis and 7.46 parts of Atractylodes macrocephala.

[0037] As an improvement of the above technical solution, the preparation method of the rhizoma alismatis decoction is as follows:

[0038] Take Alisma orientalis and Atractylodes macrocephala, soak them in water for 10 to 30 minutes, boil them over high heat for 20 to 35 minutes, and keep boiling over low heat until the amount of water used for soaking is reduced by half.

[0039] As an improvement of the above technical solution, the preparation method of the rhizoma alismatis decoction is as follows:

[0040] Take Alisma orientalis and Atractylodes macrocephala, soak them in water for 10 to 30 minutes, boil them over high heat for 20 to 35 minutes, and keep boiling over low heat until the amount of water used for soaking is reduced by half. Freeze-dry the resulting decoction.

[0041] The implementation of the present invention has the following beneficial effects:

[0042] The identification method of the present invention, based on a specific extraction method and a specific developing agent, can effectively identify Alisma orientalis and Atractylodes macrocephala at the same time, thereby reducing the inspection cost and saving the inspection time. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 is a thin layer chromatographic identification diagram of three batches of Zexie decoction in Example 2 of the present invention; wherein, 1 to 3 are freeze-dried powder preparations of Zexie decoction of batches S1 to S3, 4 is a reference medicinal material of Zexie, 5 is a reference medicinal material of Atractylodes macrocephala, 6 is a reference substance of 23-acetyl alismatol C, 7 is a reference substance of Atractylodes macrocephala lactone II, and 8 is a reference substance of Atractylodes macrocephala lactone III;

[0044] Figure 2 1 is a graph showing the results of the investigation of specific properties in Example 3 of the present invention; wherein, 1 is a negative sample of Rhizoma Alismatis, 2 is a negative sample of Rhizoma Atractylodis Macrocephalae, 3 is a freeze-dried powder preparation of Rhizoma Alismatis, 4 is a reference medicinal material of Rhizoma Alismatis, 5 is a reference medicinal material of Rhizoma Atractylodis Macrocephalae, 6 is a reference substance of 23-acetylalismol C, 7 is a reference substance of Atractylodes macrocephala lactone II, and 8 is a reference substance of Atractylodes macrocephala lactone III;

[0045] Figure 3 1-3 are freeze-dried powder preparations of Zexie decoction, and the spotting amounts are 5 μL, 10 μL, and 15 μL, respectively; 4-6 are Zexie control medicinal materials, and the spotting amounts are 10 μL, 15 μL, and 20 μL, respectively; 7-9 are Atractylodes macrocephala control medicinal materials, and the spotting amounts are 5 μL, 10 μL, and 15 μL, respectively; 10-11 are 23-acetyl alismatol C reference substances, and the spotting amounts are 1 μL and 5 μL, respectively; 12-13 are Atractylodes macrocephala lactone II reference substances, and the spotting amounts are 1 μL and 5 μL, respectively; 14-15 are Atractylodes macrocephala lactone III reference substances, and the spotting amounts are 1 μL and 5 μL, respectively;

[0046] Figure 4 1 is a graph of the investigation results at 23.4° C. in Example 3 of the present invention; wherein 1 is a negative sample of Rhizoma Alismatis, 2 is a negative sample of Rhizoma Atractylodis Macrocephalae, 3 is a freeze-dried powder preparation of Rhizoma Alismatis, 4 is a reference medicinal material of Rhizoma Alismatis, 5 is a reference medicinal material of Rhizoma Atractylodis Macrocephalae, 6 is a reference substance of 23-acetylalismol C, 7 is a reference substance of Atractylodes macrocephala lactone II, and 8 is a reference substance of Atractylodes macrocephala lactone III;

[0047] Figure 51 is a graph of the investigation results at 5.8° C. in Example 3 of the present invention; wherein 1 is a negative sample of Rhizoma Alismatis, 2 is a negative sample of Rhizoma Atractylodis Macrocephalae, 3 is a freeze-dried powder preparation of Rhizoma Alismatis, 4 is a reference medicinal material of Rhizoma Alismatis, 5 is a reference medicinal material of Rhizoma Atractylodis Macrocephalae, 6 is a reference substance of 23-acetylalismol C, 7 is a reference substance of Atractylodes macrocephala lactone II, and 8 is a reference substance of Atractylodes macrocephala lactone III;

[0048] Figure 6 1 is a graph of the investigation results when the relative humidity is 38% in Example 3 of the present invention; wherein 1 is a negative sample of Rhizoma Alismatis, 2 is a negative sample of Rhizoma Atractylodis Macrocephalae, 3 is a freeze-dried powder preparation of Rhizoma Alismatis, 4 is a reference medicinal material of Rhizoma Alismatis, 5 is a reference medicinal material of Rhizoma Atractylodis Macrocephalae, 6 is a reference substance of 23-acetylalismol C, 7 is a reference substance of Atractylodes macrocephala lactone II, and 8 is a reference substance of Atractylodes macrocephala lactone III;

[0049] Figure 7 1 is a graph of the investigation results when the relative humidity is 80% in Example 3 of the present invention; wherein 1 is a negative sample of Rhizoma Alismatis, 2 is a negative sample of Rhizoma Atractylodis Macrocephalae, 3 is a freeze-dried powder preparation of Rhizoma Alismatis, 4 is a reference medicinal material of Rhizoma Alismatis, 5 is a reference medicinal material of Rhizoma Atractylodis Macrocephalae, 6 is a reference substance of 23-acetylalismol C, 7 is a reference substance of Atractylodes macrocephala lactone II, and 8 is a reference substance of Atractylodes macrocephala lactone III;

[0050] Figure 8 1 is a graph showing the results of the investigation when the Specco silica gel G plate is used in Example 3 of the present invention; wherein, 1 is a negative sample of Rhizoma Alismatis, 2 is a negative sample of Rhizoma Atractylodis Macrocephalae, 3 is a freeze-dried powder preparation of Rhizoma Alismatis, 4 is a reference medicinal material of Rhizoma Alismatis, 5 is a reference medicinal material of Rhizoma Atractylodis Macrocephalae, 6 is a reference substance of 23-acetylalismol C, 7 is a reference substance of Atractylodes macrocephala lactone II, and 8 is a reference substance of Atractylodes macrocephala lactone III;

[0051] Fig. 9 1 is a graph of the investigation results when the Merck silica gel G plate is used in Example 3 of the present invention; wherein, 1 is a negative sample of Rhizoma Alismatis, 2 is a negative sample of Rhizoma Atractylodis Macrocephalae, 3 is a freeze-dried powder preparation of Rhizoma Alismatis, 4 is a reference medicinal material of Rhizoma Alismatis, 5 is a reference medicinal material of Rhizoma Atractylodis Macrocephalae, 6 is a reference substance of 23-acetylalismol C, 7 is a reference substance of Atractylodes macrocephala lactone II, and 8 is a reference substance of Atractylodes macrocephala lactone III;

[0052] Fig.10 1 is a graph of the investigation results when the heating time is 1 min in Example 4 of the present invention, wherein 1 is a negative sample of Rhizoma Alismatis, 2 is a negative sample of Rhizoma Atractylodis Macrocephalae, 3 is a freeze-dried powder preparation of Rhizoma Alismatis, 4 is a reference medicinal material of Rhizoma Alismatis, 5 is a reference medicinal material of Rhizoma Atractylodis Macrocephalae, 6 is a reference substance of 23-acetylalismol C, 7 is a reference substance of Atractylodes macrocephala lactone II, and 8 is a reference substance of Atractylodes macrocephala lactone III;

