Thin layer chromatography identification method for Atractylodes macrocephala formula granules and burnt Atractylodes macrocephala formula granules

Abstract

The present invention provides a thin layer chromatography identification method for Atractylodes macrocephala formula granules and burnt Atractylodes macrocephala formula granules. The method spots the test solution on a silica gel thin layer plate and develops it according to the thin layer chromatography conditions. The present invention selects petroleum ether-dichloromethane-acetone-formic acid with a volume ratio of (4-6): (1.2-1.8): (0.8-1.2): (0.08-0.12) as a developing agent, and inspects it after the development is completed. It was found that in the obtained thin layer chromatography, the Atractylodes macrocephala formula granules had spots in the range of a transfer value Rf1 of 0.32-0.51 and a transfer value Rf2 of 0.88-0.95, and the burnt Atractylodes macrocephala formula granules had no spots. Therefore, whether there are spots within the above-mentioned Rf1 range can be used as the identification point 1 of the Atractylodes macrocephala formula granules, and whether there are spots within the range of Rf2 can be used as the identification point 2 of the Atractylodes macrocephala formula granules.

Description

Technical Field

[0001] The invention belongs to the technical field of drug detection, and in particular relates to a thin layer chromatography identification method for Atractylodes macrocephala formula granules and scorched Atractylodes macrocephala formula granules. Background Art

[0002] Atractylodes macrocephala is a commonly used Chinese medicine in clinical practice. It was first recorded in Shennong's Herbal Classic and has been recorded in all herbal books of all dynasties. The origin of Atractylodes macrocephala is the dried rhizome of Atractylodes macrocephala Koidz. of the Asteraceae family. It is usually dug in winter when the lower leaves turn yellow and the upper leaves become brittle, and then the mud and sand are removed, and the roots are removed after drying in the oven or sun. Atractylodes macrocephala has the effects of strengthening the spleen and replenishing qi, drying dampness and promoting diuresis, stopping sweating, and calming the fetus. It can be used to treat symptoms such as spleen deficiency, poor appetite, abdominal distension and diarrhea, phlegm and fluid, dizziness and palpitations, edema, spontaneous sweating, or fetal movement disorder.

[0003] Scorched Atractylodes is a product made by frying Atractylodes macrocephala. The 2008 edition of the "Beijing Traditional Chinese Medicine Preparation Specifications" stipulates that the preparation method of Scorched Atractylodes macrocephala is as follows: Take Atractylodes macrocephala slices, put them in a hot pot, fry them over medium heat until the surface is brown, spray a little water, extinguish the sparks, take them out, and dry them. Scorched Atractylodes macrocephala is good at strengthening the spleen and stopping diarrhea, and is used for spleen deficiency diarrhea, chronic dysentery, and leucorrhea.

[0004] Atractylodes macrocephala mainly contains volatile oil and lactone components, and its functions and indications are different before and after processing. Generally, after processing, some of its internal chemical components may change, and its drug-causing properties are milder and less irritating, which is consistent with the traditional theory of Chinese medicine processing. Studies have confirmed that after the Atractylodes macrocephala slices are fried, the polysaccharide content is higher, the volatile oil content is significantly reduced, and the content of Atractylodes lactone I and Atractylodes lactone III are significantly increased.

[0005] At present, the decoction pieces of Atractylodes macrocephala and Jiao Baizhu can be distinguished from each other in terms of properties, but the extracts and granules made from the two are both brown in appearance and have similar characteristics of fragrance and slightly bitter taste, which can easily lead to confusion. At present, there is no report on the identification method of Atractylodes macrocephala formula granules and Jiao Baizhu formula granules. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a thin layer chromatography identification method for Atractylodes macrocephala formula granules and burnt Atractylodes macrocephala formula granules. The method can quickly and effectively identify Atractylodes macrocephala formula granules and burnt Atractylodes macrocephala formula granules by observing whether there are spots in a certain ratio shift value range in the thin layer chromatography spectrum as an identification point.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] The present invention provides a thin layer chromatography identification method for Atractylodes macrocephala formula granules and Atractylodes macrocephala formula granules, comprising the following steps:

[0009] Apply the test solution on the silica gel thin layer plate, develop it according to the thin layer chromatography conditions, and inspect it after development.

