A method for identifying different Allium macrostemon origins and genuine and counterfeit products in Allium macrostemon medicinal materials, including formulated granules, and its application.

Abstract

This invention provides a method for identifying different Allium macrostemon origins and genuine and counterfeit Allium macrostemon products, including formulated granules, and its application. The identification method includes the following steps: (1) mixing the sample to be tested with a solvent to extract and obtain a test solution; (2) mixing a reference herb with a solvent to extract and obtain a reference herb solution; (3) performing thin-layer chromatography on the test solution and the reference herb solution, and determining the origin and / or authenticity of the sample to be tested based on the detection results; the Allium macrostemon origins include Allium macrostemon (garlic root) and Allium macrostemon (Allium chinense). The identification method provided by this invention can quickly and effectively identify Allium macrostemon (garlic root) and Allium macrostemon (Allium chinense), as well as the adulterants Allium macrostemon such as Scutellaria baicalensis and Ziziphus jujuba. The detection results are accurate, and the reagents used have low toxicity and high safety, which can minimize the possibility of the experimenter being exposed to toxic reagents.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method for identifying different Allium macrostemon origins and genuine and counterfeit products in Allium macrostemon drugs, including formulated granules, and its application. Background Technology

[0002] The 2020 edition of the Chinese Pharmacopoeia records two origins of Allium macrostemon (also known as Allium macrostemon Bge.) and Allium chinense (G.Don), both belonging to the Liliaceae family. Allium macrostemon is pungent, bitter, and warm in nature. It enters the heart, lung, stomach, and large intestine meridians. Its functions are to promote yang and dissipate stagnation, regulate qi and relieve stagnation. It is used for chest pain, abdominal distension and pain, and tenesmus after diarrhea. The Chinese Pharmacopoeia only includes two origins for medicinal use. The descriptions of the medicinal materials—Allium macrostemon being oval and Allium chinense being oblong—can distinguish between the two. However, after being prepared into granules, the original characteristics of the medicinal materials are lost, making differentiation impossible. Furthermore, adulteration of Chinese medicinal materials currently exists, leading to a decline in quality and reduced or altered efficacy. Therefore, establishing a method for identifying genuine and counterfeit Allium macrostemon granules from different origins is particularly important.

[0003] CN113759066B discloses a thin-layer chromatography method for identifying Allium macrostemon (Xiebai) as a test sample, comprising: taking Allium macrostemon (Xiebai) medicinal material, water extract, or formulation granules, and preparing a test sample solution; spotting the test sample solution onto a silica gel G thin-layer plate, using petroleum ether-ethyl acetate-dichloromethane-formic acid in a volume ratio of (5-6):(3.5-4):(1-1.2):(0.8-1) as the developing solvent, developing, removing, drying, and examining under ultraviolet light. This invention uses two reagents, petroleum ether and dichloromethane, both of which are highly toxic and harmful to human health, and does not involve the identification of different origins.

[0004] CN119023873A discloses a thin-layer chromatography method for identifying Allium macrostemon (Xiebai) and distinguishing it from adulterants. The method involves preparing a test solution and a reference herb solution; spotting these solutions separately onto the same thin-layer plate, developing, removing, drying, spraying with sulfuric acid ethanol solution, and heating until the spots are clearly visible. The samples are then examined under sunlight and ultraviolet light. If the test sample chromatogram shows a main spot of the same color or a fluorescent main spot at the corresponding position as the reference herb chromatogram, then the test sample is Allium macrostemon (Xiebai). This invention does not address the identification of different Allium macrostemon (Xiebai) origins.

[0005] Therefore, how to provide a method that can effectively identify different Allium macrostemon origins and distinguish genuine Allium macrostemon from counterfeit products has become an urgent problem to be solved. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a method and its application for identifying different Allium macrostemon origins and genuine and counterfeit Allium macrostemon products, including formulated granules. The identification method provided by this invention can quickly and effectively distinguish between Allium macrostemon (small root garlic) and Allium macrostemon (Allium), as well as adulterants such as Scutellaria baicalensis and Ziziphus jujuba. The detection results are accurate, and the reagents used are low in toxicity and highly safe, minimizing the risk of exposure to toxic reagents for the experimenter.

[0007] To achieve this objective, the present invention employs the following technical solution:

[0008] On the one hand, the present invention provides a method for identifying different Allium macrostemon origins and genuine and counterfeit products in Allium macrostemon medicinal materials, including formulated granules. The identification method includes the following steps:

[0009] (1) Mix the sample to be tested with a solvent to extract the sample and obtain the test solution;

[0010] (2) The reference medicinal material was mixed with a solvent and extracted to obtain a reference medicinal material solution;

[0011] (3) Perform thin-layer chromatography on the test solution and the reference medicinal material solution, and determine the origin and / or authenticity of the sample based on the test results.