[0053] Fig.111 is a graph of the investigation results when the heating time is 5 min in Example 4 of the present invention, wherein 1 is a negative sample of Rhizoma Alismatis, 2 is a negative sample of Rhizoma Atractylodis Macrocephalae, 3 is a freeze-dried powder preparation of Rhizoma Alismatis, 4 is a reference medicinal material of Rhizoma Alismatis, 5 is a reference medicinal material of Rhizoma Atractylodis Macrocephalae, 6 is a reference substance of 23-acetylalismol C, 7 is a reference substance of Atractylodes macrocephala lactone II, and 8 is a reference substance of Atractylodes macrocephala lactone III;

[0054] Fig.12 is the result of thin layer chromatography in Example 5 of the present invention; wherein 1 is a negative sample of Rhizoma Alismatis, 2 is a negative sample of Rhizoma Atractylodis Macrocephalae, 3 is a freeze-dried powder preparation of Rhizoma Alismatis, 4 is a reference medicinal material of Rhizoma Alismatis, 5 is a reference medicinal material of Rhizoma Atractylodis Macrocephalae, 6 is a reference substance of 23-acetyl Rhizoma Alismatis B, 7 is a reference substance of 23-acetyl Rhizoma Alismatis C, 8 is a reference substance of Atractylodes lactone II, and 9 is a reference substance of Atractylodes lactone III;

[0055] Fig.13 1 is a graph of thin layer chromatography identification results in Comparative Example 1 of the present invention; wherein, 1 is a negative sample of Atractylodes macrocephala, 2-3 are freeze-dried powder preparations of Zexie decoction of batches S1 and S2; 4 is a control medicinal material of Atractylodes macrocephala; 5 is a negative sample of Zexie decoction; 6-7 are freeze-dried powder preparations of Zexie decoction of batches S1 and S2; 8 is a control medicinal material of Zexie decoction; 9 is a reference substance of 23-acetylzexie alcohol B; 10 is a reference substance of 23-acetylzexie alcohol C;

[0056] Fig.14 It is a graph of thin layer chromatography identification results in Comparative Example 2 of the present invention; wherein 1 is a negative sample of Rhizoma Alismatis, 2 is a negative sample of Atractylodes macrocephala, 3-4 are freeze-dried powder preparations of Rhizoma Alismatis decoction of batches S1-S2, 5 is a reference medicinal material of Atractylodes macrocephala, 6 is a reference medicinal material of Rhizoma Alismatis, 7 is a reference substance of 23-acetyl Rhizoma Alismatis B, and 8 is a reference substance of 23-acetyl Rhizoma Alismatis C;

[0057] Fig.15 It is a graph of thin layer chromatography identification results in Comparative Example 3 of the present invention; wherein 1 is a reference medicinal material of Rhizoma Alismatis, 2 is a freeze-dried powder preparation of Rhizoma Alismatis, 3 is a reference medicinal material of Rhizoma Atractylodis Macrocephalae, 4 is a reference substance of Atractylodes macrocephala lactone I, 5 is a reference substance of Atractylodes macrocephala lactone II, and 6 is a reference substance of Atractylodes macrocephala lactone III;

[0058] Fig.16 This is a graph of thin layer chromatography identification results in Comparative Example 4 of the present invention (inspected under 365nm ultraviolet light); wherein 1 is a control medicinal material of Atractylodes macrocephala, 2 is a freeze-dried powder preparation of Zexie decoction, 3 is a control medicinal material of Zexie, 4 is a reference substance of 23-acetyl alismatol B, and 5 is a reference substance of 23-acetyl alismatol C;

[0059] Fig.17 This is a diagram of the thin layer chromatography identification result in Comparative Example 4 of the present invention (viewed under sunlight); wherein 1 is a control medicinal material of Atractylodes macrocephala, 2 is a freeze-dried powder preparation of Zexie decoction, 3 is a control medicinal material of Zexie, 4 is a reference substance of 23-acetyl Zexie alcohol B, and 5 is a reference substance of 23-acetyl Zexie alcohol C;

[0060] Fig.18 This is a diagram of the thin layer chromatography identification result in Comparative Example 5 of the present invention; wherein 1 is a control medicinal material of Rhizoma Alismatis, 2 is a freeze-dried powder preparation of Rhizoma Alismatis decoction, and 3 is a control medicinal material of Rhizoma Atractylodis Macrocephalae. DETAILED DESCRIPTION

[0061] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings and specific implementation methods.

[0062] As the first aspect of the present invention, the present invention provides a Zexie decoction and a preparation method thereof. The present invention is based on the textual research of ancient books and documents, and the main basis for the dosage conversion result is the clinical practice since the Ming and Qing Dynasties. According to the standard of "one liang" in the Han and Tang Dynasties by Li Shizhen of the Ming Dynasty, which is approximately equivalent to "one qian is 3.73g" in the Ming Dynasty, in the original prescription, the dosage ratio of Zexie and Atractylodes is 5:2, and the conversion result is: Zexie dosage 18.65g; Atractylodes dosage 7.46g, which is more in line with the modern clinical routine dosage.

[0063] The preparation method of Zexie Decoction is as follows: take Zexie and Atractylodes macrocephala medicinal materials, and prepare them into decoction pieces; randomly formulate a prescription, take 18.65g of Zexie decoction pieces and 7.46g of Atractylodes macrocephala decoction pieces, soak them in 400mL of water, boil them over high heat, keep boiling over low heat until the medicinal liquid is about 200mL, filter to obtain Zexie Decoction decoction, transfer the decoction to a vacuum circulating pump vacuum freeze dryer for freeze drying, take it out, and obtain freeze-dried powder, which is the Zexie Decoction benchmark sample.

[0064] As a second aspect of the present invention, the present invention provides a thin layer chromatography identification method for Zexie decoction. Based on the difference of the reference substances used, it can be specifically divided into three schemes, one is to compare with the reference substance alone, the other is to compare with the reference medicinal material, and the last is to compare with the reference substance and the reference medicinal material at the same time. The following content is detailed with the technical scheme of comparing the reference substance and the reference medicinal material at the same time.

[0065] Specifically, the identification method includes the following steps:

[0066] S1: prepare test solution;

[0067] Specifically, the oriental decoction is extracted with n-hexane, the extract is dried and then dissolved with a first solvent to obtain a test solution. The oriental decoction can be a decoction, granules, powders, capsules, oral liquids, tablets or pills, but is not limited thereto. The number of extractions can be one or more, but is not limited thereto. The extraction method can be heating reflux extraction, ultrasonic extraction, shaking extraction, but is not limited thereto. The extract can be evaporated to dryness or air-dried, but is not limited thereto. The first solvent can be methanol, but is not limited thereto.

[0068] Preferably, in one embodiment of the present invention, S1 includes:

[0069] S11: Take a sample of Zexie decoction and add water to dissolve it;

[0070] The weight ratio of Zexie decoction to the volume of water added is (1-3) g: (20-50) mL, illustrative examples are 1 g: 25 mL, 1.5 g: 22 mL, 2 g: 30 mL, 2.5 g: 35 mL, but not limited thereto. Preferably, it is 2 g: 30 mL.

[0071] S12: extract the solution obtained by dissolving in step S11 with n-hexane for 1 to 4 times, combine the extracts, evaporate to dryness in a water bath at 50 to 80° C., and dissolve the residue in methanol to obtain the test solution;

[0072] The ratio of the volume of n-hexane added to the weight of Zexie decoction for each extraction is (15-30) mL: (1-3) g, exemplified by 18 mL: 1.8 g, 20 mL: 1.6 g, 25 mL: 2 g, 28 mL: 2.5 g, but not limited thereto, and preferably 20 mL: 2 g.