[0010] Preferably, the developing solvent in the thin layer chromatography conditions is petroleum ether-dichloromethane-acetone-formic acid, and the volume ratio of petroleum ether-dichloromethane-acetone-formic acid is (4-6):(1.2-1.8):(0.8-1.2):(0.08-0.12).

[0011] Preferably, the volume ratio of petroleum ether-dichloromethane-acetone-formic acid is (5-6):(1.3-1.5):1:(0.08-0.1).

[0012] Preferably, the spotting volume of the test solution is 5 to 15 μL.

[0013] Preferably, the silica gel thin layer plate is silica gel GF 254 Thin layer board.

[0014] Preferably, the distillation range of the petroleum ether in the developing agent is 60-90°C.

[0015] Preferably, the unfolding environment temperature is 4-25° C., and the environment humidity is 32-63 RH%.

[0016] Preferably, in the thin layer chromatography examined, the Atractylodes macrocephala formula granules have a specific migration value Rf 1 0.32~0.51, Rf 2 There are spots in the range of 0.88-0.95, while there are no spots in the granules of scorched white atractylodes.

[0017] Preferably, the inspection is performed under ultraviolet light.

[0018] Preferably, the wavelength of the ultraviolet lamp is 254 nm.

[0019] Preferably, the test solution is prepared according to the following method:

[0020] The test sample was mixed with water and extracted with ethyl acetate. The ethyl acetate was evaporated to dryness and methanol was added to the residue to obtain the test sample solution.

[0021] Preferably, the ethyl acetate extraction is performed 1 to 3 times;

[0022] Preferably, the mass volume ratio of the test sample to ethyl acetate in a single ethyl acetate extraction is 1:(15-25) mL.

[0023] Preferably, the method further comprises the step of spot coating the control medicinal material solution and the negative solution on a silica gel thin layer plate.

[0024] Preferably, the control medicinal material solution is a Atractylodes macrocephala control medicinal material solution.

[0025] Preferably, the negative solution is a maltodextrin solution.

[0026] Preferably, the spotting volume of the reference solution is 5 to 15 μL;

[0027] Preferably, the spotting volume of the negative solution is 5 to 15 μL.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The present invention provides a thin layer chromatography identification method for Atractylodes macrocephala formula granules and burnt Atractylodes macrocephala formula granules. The test sample solution is applied to a silica gel thin layer plate and developed according to the thin layer chromatography conditions. The present invention selects petroleum ether-dichloromethane-acetone-formic acid with a volume ratio of (4-6): (1.2-1.8): (0.8-1.2): (0.08-0.12) as a developing agent, and performs inspection after the development is completed. As a result, it is found that the Atractylodes macrocephala formula granules in the obtained thin layer chromatography have a specific transfer value Rf 1 0.32~0.51, Rf 2 There are spots in the range of 0.88 to 0.95, and there are no spots in the scorched white atractylodes formula granules. 1 Whether there are spots within the range, as the identification point 1 of the Atractylodes formula granules, in Rf 2 Whether there are spots within the range is used as the identification point of the Atractylodes macrocephala formula granules 2. It has been verified that the thin layer chromatography identification method provided by the present invention can quickly and effectively identify the Atractylodes macrocephala and the scorched Atractylodes macrocephala formula granules that have lost the morphology of the medicinal pieces. The detection method is simple to operate, has good durability and sensitivity, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the thin layer chromatogram corresponding to petroleum ether (60-90°C)-dichloromethane-acetone-formic acid (5:1.5:1:0.1) as the developing solvent;

[0031] Among them, 1 is the control herbal material of Atractylodes macrocephala; 2 is the formula granules of Atractylodes macrocephala; 3 is the formula granules of Jiao Atractylodes macrocephala;

[0032] Figure 2 This is the thin layer chromatogram corresponding to petroleum ether (60-90°C)-dichloromethane-acetone-formic acid (8:2:1.5:0.2) as the developing solvent;