[0012] Steps (1) and (2) are not in any particular order.

[0013] The basal plant origin includes Allium macrostemon (garlic root) and Allium macrostemon (Allium chinense).

[0014] The above method can quickly and effectively distinguish between Allium macrostemon (small garlic) and Allium macrostemon (Allium), as well as the adulterants Allium macrostemon such as Shallotum and Ziziphus jujuba. The test results are accurate, and the reagents used have low toxicity and high safety, which can minimize the experimenter's exposure to toxic reagents. It also avoids the situation in the preparation of test samples by acid hydrolysis that destroys glycoside components in the existing technology, thus effectively improving the identification ability.

[0015] Preferably, the Allium macrostemon medicine includes Allium macrostemon raw material, Allium macrostemon formula granules, Allium macrostemon standard decoction, Allium macrostemon slices, or a traditional Chinese medicine compound preparation containing Allium macrostemon slices.

[0016] Preferably, the solvent in step (1) is water-saturated n-butanol.

[0017] Preferably, the extraction in step (1) is performed 1-3 times.

[0018] Preferably, the ratio of the sample to the solvent in step (1) is 1:(15-25)g / mL, such as 1:15g / mL, 1:16g / mL, 1:17g / mL, 1:18g / mL, 1:19g / mL, 1:20g / mL, 1:21g / mL, 1:22g / mL, 1:23g / mL, 1:24g / mL or 1:25g / mL, but not limited to the values ​​listed above. Other values ​​not listed within the above range are also applicable.

[0019] Preferably, the thin-layer chromatography in step (3) is performed using a silica gel G thin-layer plate.

[0020] Preferably, the developing solvent for the thin-layer chromatography in step (3) is the upper layer solution of a mixed solvent of ethyl acetate, n-butanol, and water.

[0021] Preferably, the volume ratio of ethyl acetate, n-butanol, and water is (2-6):(5-9):(2-7), wherein the number of parts of ethyl acetate can be 2, 3, 4, 5, or 6, the number of parts of n-butanol can be 5, 6, 7, 8, or 9, and the number of parts of water can be 2, 3, 4, 5, 6, or 7, but is not limited to the values ​​listed above. Other unlisted values ​​within the above range are also applicable.

[0022] The specific developing solvents and their ratios mentioned above can effectively improve the separation effect of each component in thin-layer chromatography and improve the accuracy of detection. This allows the overall method to quickly and effectively identify Allium macrostemon (small garlic) and Allium macrostemon (Allium), as well as the adulterants Allium macrostemon, Shallotum and Ziziphus jujuba. At the same time, the reagents have low toxicity and high safety, effectively reducing the possibility of experimenters coming into contact with toxic reagents.

[0023] On the other hand, the present invention also provides the application of the identification method described above in the quality control of Allium macrostemon.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] This invention provides a method for identifying different Allium macrostemon origins and genuine and counterfeit Allium macrostemon products, including formulated granules. This method can quickly and effectively identify Allium macrostemon (small root garlic) and Allium macrostemon (Allium), as well as the adulterants Allium macrostemon, such as Scutellaria baicalensis and Ziziphus jujuba. The test results are accurate, and the reagents used are low in toxicity and highly safe, minimizing the risk of experimenters coming into contact with toxic reagents. It also avoids the situation in existing technologies where acid hydrolysis destroys glycoside components during sample preparation, effectively improving the identification capability. Attached Figure Description

[0026] Figure 1 These are thin-layer chromatograms of different original Allium macrostemon and adulterants in Example 1;

[0027] Figure 2These are thin-layer chromatograms of different original Allium macrostemon and its adulterants in Example 2;

[0028] Figure 3 These are thin-layer chromatograms of different original Allium macrostemon and adulterants in Example 3;

[0029] Figure 4 These are thin-layer chromatograms of different original Allium macrostemon and its adulterants in Example 4;

[0030] Figure 5 These are thin-layer chromatograms of different original Allium macrostemon and its adulterants in Example 5;

[0031] Figure 6 These are thin-layer chromatograms of different original Allium macrostemon and its adulterants in Example 6;

[0032] Figure 7 These are thin-layer chromatograms of different original Allium macrostemon and adulterants in Example 7;

[0033] Figure 8 These are thin-layer chromatograms of different original Allium macrostemon and its adulterants in Example 8;