[0073] The ratio of the volume of methanol used for dissolution to the weight of Zexie decoction is (0.5-1.5) mL: (1-3) g, exemplified by 0.8 mL: 1 g, 1 mL: 1.5 g, 1.2 mL: 2 g, 1.4 mL: 2.5 g, but not limited thereto. Preferably, it is 1 mL: 2 g.

[0074] S2: prepare reference substance solution and reference medicinal material solution;

[0075] Specifically, the reference substance is a 23-acetyl alismatol C reference substance, a white atractylodes lactone II reference substance, and a white atractylodes lactone III reference substance. The reference substance solution may be a mixed reference substance solution, specifically a mixture of three or any two. Exemplarily, in one embodiment, the reference substance solution is a mixed solution of three reference substances, wherein the concentration of each reference substance is 0.5 to 1 mg / mL. In another embodiment, the reference substance solution includes a 23-acetyl alismatol C reference substance solution and a white atractylodes lactone solution. Among them, the white atractylodes lactone solution is a mixed solution of a white atractylodes lactone II reference substance and a white atractylodes lactone III reference substance, and the concentrations of both are 0.5 to 1 mg / mL. Preferably, the reference solution includes 23-acetyl alismatol C reference solution, atractyl lactone II reference solution, and atractyl lactone III reference solution; the preparation methods are as follows: take 23-acetyl alismatol C reference, add methanol to make a solution with a concentration of 0.5-1 mg / mL, and obtain acetyl alismatol reference solution; take atractyl lactone II reference, add methanol to make a solution with a concentration of 0.5-1 mg / mL, and obtain atractyl lactone II reference solution; take atractyl lactone III reference, add methanol to make a solution with a concentration of 0.5-1 mg / mL, and obtain atractyl lactone III reference solution. Preferably, the concentrations of the three reference solutions are all 1 mg / mL.

[0076] The preparation methods of the control medicinal material solutions are:

[0077] Take 1-5 g of Rhizoma Alismatis reference medicinal material, add 30-80 mL of water, boil for 20-60 min, centrifuge and take the supernatant, extract the supernatant with n-hexane 1-4 times, combine the extracts, evaporate to dryness in a water bath at 50-80°C, add methanol to the residue to dissolve it, and use it as the Rhizoma Alismatis reference medicinal material solution.

[0078] Take 0.5-2 g of Atractylodes macrocephala reference medicinal material, add 30-80 mL of water, boil for 20-60 min, centrifuge and take the supernatant, extract the supernatant with n-hexane 1-4 times, combine the extracts, evaporate to dryness in a water bath at 50-80°C, add methanol to the residue to dissolve it, and use it as the Atractylodes macrocephala reference medicinal material solution.

[0079] It should be noted that there is no particular order for S1 and S2 in the present invention. Those skilled in the art may first implement S1 and then S2, or first implement S2 and then S1.

[0080] S3: Pipette the test solution, reference solution, Rhizoma Alismatis control medicinal material solution, and Rhizoma Atractylodis Macrocephalae control medicinal material solution onto a silica gel G thin layer plate, develop it with a mixed solution of chloroform, acetone, and formic acid in a volume ratio as a developing agent, develop the color, and inspect it.

[0081] Specifically, silica gel G thin layer plate is used for development, and a mixed solution of chloroform, acetone and formic acid is used as the developing agent. After development, it is sprayed with sulfuric acid ethanol solution and heated until the spots are clear. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatogram of the reference sample and the chromatogram of the reference medicinal material.

[0082] Among them, the spotting volume of the test solution is 5-15μL, exemplified by 6μL, 8μL, 10μL, 12μL or 14μL, but not limited thereto. Preferably, it can be 10μL. The spotting volume of the control medicinal material solution of Alisma is 5-20μL, exemplified by 6μL, 9μL, 12μL, 15μL or 18μL, but not limited thereto; preferably, it is 15μL. The spotting volume of the control medicinal material solution of Atractylodes macrocephala is 5-15μL, exemplified by 6μL, 9μL, 12μL, 15μL or 18μL, but not limited thereto; preferably, it is 10μL. The spotting volume of the reference solution is 1-3μL, exemplified by 1.2μL, 1.5μL, 2μL, 2.5μL, and preferably, it is 1μL.

[0083] The volume ratio of chloroform, acetone and formic acid in the developing solvent is (9-10): (0.1-1): (0.01-0.2), exemplified by 9:0.5:0.06, 9.2:0.8:0.02, 9.5:0.5:0.15, 9.8:0.8:0.12, but not limited thereto. Preferably, it is 9.5:0.5:0.06.

[0084] The extension distance of the silica gel G thin layer plate is 8-12 cm, preferably 8 cm.

[0085] Wherein, the heating temperature is 100-110°C, exemplified by 102°C, 105°C, 106°C, 108°C or 109°C, but not limited thereto. Preferably, it is 108°C. Specifically, the heating can be for heating the thin layer plate as a whole, or for heating a part thereof. Preferably, in one embodiment of the present invention, during heating, the heating time of the spots at a spread of 0-4 cm is ≤1 min, and the heating time of the spots at a spread of 4-8 cm is >1 min. By means of the above heating scheme, the spots in each area can be made cleaner. Preferably, the heating time of the spots at 0-4 cm is 0.5-1 min; the heating time of the spots at a spread of 4-8 cm is 2-5 min.

[0086] Specifically, the developer is a sulfuric acid ethanol solution, and the volume concentration of sulfuric acid is 5-20%, exemplified by 6%, 7.5%, 9%, 11.5%, 18%, but not limited thereto. Preferably, the volume concentration of sulfuric acid in the sulfuric acid ethanol solution is 10%.

[0087] Specifically, after heating to color development and washing, the sample is inspected under ultraviolet light, the wavelength of which is 360-370 nm, exemplified by 364 nm, 366 nm, and 368 nm, but not limited thereto, and preferably 365 nm.

[0088] It should be noted that when only the reference solution is used for comparison, only the test solution and the reference solution are drawn in the absorption step, and the remaining steps are the same as above. Correspondingly, when only the reference medicinal material solution is used for comparison, only the test solution, the oriental medicine solution, and the atractylodes macrocephala reference medicinal material solution are drawn in the absorption step, and the remaining steps are the same as above. Based on the scheme of using the reference substance and the reference medicinal material for comparison at the same time of this application, the specificity of oriental medicine and atractylodes macrocephala can be more comprehensively reflected.

[0089] The present invention will be further described below with specific embodiments:

[0090] The information of instruments, reagents, reference substances and sources of various medicinal materials of Zexie Decoction used in the present invention are shown in Table 1, Table 2, Table 3 and Table 4 respectively.

[0091] Table 1 Main instruments

[0092]

[0093] Table 2 Main reagents

[0094]

[0095] Table 3 Reference substance information

[0096]

[0097] Table 4 The batch numbers of the three batches of Zexie decoction and the corresponding origin combinations and the related single herbs and negative decoction pieces combinations

[0098]

[0099] Example 1 Lyophilized preparation of Zexie decoction and its preparation

[0100] This embodiment provides a method for preparing a lyophilized preparation of Rhizoma Alismatis Decoction, which is as follows:

[0101] Take 18.65g of Alisma orientalis slices and 7.46g of Atractylodes macrocephala slices, soak them in 400mL of water (30 minutes), boil them over high heat (30 minutes), keep boiling over low heat (300w) until the medicinal liquid is about 200mL, filter to obtain Alisma orientalis decoction; stir well and dispense into brown syringe bottles, transfer to a vacuum freeze dryer with a vacuum circulation pump for freeze drying, take out, and obtain the Alisma orientalis decoction benchmark sample preparation.

