Method for identifying different allium macrostemon bases and true and false products in allium macrostemon medicine including formula granules and application of different allium macrostemon bases and true and false products in allium macrostemon medicine

Abstract

The invention provides a method for identifying different allium macrostemon bases and true and false products in allium macrostemon medicines including formula granules and application thereof, and the method comprises the following steps: (1) mixing a sample to be detected with a solvent, and extracting to obtain a test solution; (2) mixing the reference medicinal material with a solvent, and extracting to obtain a reference medicinal material solution; and (3) carrying out thin-layer chromatography on the test solution and the reference medicinal material solution, and judging the primitive and / or authenticity of the sample to be detected according to the detection result, the allium macrostemon base source comprises allium macrostemon (small garlic roots) and allium macrostemon (allium macrostemon). According to the identification method provided by the invention, allium macrostemon (small root garlic), allium macrostemon (allium macrostemon) and allium macrostemon adulterants, namely scirpus tabernaemontani and Chinese squill, can be rapidly and effectively identified, the detection result is accurate, the used reagent is low in toxicity and high in safety, and the condition that an experimenter is in contact with the toxic reagent can be reduced to the greatest extent.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a method for identifying different Xiebai origins and genuine Xiebai drugs including formula granules, and an application thereof. Background Art

[0002] The 2020 edition of the Chinese Pharmacopoeia records that Xiebai has two origins, namely the dried bulbs of Allium macrostemon Bge. and Allium chinense G.Don, plants of the Liliaceae family. Xiebai is pungent, bitter and warm. It enters the heart, lung, stomach and large intestine meridians. Its effects are to promote yang and dissipate stagnation, and to promote qi and relieve stagnation. It is used for chest pain, abdominal distension and pain, and heaviness after diarrhea. The Chinese Pharmacopoeia only includes two origins for medicinal use. Through the description of the properties of the medicinal materials, it can be seen that Allium macrostemon is oval and Xie is long oval. This description can distinguish the two medicinal materials, but after being prepared into formula granules, the original properties of the medicinal materials are lost and the two can no longer be distinguished; and there is currently adulteration of Chinese medicinal materials, which leads to a decline in the quality of the medicinal materials; the efficacy is reduced or changed, so it is particularly important to establish a method for identifying Xiebai formula granules with different origins and their authenticity.

[0003] CN113759066B discloses a thin layer identification method for a test sample of Xiebai, comprising: taking Xiebai medicinal material, water extract or formula granules to prepare a test sample solution; spotting the test sample solution on a silica gel G thin layer plate, using petroleum ether-ethyl acetate-dichloromethane-formic acid with a volume ratio of (5-6): (3.5-4): (1-1.2): (0.8-1) as a developing agent, developing, taking out, drying, and inspecting under ultraviolet light. The inventive method uses two reagents, petroleum ether and dichloromethane, which are highly toxic and harmful to the human body, and does not involve the identification of different origins.

[0004] CN119023873A discloses a thin layer identification method of Xiebai, a method for distinguishing Xiebai from counterfeit products, preparing a test solution and a control medicinal material solution; spotting on the same thin layer plate, unfolding, taking out, drying, spraying with sulfuric acid ethanol solution, heating until the spots are clearly colored, and inspecting under sunlight and ultraviolet light respectively; when the main spot or fluorescent main spot of the same color appears in the chromatogram of the test product at the corresponding position of the chromatogram of the control medicinal material, the test product to be tested is Xiebai. The invention also does not involve the identification of different Xiebai radicals.

[0005] Therefore, how to provide a method that can effectively identify different Xiebai origins and at the same time identify the authenticity of Xiebai has become an urgent problem to be solved. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention aims to provide a method for identifying different Xiebai origins and authenticity of Xiebai medicines including formula granules and its application. The identification method provided by the present invention can quickly and effectively identify Xiebai (small root garlic) and Xiebai (xiebai) as well as Xiebai mixed with counterfeit products such as water onion and jujube, and the detection results are accurate. The reagents used are low in toxicity and high in safety, and can minimize the exposure of experimenters to toxic reagents.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In one aspect, the present invention provides a method for identifying different Xiebai origins and authenticity of Xiebai medicines including formula granules, the identification method comprising the following steps:

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

[0010] (2) extracting the control medicinal material by mixing it with a solvent to obtain a control medicinal material solution;

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

[0012] There is no order distinction between steps (1) and (2).

[0013] The Xiebai origin includes Xiebai (small garlic root) and Xiebai (xie).

[0014] The above method can quickly and effectively identify Xiebai (small root garlic) and Xiebai (xie), as well as Xiebai mixed with counterfeit products water onion and schizonepeta, and the detection results are accurate. The reagents used are low in toxicity and high in safety, which can minimize the exposure of experimenters to toxic reagents; it avoids the situation in which acid hydrolysis in the preparation of test samples in the prior art destroys glycoside components, and effectively improves the identification ability.