[0034] Figure 9 These are thin-layer chromatograms of different original Allium macrostemon and its adulterants in Example 9;

[0035] Figure 10 These are thin-layer chromatograms of different original Allium macrostemon and adulterants in Example 10;

[0036] Figure 11 This is a thin-layer chromatogram of different original Allium macrostemon and its adulterants in Comparative Example 1. Detailed Implementation

[0037] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0038] In the following examples, the instruments and reagents used are as follows:

[0039] Instruments: CAMAG thin-layer imaging system, ME104E electronic balance (Mettler Toledo), JY20002 electronic balance (Mettler Toledo), BSA124S electronic balance (Sartorius Instruments (Beijing) Co., Ltd.), BT25S electronic balance (Sartorius Instruments (Beijing) Co., Ltd.), DZKW-4 constant temperature water bath (Beijing Zhongxing Weiye Instrument Co., Ltd.), KQ-500DB ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), silicone G plate (Merck KgaA, Yinlong Chemical Plant, Jiangyou-Yantai Chemical Industry Research Institute).

[0040] Drug trials:

[0041] Allium macrostemon (small garlic) reference medicinal material (batch number: 121130-202108, China National Institutes for Food and Drug Control);

[0042] Allium macrostemon (small garlic) formula granules (202301, 202302, Beijing Kangrentang Pharmaceutical Co., Ltd.);

[0043] Allium macrostemon (Allium tuberosum) formula granules (202304, 202305, Beijing Kangrentang Pharmaceutical Co., Ltd.);

[0044] Water onion formula granules (202307, Beijing Kangrentang Pharmaceutical Co., Ltd.);

[0045] Mianzaoer Formula Granules (202308, Beijing Kangrentang Pharmaceutical Co., Ltd.);

[0046] Reagents: n-Butanol, ethyl acetate, and distilled water were all of analytical grade.

[0047] Example 1

[0048] This embodiment provides a method for identifying different Allium macrostemon origins and genuine and counterfeit products in Allium macrostemon medicinal materials. The specific steps are as follows:

[0049] Take appropriate amounts of *Allium macrostemon* (small-root garlic) formula granules, *Allium macrostemon* (scallion) formula granules, *Salvia miltiorrhiza* formula granules, and *Ziziphus jujuba* formula granules, grind them into a fine powder, take 1g, add 10mL of water to dissolve, and extract twice with water-saturated n-butanol, 20mL each time. Combine the n-butanol extracts, evaporate to dryness, and dissolve the residue in 5mL of methanol to obtain the test solution. Separately, take 2g of *Allium macrostemon* (small-root garlic) reference material, add 50mL of water, decoct for 30 minutes, filter, concentrate the filtrate to 10mL, and extract twice with water-saturated n-butanol, 20mL each time. Combine the n-butanol extracts, evaporate to dryness, and dissolve the residue in 5mL of methanol to obtain the reference material solution. The thin-layer chromatography method (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition) was followed. 5 μL of each of the two solutions were spotted separately onto the same silica gel G thin-layer plate, forming bands. The upper layer of ethyl acetate-n-butanol-water (4:7:5) was used as the developing solvent. After development, the plate was removed, dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105℃ until the spots were clearly visible. The results were then examined under ultraviolet light (365 nm). See below for details. Figure 1 (1-2: Allium macrostemon (small root garlic) formula granules, 3: Allium macrostemon (small root garlic) reference medicinal material, 4-5: Allium macrostemon (Allium macrostemon) formula granules, 6: Shallot formula granules, 7: Mianzaoer formula granules).

[0050] The images show significant differences between the *Allium chinense* and *Allium macrostemon* basidiomyces ...

[0051] Example 2

[0052] This embodiment provides a method for identifying different Allium macrostemon origins and genuine and counterfeit Allium macrostemon products. The specific steps are the same as in Example 1, except that the volume ratio of ethyl acetate-n-butanol-water is 2:5:2.

[0053] The results are as follows Figure 2 As shown (1-2: Allium macrostemon (small root garlic) formula granules, 3: Allium macrostemon (small root garlic) reference medicinal material, 4-5: Allium macrostemon (Allium macrostemon) formula granules, 6: Shallot formula granules, 7: Mulberry formula granules).

[0054] Example 3

[0055] This embodiment provides a method for identifying different Allium macrostemon origins and genuine and counterfeit products in Allium macrostemon. The specific steps are the same as in Example 1, except that the volume ratio of ethyl acetate-n-butanol-water is 6:9:7.