[0102] Specifically, the present invention prepared 3 batches of lyophilized preparations of Zexie decoction, and the specific raw materials used are shown in Table 4. In addition, Zexie negative and Atractylodes negative samples were prepared according to the above method, and the raw materials used are shown in Table 4.

[0103] Example 2 Establishment of Identification Method

[0104] 1. Preparation of test solution:

[0105] Take 2.0 g of each Zexie decoction benchmark sample from different batches, grind them into powder, add 30 mL of water, and dissolve them by ultrasonic. The solution is extracted with n-hexane by shaking for 3 times, 20 mL each time. The n-hexane solutions are combined and evaporated to dryness in a water bath at 60°C. 1 mL of methanol is added to the residue to dissolve it, which is used as the test solution.

[0106] 2. Preparation of reference substance and reference medicinal material solution:

[0107] Take 4.0 g of Rhizoma Alismatis and 1.0 g of Rhizoma Atractylodis Macrocephalae as control medicinal material, add 50 mL of water respectively, heat and boil for 30 minutes, centrifuge, take the supernatant, and prepare Rhizoma Alismatis and Rhizoma Atractylodis Macrocephalae control medicinal material solutions according to the method of step (1).

[0108] Take 23-acetyl alismatol C reference substance, atractylodes lactone II reference substance and atractylodes lactone III reference substance, add methanol to make solutions containing 1 mg per 1 mL, as reference substance solutions (23-acetyl alismatol C reference substance solution, atractylodes lactone II reference substance solution, atractylodes lactone III reference substance solution).

[0109] 3. Identification method:

[0110] 10 μL of test solution, 15 μL of orientalis control medicinal material solution, 10 μL of Atractylodes control medicinal material solution, 1 μL of 23-acetylalismol C reference solution, 1 μL of atractylodes lactone II reference solution, and 1 μL of atractylodes lactone III reference solution were respectively taken and spotted on the same silica gel G thin layer plate (Yinlong silica gel G plate), and chloroform-acetone-formic acid (9.5:0.5:0.06) was used as the developing agent. The plate was developed, taken out, dried, sprayed with 10% ethanolic sulfuric acid solution, and heated at 108°C until the spots were clearly colored. The heating time for the spots at a spread distance of 0 to 4 cm was 1 min, and the heating time for the spots at a spread distance of 4 to 8 cm was 3 min. The plates were inspected under ultraviolet light (365 nm).

[0111] The identification results of 3 batches of Zexie decoction are shown in Figure 1 As can be seen from the figure, the chromatograms of the three batches of Zexie decoction test samples and the chromatograms of the Zexie control medicinal materials, Atractylodes control medicinal materials, 23-acetylzexetol C reference material, Atractylodes lactone II reference material, and Atractylodes lactone III reference material show fluorescent spots of the same color at the corresponding positions.

[0112] Example 3 Methodology Verification

[0113] 1. Exclusiveness Investigation

[0114] 1. Prepare the Alisma negative sample and Atractylodes negative sample according to the ratio of raw materials in Table 4, and prepare the Alisma negative sample and Atractylodes negative sample respectively according to the preparation method of Example 1, and prepare the Alisma negative sample solution and Atractylodes negative sample solution according to the preparation method of the test solution in Example 2.

[0115] 2. Take 10μL of the test solution, 15μL of the control medicinal material solution of Alisma orientalis, 10μL of the control medicinal material solution of Atractylodes macrocephala, 1μL of the 23-acetyl alismatol C reference solution, 1μL of the Atractylodes macrocephala lactone II reference solution, 1μL of the Atractylodes macrocephala lactone III reference solution, 10μL of the negative sample solution of Alisma orientalis, and 15μL of the negative sample solution of Atractylodes macrocephala, and spot them on the same silica gel G thin layer plate (Silver Dragon silica gel G plate), use chloroform-acetone-formic acid (9.5:0.5:0.06) as the developing agent, develop at room temperature and humidity (T: 23°C, RH: 58%), take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 108°C until the spots are clearly colored, and inspect under ultraviolet light (365nm). Experimental results Figure 2 .

[0116] As can be seen from the figure, the chromatogram of the test sample of Zexie decoction and the chromatograms of Zexie reference medicinal materials, Atractylodes macrocephala reference medicinal materials, 23-acetylzexetol C reference material, Atractylodes macrocephala lactone II reference material, and Atractylodes macrocephala lactone III reference material show fluorescent spots of the same color at the corresponding positions, and there is no interference from the negative sample, indicating that the thin-layer method has good specificity.

[0117] 2. Investigation of different sampling quantities

[0118] 5μL, 10μL, 15μL of the test solution, 10μL, 15μL, 20μL of the control medicinal material solution of Alisma orientalis, 5μL, 10μL, 15μL of the control medicinal material solution of Atractylodes macrocephala, 1μL, 5μL of the 23-acetyl alismatol C reference solution, 1μL, 5μL of the Atractylodes macrocephala lactone II reference solution, and 1μL, 5μL of the Atractylodes macrocephala lactone III reference solution were respectively taken and spotted on the same silica gel G thin layer plate (Silver Dragon silica gel G plate), and chloroform-acetone-formic acid (9.5:0.5:0.06) was used as the developing agent. The samples were developed at room temperature and humidity (T: 23°C, RH: 58%), taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 108°C until the spots were clearly colored, and examined under ultraviolet light (365nm). The experimental results are shown in Figure 3 .

[0119] As can be seen from the figure, when the sample volume of the test solution is 10 μL, the sample volume of the control medicinal material solution of Alisma orientalis is 15 μL, the sample volume of the control medicinal material solution of Atractylodes macrocephala is 10 μL, and the sample volume of the 23-acetyl alismatol C reference solution, atractylodes lactone II reference solution, and atractylodes lactone III reference solution is 1 μL, the Rf values ​​of the test sample and the control medicinal material and reference solution spots are consistent and clear, and there is no other interference. Therefore, the sample volume is selected as 10 μL of the test sample solution, 15 μL of the control medicinal material solution of Alisma orientalis, 10 μL of the control medicinal material solution of Atractylodes macrocephala, and 1 μL each of the 23-acetyl alismatol C reference solution, atractylodes lactone II reference solution, and atractylodes lactone III reference solution.

[0120] 3. Investigation at different temperatures

[0121] 10 μL of the test solution, 15 μL of the control medicinal material solution of Alisma orientalis, 10 μL of the control medicinal material solution of Atractylodes macrocephala, 1 μL of the reference solution of 23-acetyl alismatol C, 1 μL of the reference solution of Atractylodes macrocephala lactone II, 1 μL of the reference solution of Atractylodes macrocephala lactone III, 10 μL of the negative sample solution of Alisma orientalis, and 15 μL of the negative sample solution of Atractylodes macrocephala were respectively spotted on the same silica gel G thin layer plate (Silver Dragon silica gel G plate), and chloroform-acetone-formic acid (9.5:0.5:0.06) was used as the developing agent. The plates were developed at room temperature (T: 23.4°C, RH: 58%) and low temperature (T: 5.8°C, RH: 69%), taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 108°C until the spots were clearly colored, and examined under ultraviolet light (365nm). The experimental results are shown in Figure 4-5 .

[0122] As can be seen from the figure, under normal and low temperature conditions, the separation effect of the test sample of Zexie Decoction is good, and the corresponding positions of the chromatograms of the Zexie control medicinal material, the Atractylodes macrocephala control medicinal material, the 23-acetyl alismatol C reference substance, the Atractylodes macrocephala lactone II reference substance, and the Atractylodes macrocephala lactone III reference substance show the same color fluorescent spots. The experimental results show that temperature has no obvious effect on the thin layer identification of Zexie and Atractylodes macrocephala in Zexie Decoction, indicating that the thin layer identification method has good durability at different temperatures.