[0033] Among them, 1 is the control herbal material of Atractylodes macrocephala; 2 is the formula granules of Atractylodes macrocephala; 3 is the formula granules of Jiao Atractylodes macrocephala;

[0034] Figure 3 It is the thin layer chromatogram corresponding to n-hexane-chloroform-ethyl acetate-formic acid (5:1.5:1:0.1) as the developing solvent;

[0035] Among them, 1 is the control herbal material of Atractylodes macrocephala; 2 is the formula granules of Atractylodes macrocephala; 3 is the formula granules of Jiao Atractylodes macrocephala;

[0036] Figure 4 The thin layer chromatograms at different sample loadings are shown;

[0037] Among them, 1-3 are the sample volumes of Atractylodes macrocephala control medicinal materials, 5 μL, 10 μL, and 15 μL; 4-6 are the sample volumes of Atractylodes macrocephala formula granules, 5 μL, 10 μL, and 15 μL; 7-9 are the sample volumes of Atractylodes macrocephala formula granules, 5 μL, 10 μL, and 15 μL;

[0038] Figure 5 The thin layer chromatograms obtained by investigating the specificity of negative solution, control medicinal material solution, Atractylodes macrocephala formula granule solution, and Scorched Atractylodes macrocephala formula granule solution;

[0039] Among them, 1 is a negative solution; 2 is a control herbal material of Atractylodes macrocephala; 3 is a formula granule of Atractylodes macrocephala; 4 is a formula granule of scorched Atractylodes macrocephala;

[0040] Figure 6 Qingdao Ocean's prefabricated silica gel GF 254 Thin layer chromatogram obtained from the plate;

[0041] Among them, 1 is the control herbal material of Atractylodes macrocephala; 2 is the formula granules of Atractylodes macrocephala; 3 is the formula granules of Jiao Atractylodes macrocephala;

[0042] Figure 7 Tianjin Silida's prefabricated silicone GF 254 Thin layer chromatogram obtained from the plate.

[0043] Among them, 1 is the control herbal material of Atractylodes macrocephala; 2 is the formula granules of Atractylodes macrocephala; 3 is the formula granules of Jiao Atractylodes macrocephala;

[0044] Figure 8 This is a thin layer chromatogram obtained at a low temperature of 4°C;

[0045] Among them, 1 is the control herbal material of Atractylodes macrocephala; 2 is the formula granules of Atractylodes macrocephala; 3 is the formula granules of Jiao Atractylodes macrocephala;

[0046] Fig. 9 This is the thin layer chromatogram obtained at room temperature 25°C;

[0047] Among them, 1 is the control herbal material of Atractylodes macrocephala; 2 is the formula granules of Atractylodes macrocephala; 3 is the formula granules of Jiao Atractylodes macrocephala;

[0048] Fig.10 is the thin layer chromatogram obtained at 63 RH%;

[0049] Among them, 1 is the control herbal material of Atractylodes macrocephala; 2 is the formula granules of Atractylodes macrocephala; 3 is the formula granules of Jiao Atractylodes macrocephala;

[0050] Fig.11 is the thin layer chromatogram obtained at 32 RH%;

[0051] Among them, 1 is the control herbal material of Atractylodes macrocephala; 2 is the formula granules of Atractylodes macrocephala; 3 is the formula granules of Jiao Atractylodes macrocephala;

[0052] Fig.12 Thin layer chromatograms for verifying the control medicinal material solution, and multiple batches of Atractylodes macrocephala formula granule solutions and multiple batches of Jiao Atractylodes macrocephala formula granule solutions;

[0053] Among them, 1 is the control medicinal material of Atractylodes macrocephala; 2-4 are the formula granules of Atractylodes macrocephala BZ2307011; BZ2307012; BZ2307013; 5-7 are the scorched Atractylodes macrocephala formula granules JBZ2308003; JBZ2307004; JBZ2307005. DETAILED DESCRIPTION

[0054] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] In view of the fact that there is no effective identification method based on Atractylodes macrocephala formula granules and burnt Atractylodes macrocephala formula granules in the prior art, the present invention provides a thin layer chromatography identification method of Atractylodes macrocephala formula granules and burnt Atractylodes macrocephala formula granules, characterized in that it comprises the following steps:

[0056] Apply the test solution on the silica gel thin layer plate, develop it according to the thin layer chromatography conditions, and inspect it after development.