[0056] The results are as follows Figure 3 As shown (1-2: Allium macrostemon (small root garlic) formula granules, 3: Allium macrostemon (small root garlic) reference medicinal material, 4-5: Allium macrostemon (Allium macrostemon) formula granules, 6: Shallot formula granules, 7: Mulberry formula granules).

[0057] Example 4

[0058] This embodiment provides a method for identifying different Allium macrostemon origins and genuine and counterfeit Allium macrostemon products in Allium macrostemon medicine. The specific steps are the same as in Example 1, except that the volume ratio of ethyl acetate-n-butanol-water is 3:6:4.

[0059] The results are as follows Figure 4 As shown (1-2: Allium macrostemon (small root garlic) formula granules, 3: Allium macrostemon (small root garlic) reference medicinal material, 4-5: Allium macrostemon (Allium macrostemon) formula granules, 6: Shallot formula granules, 7: Mulberry formula granules).

[0060] Example 5

[0061] This embodiment provides a method for identifying different Allium macrostemon origins and genuine and counterfeit Allium macrostemon products. The specific steps are the same as in Example 1, except that the volume ratio of ethyl acetate-n-butanol-water is 6:8:7.

[0062] The results are as follows Figure 5As shown (1-2: Allium macrostemon (small root garlic) formula granules, 3: Allium macrostemon (small root garlic) reference medicinal material, 4-5: Allium macrostemon (Allium macrostemon) formula granules, 6: Shallot formula granules, 7: Mulberry formula granules).

[0063] Figure 2-5 The results showed that by adjusting the ratio of the developing solvent, the spots in the resulting chromatograms were clearer and better separated, and all of them could distinguish different origins and adulterants of Allium macrostemon.

[0064] Example 6

[0065] This embodiment provides a method for identifying different Allium macrostemon origins and genuine and counterfeit products in Allium macrostemon. The specific steps are the same as in Example 1, except that the developing solvent is the upper layer solution of n-hexane:n-butanol:water (4:7:5).

[0066] The results are as follows Figure 6 As shown (1-2: Allium macrostemon (small root garlic) formula granules, 3: Allium macrostemon (small root garlic) reference medicinal material, 4-5: Allium macrostemon (Allium macrostemon) formula granules, 6: Shallot formula granules, 7: Mulberry formula granules).

[0067] Example 7

[0068] This embodiment provides a method for identifying different Allium macrostemon origins and genuine and counterfeit products in Allium macrostemon. The specific steps are the same as in Example 1, except that the developing solvent is ethyl acetate:methanol:water (4:7:5).

[0069] The results are as follows Figure 7 As shown (1-2: Allium macrostemon (small root garlic) formula granules, 3: Allium macrostemon (small root garlic) reference medicinal material, 4-5: Allium macrostemon (Allium macrostemon) formula granules, 6: Shallot formula granules, 7: Mulberry formula granules).

[0070] Example 8

[0071] This embodiment provides a method for identifying different Allium macrostemon origins and genuine and counterfeit Allium macrostemon products. The specific steps are the same as in Example 1, except that the developing solvent is dichloromethane:methanol (4:7).

[0072] The results are as follows Figure 8 As shown (1-2: Allium macrostemon (small root garlic) formula granules, 3: Allium macrostemon (small root garlic) reference medicinal material, 4-5: Allium macrostemon (Allium macrostemon) formula granules, 6: Shallot formula granules, 7: Mulberry formula granules).

[0073] Example 9

[0074] This embodiment provides a method for identifying different Allium macrostemon origins and genuine and counterfeit Allium macrostemon products in Allium macrostemon medicine. The specific steps are the same as in Example 1, except that the developing solvent is ethyl acetate: n-butanol (4:7).

[0075] The results are as follows Figure 9 As shown (1-2: Allium macrostemon (small root garlic) formula granules, 3: Allium macrostemon (small root garlic) reference medicinal material, 4-5: Allium macrostemon (Allium macrostemon) formula granules, 6: Shallot formula granules, 7: Mulberry formula granules).

[0076] Example 10

[0077] This embodiment provides a method for identifying different Allium macrostemon origins and genuine and counterfeit Allium macrostemon products in Allium macrostemon medicine. The specific steps are the same as in Example 1, except that the developing solvent is the upper layer solution of ethyl acetate:water (7:5).

[0078] The results are as follows Figure 10 As shown (1-2: Allium macrostemon (small root garlic) formula granules, 3: Allium macrostemon (small root garlic) reference medicinal material, 4-5: Allium macrostemon (Allium macrostemon) formula granules, 6: Shallot formula granules, 7: Mulberry formula granules).