[0015] Preferably, the Xiebai medicine includes Xiebai medicinal materials, Xiebai formula granules, Xiebai standard decoction, Xiebai decoction pieces or a traditional Chinese medicine compound preparation containing Xiebai decoction pieces.

[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 solid-liquid ratio of the sample to be tested and the solvent in step (1) is 1:(15-25) g / mL, for example 1:15 g / mL, 1:16 g / mL, 1:17 g / mL, 1:18 g / mL, 1:19 g / mL, 1:20 g / mL, 1:21 g / mL, 1:22 g / mL, 1:23 g / mL, 1:24 g / mL or 1:25 g / mL, but is not limited to the values ​​listed above, and other values ​​not listed within the above numerical range are equally 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 an 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 are not limited to the values ​​listed above, and other values ​​not listed within the above numerical range are also applicable.

[0022] The specific developing agent and its ratio can effectively improve the separation effect of each component of thin layer chromatography and improve the accuracy of detection, so that the overall method can quickly and effectively identify Xiebai (small root garlic) and Xiebai (xie) as well as Xiebai mixed with counterfeit products water onion and jujube. At the same time, the reagent has low toxicity and high safety, which effectively reduces the exposure of experimenters to toxic reagents.

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

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

[0025] The invention provides a method for identifying different Xiebai origins and genuine and fake Xiebai drugs including formula granules. The method can quickly and effectively identify Xiebai (small root garlic) and Xiebai (xiebai) as well as Xiebai mixed fakes such as water scallion and Scilla scoparia. The detection result is accurate, and the reagents used have low toxicity and high safety, which can minimize the situation that experimenters are exposed to toxic reagents; the situation that glycoside components are destroyed by acid hydrolysis in the preparation of test samples in the prior art is avoided, and the identification ability is effectively improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the result of thin layer chromatography of different origin Xiebai and mixed products in Example 1;

[0027] Figure 2It is the result of thin layer chromatography of different origin Xiebai and mixed products in Example 2;

[0028] Figure 3 It is the result of thin layer chromatography of different origin Xiebai and mixed counterfeit products in Example 3;

[0029] Figure 4 It is the result of thin layer chromatography of different origin Xiebai and mixed counterfeit products in Example 4;

[0030] Figure 5 It is the result of thin layer chromatography of different origin Xiebai and mixed counterfeit products in Example 5;

[0031] Figure 6 It is the result of thin layer chromatography of different origin Xiebai and mixed counterfeit products in Example 6;

[0032] Figure 7 It is the result of thin layer chromatography of different origin Xiebai and mixed counterfeit products in Example 7;

[0033] Figure 8 It is the result of thin layer chromatography of different origin Xiebai and mixed counterfeit products in Example 8;

[0034] Fig. 9 It is the result of thin layer chromatography of different origin Xiebai and mixed counterfeit products in Example 9;

[0035] Fig.10 1 is a graph showing the results of thin layer chromatography of different origin Xiebai and mixed products in Example 10;

[0036] Fig.11 This is a graph showing the thin layer chromatography results of Xiebai with different origins and mixed products in Comparative Example 1. DETAILED DESCRIPTION

[0037] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[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 Scientific Instrument (Beijing) Co., Ltd.), BT25S electronic balance (Sartorius Scientific Instrument (Beijing) Co., Ltd.), DZKW-4 constant temperature water bath (Beijing Zhongxing Weiye Instrument Co., Ltd.), KQ-500DB ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), silica gel G plate (Merck KgaA, Yinlong Chemical Plant, Jiangyou-Yantai Chemical Industry Research Institute).

[0040] Drug testing:

[0041] Xiebai (small root garlic) control medicinal material (batch number: 121130-202108, China Food and Drug Inspection Institute);

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

[0043] Xiebai (Xiebai) 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 Xiebai origins and authenticity of Xiebai medicines, and the specific steps are as follows:

[0049] Take appropriate amount of Xiebai (small root garlic) formula granules, Xiebai (xie) formula granules, water onion formula granules and schizonepeta formula granules, grind to take 1g, add 10mL of water to dissolve, shake and extract twice with water-saturated n-butanol, 20mL each time, combine the n-butanol solution, evaporate to dryness, add 5mL of methanol to the residue and dissolve it as the test solution. Take another 2g of Xiebai (small root garlic) control medicinal material, add 50mL of water, decoct for 30 minutes, filter, concentrate the filtrate to 10mL, shake and extract twice with water-saturated n-butanol, 20mL each time, combine the n-butanol solution, evaporate to dryness, add 5mL of methanol to the residue and dissolve it as the control medicinal material solution. According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), 5 μL of each of the above two solutions were taken and spotted on the same silica gel G thin layer plate to form strips. The upper layer solution of ethyl acetate-n-butanol-water (4:7:5) was used as the developing agent. The plate was developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and examined under ultraviolet light (365nm). The results are shown in Figure 1 (1-2: Xiebai (small root garlic) formula granules, 3: Xiebai (small root garlic) control medicinal material, 4-5: Xiebai (xie) formula granules, 6: Water onion formula granules, 7: Ziziphus scoparia formula granules).