[0057] In the present invention, the test solution is a solution of Atractylodes macrocephala granules or a solution of Atractylodes macrocephala granules. In some embodiments, it is preferably prepared according to the following method:

[0058] The test sample (i.e., Atractylodes macrocephala formula granules or burnt Atractylodes macrocephala formula granules) was mixed with water and extracted with ethyl acetate. The ethyl acetate solution was evaporated to dryness, and methanol was added to the residue to obtain a test sample solution.

[0059] According to the present invention, firstly, a test sample (i.e., Atractylodes macrocephala formula granules or charred Atractylodes macrocephala formula granules) is mixed with water, and the mixing is preferably carried out under ultrasonic conditions. After the test sample is completely dissolved, ethyl acetate is added and shaken for extraction 1 to 3 times, preferably 2 times. The mass volume ratio of the test sample to ethyl acetate in a single ethyl acetate extraction is preferably 1:(15 to 25) mL, that is, for 1 g of the test sample, the amount of ethyl acetate used in a single extraction is 15 to 25 mL, preferably 20 mL. Then, the ethyl acetate solutions are combined, and methanol is added to the residue after evaporation to obtain a test sample solution.

[0060] In some embodiments of the present invention, after the ethyl acetate solution is combined, it is preferably washed with water, the water layer is discarded, evaporated to dryness, and methanol is added to the residue to dissolve it as the test solution.

[0061] In a specific embodiment of the present invention, the test solution is prepared according to the following method:

[0062] Take an appropriate amount of this product (Atractylodes macrocephala granules or scorched Atractylodes macrocephala granules), grind it into powder, take 1g, add 20mL of water, ultrasonicate for 15min to dissolve it, add ethyl acetate and shake to extract twice, 20mL each time, combine the ethyl acetate liquid, add 20mL of water to wash, discard the water layer, evaporate to dryness, add 1mL of methanol to dissolve the residue, and use them as the test solution of Atractylodes macrocephala granules and scorched Atractylodes macrocephala granules, respectively.

[0063] According to the present invention, after obtaining the test solution, the test solution is applied to a silica gel thin layer plate, developed according to thin layer chromatography conditions, and inspected after the development. Among them, the silica gel thin layer plate is preferably silica gel GF 254 Thin layer board, because of the use of silicone GF 254 The thin layer plate is spotted and unfolded, and the spot is cleaned and distinguishable when examined at 254nm. If other silica gel thin layer plates are used for examination, the effect is relatively poor. The present invention has been studied for durability and found that the silica gel thin layer plates produced by different manufacturers can achieve the corresponding effect, indicating that the method has good adaptability to different thin layer plates, so there is no special restriction on the source of the silica gel thin layer plate. At the same time, after the spotting amount is investigated, it is found that when the spotting amount of the test solution is 5 to 15 μL, the spots in the obtained thin layer chromatography are clearly colored and have good separation, so the spotting amount of the test solution is preferably 5 to 15 μL, and more preferably 8 to 12 μL.

[0064] It should be noted that not all developing agents can make the Atractylodes macrocephala formula granules and the charred Atractylodes macrocephala formula granules have a developing effect. The present invention uses petroleum ether (60-90°C)-dichloromethane-acetone-formic acid (5:1.5:1:0.1) (developing agent 1), petroleum ether (60-90°C)-dichloromethane-acetone-formic acid (8:2:1.5:0.2) (developing agent 2), and n-hexane-trichloromethane-ethyl acetate-formic acid (5:1.5:1:0.1) (developing agent 3) as developing agents. After development, it was found that only when the developing agent 1, i.e., petroleum ether (60-90°C)-dichloromethane-acetone-formic acid (5:1.5:1:0.1), was used, the spots were clear. Compared with the Atractylodes macrocephala control medicinal material atlas, the ratio shift values ​​(Rf) were 0.32 and 0.89, the Atractylodes macrocephala formula granules had the same spots, while the charred Atractylodes macrocephala formula granules had no spots. Therefore, the present invention uses petroleum ether-dichloromethane-acetone-formic acid as a developing agent, and preferably the volume ratio of the petroleum ether-dichloromethane-acetone-formic acid is (4-6):(1.2-1.8):(0.8-1.2):(0.08-0.12), more preferably (5-6):(1.3-1.5):1:(0.08-0.1), and most preferably 5:1.5:1:0.1.