[0079] Figure 6-10 The results showed that using other developing solvent combinations resulted in poor separation of the chromatograms, with unclear spots or stripes after development; the basic requirements for thin-layer identification could not be met.

[0080] Comparative Example 1

[0081] This comparative example provides a method for identifying different Allium macrostemon origins and genuine and counterfeit products in Allium macrostemon medicinal materials. For specific steps, please refer to CN202110735282.X:

[0082] Take an appropriate amount (approximately 0.5g) of the following herbal formulas: Allium macrostemon (small-rooted garlic), Allium macrostemon (scallion), Shallotum spp., and Ziziphus jujuba var. spinosa. Grind them into a fine powder, add 30mL of water and 3mL of hydrochloric acid, heat under reflux for 1 hour, cool, and extract twice with 30mL of ethyl acetate each time. Combine the ethyl acetate extracts and concentrate to 5mL to obtain the test solution. Separately, take 1.5g of Allium macrostemon (small-rooted garlic) reference material, add 50mL of water and 3mL of hydrochloric acid, heat under reflux for 1 hour, cool, and prepare the reference material solution using the same method. Perform the thin-layer chromatography test (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition). Apply 2 μL of the above test solution and 1 μL of the reference herb solution separately to the same silica gel G thin-layer plate. Use petroleum ether (60–90℃)-ethyl acetate-dichloromethane-formic acid (5:3.5:1:0.8) as the developing solvent. Develop, remove, air-dry, and examine under ultraviolet light (365 nm). See [link to relevant documentation]. Figure 11 (1-2: Allium macrostemon (small root garlic) formula granules, 3: Allium macrostemon (small root garlic) reference medicinal material, 4-5: Allium macrostemon (Allium macrostemon) formula granules, 6: Shallot formula granules, 7: Mianzaoer formula granules).

[0083] As can be seen from the results in the figure, whether it is Allium macrostemon of different origins or adulterants of Allium macrostemon, the glycoside components are destroyed after hydrolysis with hydrochloric acid. The only difference shown is the slight difference in the depth of the spot color, but the basic spot information is the same. This indicates that this method cannot be used to identify the origin and adulterants.

[0084] The results above show that the identification method provided by the present invention can effectively improve the separation effect of each component in thin-layer chromatography and improve the accuracy of detection. The overall method can quickly and effectively identify Allium macrostemon (small garlic) and Allium macrostemon (Allium), as well as the adulterants Allium macrostemon, Shallotum and Ziziphus jujuba. Comparing Examples 1-7, it can be found that the present invention can further improve the accuracy of detection by selecting a specific combination of developing solvents, and realize the identification of different Allium macrostemon origins and genuine and counterfeit products.

[0085] The applicant declares that this invention illustrates, through the above embodiments, the method for identifying different Allium macrostemon origins and genuine and counterfeit products in Allium macrostemon medicines, including formulated granules, and its application. However, this invention is not limited to the above embodiments, meaning that this invention does not necessarily rely on the above embodiments for implementation. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials in the product of this invention, addition of auxiliary components, and selection of specific methods, all fall within the protection and disclosure scope of this invention.

[0086] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0087] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A method for identifying different Allium macrostemon origins in Allium macrostemon medicinal materials, characterized in that, The identification method includes the following steps: (1) Mix the sample to be tested with water, then extract with water-saturated n-butanol, evaporate the n-butanol solution to dryness, dissolve the residue in methanol to obtain the test solution; (2) The reference medicinal material was mixed with a solvent and extracted to obtain a reference medicinal material solution; (3) Perform thin-layer chromatography on the test solution and the reference medicinal material solution, and determine the origin of the sample to be tested based on the detection results; Steps (1) and (2) are not in any particular order; The allium macrostemon gene includes garlic root and allium macrostemon; The Allium macrostemon medicine refers to Allium macrostemon raw material, Allium macrostemon formula granules, Allium macrostemon standard decoction, or Allium macrostemon slices; The thin-layer chromatography described in step (3) is performed using silica gel G thin-layer plates; The developing solvent for the thin-layer chromatography in step (3) is the upper layer solution of a mixed solvent of ethyl acetate, n-butanol, and water; The volume ratio of ethyl acetate, n-butanol, and water is (2-6):(5-9):(2-7).

2. The identification method according to claim 1, characterized in that, The number of extractions in step (1) is 1-3 times.

3. The identification method according to claim 1, characterized in that, In step (1), the ratio of the sample to the solvent is 1:(15-25) g / mL.

4. The application of an identification method according to any one of claims 1-3 in the quality control of Allium macrostemon.

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