[0123] 4. Investigation of different humidity

[0124] 10 μL of the test solution, 15 μL of the control medicinal material solution of Alisma orientalis, 10 μL of the control medicinal material solution of Atractylodes macrocephala, 1 μL of the 23-acetyl alismatol C reference solution, 1 μL of the reference solution of Atractylodes macrocephala lactone II, 1 μL of the reference solution of Atractylodes macrocephala lactone III, 10 μL of the negative sample solution of Alisma orientalis, and 15 μL of the negative sample solution of Atractylodes macrocephala were respectively spotted on the same silica gel G thin layer plate (Silver Dragon silica gel G plate), and chloroform-acetone-formic acid (9.5:0.5:0.06) was used as the developing agent. The plates were developed under high humidity (T: 23°C, RH: 80%) and low humidity (T: 23°C, RH: 38%) conditions, respectively. The plates were taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 108°C until the spots were clearly colored, and examined under ultraviolet light (365nm). Experimental results Figure 6-7 .

[0125] As can be seen from the figure, under high and low humidity conditions, the separation effect of the test sample of Zexie Decoction is good, and the corresponding positions of the chromatograms of Zexie Decoction and the chromatograms of Zexie reference materials, Atractylodes macrocephala reference materials, 23-acetyl alismatol C reference materials, Atractylodes macrocephala lactone II reference materials, and Atractylodes macrocephala lactone III reference materials show the same color of fluorescent spots. The experimental results show that humidity has no effect on the thin-layer identification of Zexie and Atractylodes macrocephala in Zexie Decoction, indicating that the thin-layer identification method has good durability under different humidity.

[0126] 5. Inspection of thin layer boards from different manufacturers

[0127] 10 μL of the test solution, 15 μL of the control medicinal material solution of Alisma orientalis, 10 μL of the control medicinal material solution of Atractylodes macrocephala, 1 μL of the reference solution of 23-acetyl alismatol C, 1 μL of the reference solution of Atractylodes macrocephala lactone II, 1 μL of the reference solution of Atractylodes macrocephala lactone III, 10 μL of the negative sample solution of Alisma orientalis, and 15 μL of the negative sample solution of Atractylodes macrocephala were respectively spotted on the same silica gel G thin layer plate (Merck silica gel G plate, Spectrum silica gel G plate), and developed with chloroform-acetone-formic acid (9.5:0.5:0.06) as the developing agent at room temperature and humidity (T: 23°C, RH: 58%), taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 108°C until the spots were clearly colored, and inspected under ultraviolet light (365nm). Experimental results Figure 8-Figure 9 .

[0128] As can be seen from the figure, silica gel G thin layer plates from different manufacturers have no significant effect on the thin layer identification of Alisma orientalis and Atractylodes macrocephala in Alisma orientalis decoction. The chromatographic color development and fluorescent spot presentation of the chromatographic material of Alisma orientalis, Atractylodes macrocephala, 23-acetylalaxol C reference substance, Atractylodes macrocephala lactone II reference substance and Atractylodes macrocephala lactone III reference substance are clear and stable, indicating that the thin layer identification method has good durability for silica gel G thin layer plates from different manufacturers.

[0129] Example 4 Investigation of heating time

[0130] 10 μL of test solution, 15 μL of oriental rhizome control medicinal material solution, 10 μL of atractylodes macrocephala control medicinal material solution, 1 μL of 23-acetyl alismatol C reference solution, 1 μL of atractylodes lactone II reference solution, 1 μL of atractylodes lactone III reference solution, 10 μL of oriental rhizome negative sample solution and 15 μL of atractylodes macrocephala negative sample solution were respectively spotted on the same silica gel G thin layer plate (Silver Dragon silica gel G plate), and chloroform-acetone-formic acid (9.5:0.5:0.06) was used as the developing agent. The plate was developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 108°C for 1 min, and examined under ultraviolet light (365 nm). The experimental results are shown in Fig.10 ; Heat for another 5 minutes and inspect under ultraviolet light (365nm). Fig.11 .

[0131] Depend on Fig.11 It can be seen that after heating for 1 minute, the same color fluorescent spots appeared in the chromatogram of the test sample at the corresponding positions of the chromatogram of the control medicinal material and the chromatogram of the control substance, but the chromatographic spots of Atractylodes lactone II were unclear. After heating for another 5 minutes, the same color fluorescent spots appeared in the chromatogram of the test sample at the corresponding positions of the chromatogram of the control medicinal material and the chromatogram of the control substance, but the chromatographic spots of the control medicinal material of Alisma, the control medicinal material of Atractylodes, and 23-acetyl alismatol B turned red at Rf values ​​less than 0.5, but the chromatographic spots of Atractylodes lactone II appeared bright blue. Therefore, when heating after color development, the heating time of the spots at a spread of 0-4 cm should be within 1 minute, and the heating time of the spots at a spread of 4-8 cm should be greater than 1 minute.

[0132] Example 5 Investigation of Spread

[0133] 1. Preparation of test solution:

[0134] Take 2.0 g of each Zexie decoction benchmark sample from different batches, grind them into powder, add 30 mL of water, and dissolve them by ultrasonic. The solution is extracted with n-hexane by shaking for 3 times, 20 mL each time. The n-hexane solutions are combined and evaporated to dryness in a water bath at 60°C. 1 mL of methanol is added to the residue to dissolve it, which is used as the test solution.

[0135] 2. Preparation of reference substance and reference medicinal material solution:

[0136] Take 4.0 g of Rhizoma Alismatis and 1.0 g of Rhizoma Atractylodis Macrocephalae as control medicinal material, add 50 mL of water respectively, heat and boil for 30 minutes, centrifuge, take the supernatant, and prepare Rhizoma Alismatis and Rhizoma Atractylodis Macrocephalae control medicinal material solutions according to the method of step (1).

[0137] Take 23-acetyl alismatol B reference substance, 23-acetyl alismatol C reference substance, atractylodes lactone II reference substance and atractylodes lactone III reference substance, add methanol to make a solution containing 1 mg per 1 mL, as the reference substance solution.

[0138] 3. Preparation of negative sample solution

[0139] According to the raw materials of the negative sample ratio of Alisma orientalis in Table 4, and according to the preparation method of Example 1, the negative sample of Alisma orientalis and the negative sample of Atractylodes macrocephala were prepared respectively, and according to the preparation method of the test solution in Example 2, the negative sample solution of Alisma orientalis and the negative sample solution of Atractylodes macrocephala were prepared.

[0140] 4. Identification method

[0141] 10 μL of Zexie decoction test solution, 15 μL of Zexie control medicinal material solution, 10 μL of Atractylodes macrocephala control medicinal material solution, 1 μL of 23-acetyl Zexie alcohol B reference solution, 1 μL of 23-acetyl Zexie alcohol C reference solution, 1 μL of Atractylodes macrocephala lactone II reference solution, 1 μL of Atractylodes macrocephala lactone III reference solution, 10 μL of Zexie negative sample solution and 15 μL of Atractylodes macrocephala negative sample solution were respectively spotted on the same silica gel G thin layer plate (Silver Dragon silica gel G plate), and chloroform-acetone-formic acid (9.5:0.5:0.06) was used as the developing agent. The plate was developed for about 12 cm, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 108°C until the spots were clearly colored, and examined under ultraviolet light (365nm). The experimental results are shown in the table. Fig.12 .