[0065] It should be noted that, in the present invention, after screening, the distillation range of the petroleum ether of the developing agent is preferably 60-90°C.

[0066] The present invention performs thin layer chromatography separation on the above-mentioned test solution under development environments of different temperatures and different humidities, and it is found that the method has strong adaptability to temperature and humidity. The development environment temperature can be in the range of 4 to 25°C, specifically 4°C, 8°C, 10°C, 15°C, 20°C or 25°C, etc.; the environment humidity can be in the range of 32 to 63RH%, specifically 32RH%, 35RH%, 40RH%, 45RH%, 50RH%, 55RH%, 60RH% or 63RH%, etc.

[0067] The present invention has found that the white atractylodes formula granules have a specific transfer value Rf 1 0.32~0.51, Rf 2 There are spots in the range of 0.88 to 0.95, and there are no spots in the scorched white atractylodes formula granules. 1 Whether there are spots within the range, as the identification point 1 of the Atractylodes formula granules, in Rf 2 Whether there are spots within the range is used as the identification point 2 of the Atractylodes macrocephala formula granules. In the present invention, the above-mentioned thin layer chromatography method also includes the step of applying the control medicinal material solution and the negative solution on the silica gel thin layer plate. Among them, the control medicinal material solution is used for comparative analysis with the test solution, and the negative solution is used to eliminate interference with the sample.

[0068] In some specific embodiments of the present invention, the control medicinal material solution is preferably prepared according to the following steps:

[0069] Take 1 g of Atractylodes macrocephala control medicinal material, add 50 mL of water, heat and reflux for 30 minutes, centrifuge for 3 minutes (speed 6000 rpm), take the supernatant, concentrate it in a water bath to about 20 mL, add ethyl acetate and shake to extract twice, 20 mL each time, combine the ethyl acetate liquid, add 20 mL of water to wash, discard the water layer, evaporate to dryness, add 1 mL of methanol to the residue to dissolve it, and obtain the product.

[0070] The negative solution can be prepared according to the following method:

[0071] Take 1 g of maltodextrin, add 20 mL of water, ultrasonicate for 15 minutes, add ethyl acetate and shake to extract twice, 20 mL each time, combine the ethyl acetate liquid, add 20 mL of water to wash, discard the water layer, evaporate to dryness, add 1 mL of methanol to dissolve the residue, and use it as the negative solution.

[0072] It should be noted that in the present invention, after research, the spotting amount of the control medicinal solution is preferably 5-15 μL, more preferably 8-12 μL, and most preferably 10 μL; the spotting amount of the negative solution is preferably 5-15 μL, more preferably 8-12 μL, and most preferably 10 μL.

[0073] It has been verified that the thin layer chromatography identification method provided by the present invention can quickly and effectively identify the Atractylodes macrocephala and Scorched Atractylodes macrocephala formula granules that have lost their medicinal piece form. The detection method is easy to operate, has good durability and sensitivity, and has good application prospects.

[0074] In order to further illustrate the present invention, the following examples are provided for detailed description. The instruments, reagents and reagents used in the following examples of the present invention are as follows:

[0075] 1.1 Instrument

[0076] Heating plate, mortar, thin layer imaging system: CAMAG TLC Visualizer, silica gel GF 254 Thin layer plate (Merck, Germany, batch number: HX87183353, Tianjin Slida Technology Co., Ltd., batch number: 191016, Qingdao Ocean Chemical Plant, batch number: 20180527)

[0077] 1.2 Reagents

[0078] Ethyl acetate, petroleum ether (60-90°C), dichloromethane, acetone, formic acid, n-hexane, and chloroform were all of analytical grade, and water was ultrapure water (prepared in the laboratory).