[0050] It can be found from the figure that the difference between the Allium sibiricum and Xiebai sibiricum is obvious in the identification diagram, and the spots of the Allium sibiricum sample and Xiebai (Allium sibiricum) are consistent, with good correspondence. The two counterfeit products, Zaoer and Shuishang, are obviously different from Xiebai (Allium sibiricum) in the identification diagram, with fewer spots and inconsistent correspondence; therefore, Xiebai of different origins and Xiebai's easily confused counterfeit products can be identified through thin layer identification.

[0051] Example 2

[0052] This embodiment provides a method for identifying different Xiebai radicals and authenticity in Xiebai medicines. The specific steps are consistent with those 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: Xiebai (small root garlic) formula granules, 3: Xiebai (small root garlic) control medicinal material, 4-5: Xiebai (xie) formula granules, 6: Water onion formula granules, 7: Ziziphus chinensis formula granules).

[0054] Example 3

[0055] This embodiment provides a method for identifying different Xiebai radicals and authenticity in Xiebai medicines. The specific steps are consistent with those 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: Xiebai (small root garlic) formula granules, 3: Xiebai (small root garlic) control medicinal material, 4-5: Xiebai (xie) formula granules, 6: Water onion formula granules, 7: Ziziphus chinensis formula granules).

[0057] Example 4

[0058] This embodiment provides a method for identifying different Xiebai radicals and authenticity in Xiebai medicines. The specific steps are consistent with those 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: Xiebai (small root garlic) formula granules, 3: Xiebai (small root garlic) control medicinal material, 4-5: Xiebai (xie) formula granules, 6: Water onion formula granules, 7: Ziziphus chinensis formula granules).

[0060] Example 5

[0061] This embodiment provides a method for identifying different Xiebai radicals and authenticity in Xiebai medicines. The specific steps are consistent with those 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: Xiebai (small root garlic) formula granules, 3: Xiebai (small root garlic) control medicinal material, 4-5: Xiebai (xie) formula granules, 6: Water onion formula granules, 7: Ziziphus chinensis formula granules).

[0063] Figure 2-5 The results showed that the proportion of the developing agent was adjusted on the basis of the original developing agent. The chromatogram obtained by the adjusted developing agent had clear spots and better separation, and could distinguish different origins of Allium macrostemon and mixed products.

[0064] Example 6

[0065] This embodiment provides a method for identifying different Xiebai radicals and authenticity in Xiebai medicines. The specific steps are consistent with those in Example 1 except that the developing agent is an upper layer solution of n-hexane: n-butanol: water (4:7:5).

[0066] The results are as follows Figure 6 As shown (1-2: Xiebai (small root garlic) formula granules, 3: Xiebai (small root garlic) control medicinal material, 4-5: Xiebai (xie) formula granules, 6: Water onion formula granules, 7: Ziziphus chinensis formula granules).

[0067] Example 7

[0068] This embodiment provides a method for identifying different Xiebai radicals and authenticity in Xiebai medicines. The specific steps are consistent with those 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: Xiebai (small root garlic) formula granules, 3: Xiebai (small root garlic) control medicinal material, 4-5: Xiebai (xie) formula granules, 6: Water onion formula granules, 7: Ziziphus chinensis formula granules).

[0070] Example 8

[0071] This embodiment provides a method for identifying different Xiebai radicals and authenticity in Xiebai medicines. The specific steps are consistent with those 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: Xiebai (small root garlic) formula granules, 3: Xiebai (small root garlic) control medicinal material, 4-5: Xiebai (xie) formula granules, 6: Water onion formula granules, 7: Ziziphus chinensis formula granules).

[0073] Example 9

[0074] This embodiment provides a method for identifying different Xiebai radicals and authenticity in Xiebai medicines. The specific steps are consistent with those in Example 1 except that the developing solvent is ethyl acetate: n-butanol (4:7).

[0075] The results are as follows Fig. 9 As shown (1-2: Xiebai (small root garlic) formula granules, 3: Xiebai (small root garlic) control medicinal material, 4-5: Xiebai (xie) formula granules, 6: Water onion formula granules, 7: Ziziphus chinensis formula granules).

[0076] Example 10

[0077] This embodiment provides a method for identifying different Xiebai radicals and authenticity in Xiebai medicines. The specific steps are consistent with those in Example 1 except that the developing agent is an upper layer solution of ethyl acetate: water (7:5).