[0142] As can be seen from the figure, the chromatogram of the test sample of Zexie decoction developed at 12 cm and the corresponding positions of the chromatograms of Zexie control medicinal materials, Atractylodes macrocephala control medicinal materials, 23-acetylzexetol C control substance, Atractylodes macrocephala lactone II control substance, and Atractylodes macrocephala lactone III control substance show fluorescent spots of the same color, and there is no interference from the negative sample, but the chromatographic spots of the 23-acetylzexetol B control substance have spots with similar Rf values ​​and consistent colors in the chromatogram of the test sample, which are easily interfered, and increasing the development distance also fails to distinguish the two chromatographic spots well, so the identification of the 23-acetylzexetol B control substance is not included in the method. The development distance is extended and the development time is also longer, so a thin layer plate of silica gel G with a height of 10 cm can be used for development.

[0143] Comparative Example 1

[0144] This comparative example provides a thin layer chromatography identification method for Zexie decoction, which is as follows:

[0145] 1. Preparation of test solution:

[0146] Take 2.0 g of each Zexie decoction benchmark sample from different batches, grind them into powder, add 30 mL of water, and dissolve them by ultrasonic. The solution is extracted by shaking with water-saturated n-butanol for 3 times, 20 mL each time. The n-butanol solutions are combined and evaporated to dryness in a water bath at 60°C. 1 mL of methanol is added to the residue to dissolve it, which is used as the test solution.

[0147] 2. Preparation of reference substance and reference medicinal material solution:

[0148] Take 4.0 g of Rhizoma Alismatis and 1.0 g of Rhizoma Atractylodis Macrocephalae as control medicinal material, add 50 mL of water respectively, heat and boil for 30 minutes, centrifuge, take the supernatant, and prepare Rhizoma Alismatis and Rhizoma Atractylodis Macrocephalae control medicinal material solutions according to the method of step (1).

[0149] Take 23-acetyl alismatol C reference substance and 23-acetyl alismatol B reference substance, add methanol to prepare solutions containing 1 mg per 1 mL, as reference substance solutions (23-acetyl alismatol C reference substance solution, 23-acetyl alismatol B solution).

[0150] 3. Preparation of negative sample solution

[0151] According to the raw materials of the negative sample ratio of Alisma orientalis in Table 4, and according to the preparation method of Example 1, the negative sample of Alisma orientalis and the negative sample of Atractylodes macrocephala were prepared respectively, and the negative sample solution of Alisma orientalis and the negative sample solution of Atractylodes macrocephala were prepared according to the preparation method of the above-mentioned test solution.

[0152] 4. Identification method:

[0153] 15 μL of Atractylodes macrocephala negative sample solution, 10 μL and 10 μL of test solution, 10 μL of Atractylodes macrocephala control medicinal material solution, 10 μL of Alisma orientalis negative sample solution, 10 μL and 10 μL of test solution, 15 μL of Alisma orientalis control medicinal material solution, 1 μL of 23-acetyl alismatol C reference solution, and 1 μL of 23-acetyl alismatol B reference solution were respectively spotted on the same silica gel G thin layer plate (Silver Dragon silica gel G plate), and developed with chloroform-acetone-formic acid (9.5:0.5:0.06) as the developing agent, developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 108°C until the spots were clearly colored, and inspected under ultraviolet light (365nm). Experimental results Fig.13 .

[0154] As can be seen from the figure, the chromatographic spot of the test sample of Zexie decoction corresponds one-to-one with a blue fluorescent chromatographic spot of the reference herb Atractylodes macrocephala, and there is no interference in the negative test. Therefore, this method can be applied to the identification of Atractylodes macrocephala in Zexie decoction. The chromatographic spot of the test sample corresponds one-to-one with a blue fluorescent chromatographic spot of the reference herb 23-acetylzexetol C and the reference herb Zexie macrocephala, and there is no interference in the negative test. Therefore, this method can be applied to the identification of Zexie macrocephala in Zexie decoction, but the background interference of this spot is relatively serious.

[0155] Compared with Example 2, the spots in Example 2 are more regular and clear, and there are more spots on Atractylodes macrocephala, which can reflect the specific components of Atractylodes macrocephala medicinal taste, Atractylodes lactone II and Atractylodes lactone III reference substances, that is, the extraction effect of n-hexane is better than that of water-saturated n-butanol.

[0156] Comparative Example 2

[0157] This comparative example provides a thin layer chromatography identification method for Zexie decoction, which is as follows:

[0158] 1. Preparation of test solution

[0159] Take 2.0 g of each Zexie decoction benchmark sample from different batches, grind them into powder, add 30 mL of water, and dissolve them by ultrasonic. The solution is extracted with ethyl acetate by shaking for 3 times, 20 mL each time. The ethyl acetate solutions are combined and evaporated to dryness in a water bath. 1 mL of methanol is added to the residue to dissolve it, which is used as the test solution.

[0160] 2. Preparation of reference substance and reference medicinal material solution:

[0161] Take 4.0 g of Rhizoma Alismatis and 1.0 g of Rhizoma Atractylodis Macrocephalae as control medicinal material, add 50 mL of water respectively, heat and boil for 30 minutes, centrifuge, take the supernatant, and prepare Rhizoma Alismatis and Rhizoma Atractylodis Macrocephalae control medicinal material solutions according to the method of step (1).

[0162] Take 23-acetyl alismatol B reference substance and 23-acetyl alismatol C reference substance, add methanol to make solutions containing 1 mg per 1 mL, as reference substance solutions (23-acetyl alismatol B reference substance solution, 23-acetyl alismatol C reference substance solution).

[0163] 3. Preparation of negative sample solution

[0164] According to the raw materials of the negative sample ratio of Alisma orientalis in Table 4, and according to the preparation method of Example 1, the negative sample of Alisma orientalis and the negative sample of Atractylodes macrocephala were prepared respectively, and the negative sample solution of Alisma orientalis and the negative sample solution of Atractylodes macrocephala were prepared according to the preparation method of the above-mentioned test solution.

[0165] 4. Identification method:

[0166] 10 μL of Alisma negative control solution, 15 μL of Atractylodes macrocephala negative control solution, 10 μL of test solution, 10 μL of Atractylodes macrocephala control medicinal material solution, 15 μL of Alisma control medicinal material solution, 1 μL of 23-acetyl alismatol B reference solution, and 1 μL of 23-acetyl alismatol C reference solution were respectively taken and spotted on the same silica gel G thin layer plate (Silver Dragon silica gel G plate), and chloroform-acetone-formic acid (9.5:0.5:0.06) was used as the developing agent. The plate was developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 108°C until the spots were clearly colored, and examined under ultraviolet light (365nm). Experimental results Fig.14 .

[0167] As can be seen from the figure, the chromatographic spots of the test sample of Zexie decoction correspond one-to-one with the two blue fluorescent chromatographic spots of the Atractylodes macrocephala reference medicinal material, and there is no interference in the negative, so this method can be applied to the identification of Atractylodes macrocephala in Zexie decoction. The chromatographic spots of the test sample correspond one-to-one with the two chromatographic spots of the Atractylodes macrocephala reference medicinal material and correspond to the 23-acetyl alismatol B reference substance and the 23-acetyl alismatol C reference substance spots, and there is no interference in the negative, so this method can be applied to the identification of Atractylodes macrocephala in Zexie decoction, but the Rf values ​​of the two reference substance spots are relatively close, and the spot color of 23-acetyl alismatol B is similar to the background color, so the identification effect is poor. Compared with Example 2, the spots of Example 2 are more regular and clear, and there are more spots of Atractylodes macrocephala, which can reflect the specific components of the taste of Atractylodes macrocephala, Atractylodes macrocephala lactone II and Atractylodes macrocephala lactone III reference substances, that is, the extraction effect of n-hexane is better than that of ethyl acetate.