[0079] 1.3 Drug testing

[0080] Atractylodes macrocephala control medicinal materials (China Food and Drug Inspection Institute, batch number: 120925-202114), Atractylodes macrocephala formula granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch number: BZ2307011; BZ2307012; BZ2307013) Jiao Atractylodes macrocephala formula granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch number: JBZ2308003; JBZ2307004; JBZ2307005).

[0081] Preparation Example 1

[0082] This preparation example provides a test solution of Atractylodes macrocephala formula granules and Atractylodes macrocephala formula granules, and the preparation method thereof is as follows:

[0083] Take an appropriate amount of this product (Atractylodes macrocephala granules or scorched Atractylodes macrocephala granules), grind it into powder, take 1g, add 20mL of water, ultrasonicate for 15min to dissolve it, add ethyl acetate and shake to extract twice, 20mL each time, combine the ethyl acetate liquid, add 20mL of water to wash, discard the water layer, evaporate to dryness, add 1mL of methanol to dissolve the residue, and use them as the test solution of Atractylodes macrocephala granules and scorched Atractylodes macrocephala granules, respectively.

[0084] Preparation Example 2

[0085] This preparation example provides a control medicinal material solution, wherein the control medicinal material solution is a control medicinal material solution of Atractylodes macrocephala. The specific preparation method is: take 1 g of Atractylodes macrocephala control medicinal material, add 50 mL of water, heat and reflux for 30 minutes, centrifuge for 3 minutes (speed 6000 rpm), take the supernatant, concentrate it in a water bath to about 20 mL, add ethyl acetate and shake to extract twice, 20 mL each time, combine the ethyl acetate liquid, add 20 mL of water to wash, discard the water layer, evaporate to dryness, add 1 mL of methanol to the residue to dissolve it, and obtain the product.

[0086] Preparation Example 3

[0087] This preparation example provides a negative solution, which is a maltodextrin solution. The specific preparation method is: take 1 g of maltodextrin, add 20 mL of water, ultrasonicate for 15 minutes, add ethyl acetate and shake to extract twice, 20 mL each time, combine the ethyl acetate solution, add 20 mL of water to wash, discard the water layer, evaporate to dryness, add 1 mL of methanol to the residue to dissolve it, and use it as the negative solution.

[0088] Test example (developer investigation)

[0089] Take the test solution of the Atractylodes macrocephala formula granules and the test solution of the scorched Atractylodes macrocephala formula granules obtained in Preparation Example 1, and take 1 g of Atractylodes macrocephala control medicinal material, add 50 mL of water, heat and reflux for 30 min, centrifuge for 3 min (speed 6000 rpm), take the supernatant, concentrate it in a water bath to about 20 mL, and prepare the control medicinal material solution in the same way as "add ethyl acetate and shake to extract twice" in the preparation process of the test solution.

[0090] According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part 1 General Rule 0502), 10 μL of the test solution of the above-mentioned Atractylodes macrocephala formula granules, the test solution of the scorched Atractylodes macrocephala formula granules, and the control medicinal material solution were taken and spotted on the same silica gel GF 254 On the thin layer plate, petroleum ether (60-90°C)-dichloromethane-acetone-formic acid (5:1.5:1:0.1) (developer 1), petroleum ether (60-90°C)-dichloromethane-acetone-formic acid (8:2:1.5:0.2) (developer 2), and n-hexane-trichloromethane-ethyl acetate-formic acid (5:1.5:1:0.1) (developer 3) were used as developing agents, respectively. The plates were developed, taken out, dried, and examined under ultraviolet light (254nm).

[0091] The obtained thin layer chromatogram is as follows Figures 1 to 3 As shown, Figure 1 is the thin layer chromatogram corresponding to developing agent 1; Figure 2 is the thin layer chromatogram corresponding to developing agent 2; Figure 3 The figure is the thin layer chromatogram corresponding to the developing agent 3. The results show that when the developing agent 1, i.e., petroleum ether (60-90°C)-dichloromethane-acetone-formic acid (5:1.5:1:0.1), is used, the spots are clear. Compared with the Atractylodes macrocephala reference medicinal material spectrum, the ratio shift value (Rf) is 0.32 and 0.89, the Atractylodes macrocephala formula granules have the same spots, while the Jiao Baizhu formula granules have no spots. This method can be used to achieve the qualitative identification of the Atractylodes macrocephala formula granules and Jiao Baizhu formula granules.