[0078] The results are as follows Fig.10 As shown (1-2: Xiebai (small root garlic) formula granules, 3: Xiebai (small root garlic) control medicinal material, 4-5: Xiebai (xie) formula granules, 6: Water onion formula granules, 7: Ziziphus chinensis formula granules).

[0079] Figure 6-10 The results showed that the separation effect of the obtained spectra was poor when other developing agent combinations were used for development, the spots were not clear or they became strips after development; the basic requirements of thin layer identification could not be met.

[0080] Comparative Example 1

[0081] This comparative example provides a method for identifying different Xiebai origins and authenticity of Xiebai medicines. For specific steps, refer to CN202110735282.X:

[0082] Take appropriate amount of Xiebai (small root garlic) formula granules, Xiebai (xie) formula granules, water onion formula granules and schizonepeta formula granules, take about 0.5g, grind, add 30mL of water, 3mL of hydrochloric acid, heat and reflux for 1 hour, cool, shake and extract with ethyl acetate twice, 30mL each time, combine the ethyl acetate solution, concentrate to 5mL, as the test solution. Take another 1.5g of Xiebai (small root garlic) control medicinal material, add 50mL of water, 3mL of hydrochloric acid, heat and reflux for 1 hour, cool, and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), 2 μL of the test solution and 1 μL of the control medicinal material solution were taken and spotted on the same silica gel G thin layer plate, and petroleum ether (60-90°C)-ethyl acetate-dichloromethane-formic acid (5:3.5:1:0.8) was used as the developing solvent. After development, the plate was taken out, dried, and examined under ultraviolet light (365 nm). Fig.11 (1-2: Xiebai (small root garlic) formula granules, 3: Xiebai (small root garlic) control medicinal material, 4-5: Xiebai (xie) formula granules, 6: Water onion formula granules, 7: Ziziphus scoparia formula granules).

[0083] From the results in the figure, it can be seen that no matter it is Allium macrostemon from different origins or mixed and counterfeit products of Allium macrostemon, the glycoside components are destroyed after hydrolysis with hydrochloric acid, and the only information displayed is that the depth of the spots is slightly different, but the basic spot information is consistent, indicating that this method cannot be used to identify the origin and mixed and counterfeit products.

[0084] From the above results, it can be found that the identification method provided by the present invention can effectively improve the separation effect of each component of thin layer chromatography, improve the accuracy of detection, so that the overall method can quickly and effectively identify Xiebai (small root garlic) and Xiebai (xie), as well as Xiebai mixed with counterfeit products water onion and schizonepeta; by comparing Examples 1-7, it can be found that the present invention can further improve the accuracy of detection by selecting a specific developing agent combination, and realize the identification of different Xiebai origins and authenticity.

[0085] The applicant declares that the present invention uses the above-mentioned embodiments to illustrate the identification method and application of different Xiebai origin and authenticity in Xiebai medicines including formula granules of the present invention, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0086] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to 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 further describe various possible combinations.

Claims

1. A method for identifying different Xiebai origins and authenticity of Xiebai medicines including formulated granules, characterized in that: The identification method comprises the following steps: (1) Mixing the sample to be tested with a solvent and extracting to obtain a test solution; (2) extracting the control medicinal material by mixing it with a solvent to obtain a control medicinal material solution; (3) subjecting the test sample solution and the control medicinal material solution to thin layer chromatography, and determining the origin and / or authenticity of the test sample based on the test results; Steps (1) and (2) are not sequential; The Xiebai origin includes Xiebai (small garlic root) and Xiebai (xie).

2. The identification method according to claim 1, characterized in that: The Xiebai medicine includes Xiebai medicinal materials, Xiebai formula granules, Xiebai standard decoction, Xiebai decoction pieces or traditional Chinese medicine compound preparations containing Xiebai decoction pieces.

3. The identification method according to claim 1 or 2, characterized in that: The solvent in step (1) is water-saturated n-butanol.

4. The identification method according to any one of claims 1 to 3, characterized in that: The extraction times in step (1) is 1 to 3 times.

5. The identification method according to any one of claims 1 to 4, characterized in that: The solid-liquid ratio of the sample to be tested to the solvent in step (1) is 1:(15-25) g / mL.

6. The identification method according to any one of claims 1 to 5, characterized in that: The thin layer chromatography in step (3) is performed using a silica gel G thin layer plate.

7. The identification method according to any one of claims 1 to 6, characterized in that: The developing solvent of the thin layer chromatography in step (3) is an upper layer solution of a mixed solvent of ethyl acetate, n-butanol and water.

8. The identification method according to claim 7, characterized in that: The volume ratio of ethyl acetate, n-butanol and water is (2-6):(5-9):(2-7).

9. Use of the identification method according to any one of claims 1 to 8 in the quality control of Xiebai medicine.

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