[0168] Comparative Example 3

[0169] This comparative example provides a thin layer chromatography identification method for Zexie decoction, which is as follows:

[0170] 1. Preparation of test solution:

[0171] Take 2.0 g of each Zexie decoction benchmark sample from different batches, grind them into powder, add 30 mL of water, and dissolve them by ultrasonic. The solution is extracted with n-hexane by shaking for 3 times, 20 mL each time. The n-hexane solutions are combined and evaporated to dryness in a water bath at 60°C. 1 mL of methanol is added to the residue to dissolve it, which is used as the test solution.

[0172] 2. Preparation of reference substance and reference medicinal material solution:

[0173] Take 4.0 g of Rhizoma Alismatis and 1.0 g of Rhizoma Atractylodis Macrocephalae as control medicinal material, add 50 mL of water respectively, heat and boil for 30 minutes, centrifuge, take the supernatant, and prepare Rhizoma Alismatis and Rhizoma Atractylodis Macrocephalae control medicinal material solutions according to the method of step (1).

[0174] Take Atractylodes lactone I reference substance, Atractylodes lactone II reference substance and Atractylodes lactone III reference substance, add methanol to make a solution containing 1 mg per 1 mL, as the reference substance solution.

[0175] 3. Identification method:

[0176] 15 μL of Alisma orientalis control medicinal material solution, 10 μL of test solution, 10 μL of Atractylodes macrocephala control medicinal material solution, 1 μL of Atractylodes macrocephala lactone I reference solution, 1 μL of Atractylodes macrocephala lactone II reference solution, and 1 μL of Atractylodes macrocephala lactone III reference solution were respectively taken and spotted on the same silica gel G thin layer plate (Yinlong silica gel G plate), and cyclohexane-isopropanol (8:1) was used as the developing agent. The plate was developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 108°C for 1 min, and examined under ultraviolet light (365nm). The experimental results are shown in the table. Fig.15 .

[0177] As can be seen from the figure, the spots are relatively concentrated but the background interference is large. The chromatographic spots of the test sample correspond one to one with the two chromatographic spots of the Atractylodes macrocephala control medicinal material and the chromatographic spots of Atractylodes lactone II and Atractylodes lactone III reference materials. However, the Alisma orientalis control medicinal material presents a spot of the same color at the same position as the spot of Atractylodes lactone III reference material. Therefore, this developing agent is not suitable for the identification of Atractylodes macrocephala and Alisma orientalis in Alisma orientalis decoction.

[0178] The results of comparative examples 1, 2 and 3 show that the present invention can simultaneously identify 23-acetyl alismatol C, atractylodes lactone II and atractylodes lactone III in orientalis and atractylodes macrocephala by thin layer chromatography only when n-hexane is used as the extractant and chloroform-acetone-formic acid is used as the developing solvent, and the separation effect is good.

[0179] Comparative Example 4

[0180] According to the identification method under Alisma orientalis in the 2020 edition of the Chinese Pharmacopoeia, samples were prepared and spotted. The results are as follows Fig.16 , 17 shown.

[0181] Depend on Fig.16 It can be seen that the chromatographic spots of the test sample correspond one-to-one with the three blue fluorescent chromatographic spots of the Atractylodes macrocephala control medicinal material, and there is no such spot in the chromatographic spots of the Alisma orientalis control medicinal material. Therefore, this method can be applied to the identification of Atractylodes macrocephala in Alisma orientalis decoction; the chromatographic spots of the test sample correspond one-to-one with the chromatographic spots of the Alisma orientalis control medicinal material and the chromatographic spots of 23-acetylalaxol C, but the edge effect of the developing agent is obvious during the development process. After repeated experiments, the 23-acetylalaxol C reference material spots are prone to tilt. Although the chromatographic spots presented by the Alisma orientalis medicinal flavor in this identification method are relatively abundant, the spot background interference is large and the chromatographic spot separation is poor. Fig.17It can be seen that the chromatographic spots of the test sample correspond one to one with the chromatographic spots of the Alisma orientalis reference medicinal material solution and the chromatographic spots of 23-acetylalus orientalis alcohol B, and there is no such spot in the chromatographic spots of the Atractylodes macrocephala reference medicinal material. Therefore, this method can be applied to the identification of Alisma orientalis in Alisma orientalis decoction.

[0182] Compared with Example 2, the sample processing method of Example 2 is relatively simple, avoiding tedious processing such as passing through a macroporous resin column, simplifying the inspection steps, and improving the inspection efficiency. In addition, the chromatographic spots presented in Example 2 are more regular and clear, the development is stable, and there is no edge effect. At the same time, it can better reflect the specific components of the medicinal taste of Atractylodes macrocephala, Atractylodes lactone II and Atractylodes lactone III reference substances.

[0183] Comparative Example 5

[0184] According to the identification method under Atractylodes macrocephala in the 2020 edition of the Chinese Pharmacopoeia, samples were prepared and spotted. The results were as follows Fig.18 shown.

[0185] As can be seen from the figure, the TLC identification method failed to show TLC spots and could not identify the Atractylodes macrocephala and Alisma orientalis components in Zexie Decoction. The possible reason is that this method is aimed at the identification of low-polar components, while the transfer of low-polar components in Zexie Decoction is less.

[0186] The results of comparative examples 4 and 5 show that the two methods for identifying Alisma orientalis and Atractylodes macrocephala in the pharmacopoeia cannot simultaneously identify the two medicinal materials in Alisma orientalis decoction, or the identification effect is very poor, indicating that the main active ingredients of the two medicinal materials have different physical and chemical properties. Therefore, it requires a certain amount of creative labor to perform thin layer identification of the two medicinal materials on a thin layer plate at the same time. It also shows that the fast and simple thin layer identification method for simultaneously detecting two medicinal materials provided by the present invention presents a better technical effect.

[0187] The above is a preferred embodiment of the invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the principle of the invention. These improvements and modifications are also considered to be within the scope of protection of the invention.

Claims

1. A thin layer chromatography identification method for Zexie decoction, characterized in that: include: (1) Extract the oriental decoction with n-hexane, dry the extract and dissolve it with methanol to obtain a test solution; (2) Take 1-5 g of the control medicinal material of Alisma orientalis, add 30-80 mL of water, boil for 20-60 min, centrifuge and take the supernatant, extract the supernatant with n-hexane for 1-4 times, combine the extracts, evaporate to dryness in a water bath at 50-80 °C, add methanol to dissolve the residue, and use it as the control medicinal material solution of Alisma orientalis; take 0.5-2 g of the control medicinal material of Atractylodes macrocephala, add 30-80 mL of water, boil for 20-60 min, centrifuge and take the supernatant, extract the supernatant with n-hexane for 1-4 times, combine the extracts, evaporate to dryness in a water bath at 50-80 °C, add methanol to dissolve the residue, and use it as the control medicinal material solution of Atractylodes macrocephala; (3) Pipette the test sample solution, the control medicinal material solution of Alisma orientalis, and the control medicinal material solution of Atractylodes macrocephala onto a silica gel G thin layer plate, and develop it with a mixed solution of chloroform, acetone, and formic acid in a volume ratio of (9-10): (0.1-1): (0.01-0.2) as the developing agent. After development, take out, dry, spray with a color developer solution, heat at 100-110°C until the color is clear, and then examine under ultraviolet light; Wherein, step (1) comprises: (1.1) Take Zexie decoction and add water to dissolve it. The ratio of the weight of Zexie decoction to the volume of water added is (1-3) g: (20-50) mL. (1.2) The solution obtained by dissolving in step (1.1) is extracted 1 to 4 times with n-hexane, the extracts are combined, evaporated to dryness in a water bath at 50 to 80°C, and methanol is added to the residue to dissolve it as the test solution; wherein the ratio of the volume of n-hexane added for each extraction to the weight of the Zexie decoction is (15 to 30) mL: (1 to 3) g; the ratio of the volume of methanol used for dissolution to the weight of the Zexie decoction is (0.5 to 1.5) mL: (1 to 3) g.

2. A thin layer chromatography identification method for Zexie decoction, characterized in that: include: (1) Extract the oriental decoction with n-hexane, dry the extract and dissolve it with methanol to obtain a test solution; (2) Take 23-acetyl alismatol C reference substance, add methanol to make a solution with a concentration of 0.5~1 mg / mL, and obtain acetyl alismatol reference substance solution; take atractyl lactone II reference substance, add methanol to make a solution with a concentration of 0.5~1 mg / mL, and obtain atractyl lactone II reference substance solution; take atractyl lactone III reference substance, add methanol to make a solution with a concentration of 0.5~1 mg / mL, and obtain atractyl lactone III reference substance solution; (3) Pipette the test solution, 23-acetyl alismatol C reference solution, atractylodes lactone II reference solution, and atractylodes lactone III reference solution onto a silica gel G thin layer plate, and develop it with a mixed solution of chloroform, acetone, and formic acid in a volume ratio of (9-10): (0.1-1): (0.01-0.2) as the developing agent. After development, take out, dry, spray with a color developer solution, heat at 100-110°C until the color is clear, and then inspect under ultraviolet light; Wherein, step (1) comprises: (1.1) Take Zexie decoction and add water to dissolve it. The ratio of the weight of Zexie decoction to the volume of water added is (1-3) g: (20-50) mL. (1.2) The solution obtained by dissolving in step (1.1) is extracted 1 to 4 times with n-hexane, the extracts are combined, evaporated to dryness in a water bath at 50 to 80°C, and methanol is added to the residue to dissolve it as the test solution; wherein the ratio of the volume of n-hexane added for each extraction to the weight of the Zexie decoction is (15 to 30) mL: (1 to 3) g; the ratio of the volume of methanol used for dissolution to the weight of the Zexie decoction is (0.5 to 1.5) mL: (1 to 3) g.

3. A thin layer chromatography identification method for Zexie decoction, characterized in that: include: (1) Extract the oriental decoction with n-hexane, dry the extract and dissolve it with methanol to obtain a test solution; (2) Take 1-5 g of Rhizoma Alismatis and add 30-80 mL of water, boil for 20-60 min, centrifuge and take the supernatant, extract the supernatant 1-4 times with n-hexane, combine the extracts, evaporate to dryness in a water bath at 50-80 °C, add methanol to dissolve the residue, and use this as the Rhizoma Alismatis and reference medicinal solution; take 0.5-2 g of Rhizoma Atractylodis Macrocephala and add 30-80 mL of water, boil for 20-60 min, centrifuge and take the supernatant, extract the supernatant 1-4 times with n-hexane, combine the extracts, evaporate to dryness in a water bath at 50-80 °C, add methanol to dissolve the residue, and use this as the Rhizoma Alismatis and reference medicinal solution. Evaporate to dryness in a water bath at 80°C, add methanol to the residue to dissolve it, and use it as a reference medicinal material solution of Atractylodes macrocephala; take 23-acetyl alismatol C reference substance, add methanol to make a solution with a concentration of 0.5~1 mg / mL, and obtain acetyl alismatol reference substance solution; take atractylolactone II reference substance, add methanol to make a solution with a concentration of 0.5~1 mg / mL, and obtain atractylolactone II reference substance solution; take atractylolactone III reference substance, add methanol to make a solution with a concentration of 0.5~1 mg / mL, and obtain atractylolactone III reference substance solution; (3) Pipette the test solution, the control medicinal material solution of Alisma orientalis, the control medicinal material solution of Atractylodes macrocephala, the control medicinal material solution of 23-acetyl alismatol C, the control substance solution of Atractylodes macrocephala lactone II, and the control substance solution of Atractylodes macrocephala lactone III onto a silica gel G thin layer plate, and develop it with a mixed solution of chloroform, acetone, and formic acid in a volume ratio of (9-10): (0.1-1): (0.01-0.2) as a developing agent. After development, take out, dry, spray with a color developer solution, heat at 100-110°C until the color is clear, and then inspect under ultraviolet light; Wherein, step (1) comprises: (1.1) Take Zexie decoction and add water to dissolve it. The ratio of the weight of Zexie decoction to the volume of water added is (1-3) g: (20-50) mL. (1.2) The solution obtained by dissolving in step (1.1) is extracted 1 to 4 times with n-hexane, the extracts are combined, evaporated to dryness in a water bath at 50 to 80°C, and methanol is added to the residue to dissolve it as the test solution; wherein the ratio of the volume of n-hexane added for each extraction to the weight of the Zexie decoction is (15 to 30) mL: (1 to 3) g; the ratio of the volume of methanol used for dissolution to the weight of the Zexie decoction is (0.5 to 1.5) mL: (1 to 3) g.

4. The thin layer chromatography identification method of Zexie decoction according to any one of claims 1 to 3, characterized in that: In step (3), the volume ratio of chloroform, acetone and formic acid is 9.5:0.5:0.

06.

5. The thin layer chromatography identification method of Zexie decoction according to claim 4, characterized in that: The developer solution is a sulfuric acid ethanol solution, the volume concentration of sulfuric acid is 5-20%, and the wavelength of the ultraviolet light is 360-370nm.

6. The thin layer chromatography identification method of Zexie decoction according to any one of claims 1 to 3, characterized in that: The spread distance of the silica gel G thin layer plate is 8-12 cm.

7. The thin layer chromatography identification method of Zexie decoction according to claim 1, characterized in that: During heating, the heating time of the spot at a spread distance of 0 to 4 cm is ≤ 1 min, and the heating time of the spot at a spread distance of 4 to 8 cm is > 1 min.

8. The thin layer chromatography identification method of Zexie decoction according to claim 3, characterized in that: In step (3), the spotting volume of the test solution is 5-15 μL, the spotting volume of the Rhizoma Alismatis reference medicinal material solution is 5-20 μL, the spotting volume of the Rhizoma Atractylodis Macrocephala reference medicinal material solution is 5-15 μL, and the spotting volumes of the 23-acetyl Rhizoma Alismatis C reference solution, the Rhizoma Atractylodis Macrocephala lactone II reference solution, and the Rhizoma Atractylodis Macrocephala lactone III reference solution are all 1-3 μL.

9. The thin layer chromatography identification method of Zexie decoction according to any one of claims 1 to 3, characterized in that: The decoction is composed of the following raw materials in parts by weight: There are 18.65 parts of Alisma orientalis and 7.46 parts of Atractylodes macrocephala.

10. The thin layer chromatography identification method of Zexie decoction according to any one of claims 1 to 3, characterized in that: The preparation method of the rhizoma alismatis decoction is as follows: Take Alisma orientalis and Atractylodes macrocephala, soak them in water for 10-30 minutes, boil them over high heat for 20-35 minutes, and keep boiling over low heat until the amount of water used for soaking is reduced by half.

11. The thin layer chromatography identification method of Zexie decoction according to any one of claims 1 to 3, characterized in that: The preparation method of the rhizoma alismatis decoction is as follows: Take Alisma orientalis and Atractylodes macrocephala, soak them in water for 10-30 minutes, boil them over high heat for 20-35 minutes, and keep boiling over low heat until the amount of water used for soaking is reduced by half. Freeze-dry the resulting decoction.

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