[0092] Therefore, in the subsequent examples, petroleum ether (60-90° C.)-dichloromethane-acetone-formic acid (5:1.5:1:0.1) was used as the developing solvent for investigation.

[0093] Establishment of thin layer identification method

[0094] Take the test solution of the Atractylodes macrocephala formula granules and the test solution of the scorched Atractylodes macrocephala formula granules obtained in Preparation Example 1, and take 1 g of Atractylodes macrocephala control medicinal material, add 50 mL of water, heat and reflux for 30 min, centrifuge for 3 min (speed 6000 rpm), take the supernatant, concentrate it in a water bath to about 20 mL, and prepare the control medicinal material solution in the same way as "add ethyl acetate and shake to extract twice" in the preparation process of the test solution.

[0095] According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part 1 General Rule 0502), 10 μL of the test solution of Atractylodes macrocephala formula granules, the test solution of Atractylodes macrocephala formula granules, and the control medicinal material solution were taken and spotted on the same silica gel GF 254On the thin layer plate, use petroleum ether (60-90°C)-dichloromethane-acetone-formic acid (5:1.5:1:0.1) as the developing agent, take out, dry, and examine under ultraviolet light (254nm).

[0096] Example 1

[0097] This embodiment provides a thin layer identification chromatography method for Atractylodes macrocephala formula granules and Atractylodes macrocephala formula granules, and investigates the spotting amount, comprising the following steps:

[0098] S1: According to the method of Preparation Examples 1-2, provide a test solution of Atractylodes macrocephala granules, a test solution of Jiao Atractylodes macrocephala granules and a control medicinal material solution;

[0099] S2: Take 5 μL, 10 μL and 15 μL of the control drug solution and the test solution respectively and spot them on the same silica gel GF 254 On the thin layer plate, develop and inspect according to the thin layer chromatography conditions described in the above established thin layer identification method.

[0100] The results are as follows Figure 4 As shown, it can be seen that when the spotting volume is 5-15 μL, the TLC spots are clearly colored. Compared with the Atractylodes macrocephala reference medicinal material spectrum, the Atractylodes macrocephala formula granules have the same spots at the Rf values ​​of 0.41 and 0.95, while the Jiao Atractylodes macrocephala formula granules have no spots. Therefore, the spotting volume of the reference medicinal material solution and the test solution is determined to be 5-15 μL, preferably 10 μL.

[0101] Example 2

[0102] This embodiment provides a thin layer chromatography method for identifying the Atractylodes macrocephala formula granules and the Atractylodes macrocephala formula granules, and investigates the specificity, comprising the following steps:

[0103] S1: According to the methods of Preparation Examples 1 to 3, provide a test solution of Atractylodes macrocephala granules, a test solution of Atractylodes macrocephala granules, a control medicinal material solution, and a negative solution;

[0104] S2: Take 10 μL of the test solution of Atractylodes macrocephala formula granules and the test solution of Atractylodes macrocephala formula granules, 10 μL of the control medicinal material solution, and 10 μL of the negative solution, and spot them on the same silica gel GF. 254 The samples were developed and inspected on a thin layer plate (manufacturer: Merck, Germany) according to the thin layer chromatography conditions described in the above established thin layer identification method.

[0105] The results are as follows Figure 5As shown, it can be seen that compared with the Atractylodes macrocephala control medicinal material spectrum, the Atractylodes macrocephala formula granules have the same spots at the ratio shift value (Rf) of 0.32 and 0.90, while the Jiao Baishu formula granules have no spots. Therefore, the negative sample has no interference with the test sample of the Atractylodes macrocephala formula granules and the Jiao Baishu formula granules, and the method has good specificity.

[0106] Example 3

[0107] This embodiment provides a thin layer chromatography method for identifying the Atractylodes macrocephala formula granules and the Atractylodes macrocephala formula granules, and investigates the durability (different thin layer plates), comprising the following steps:

[0108] S1: According to the method of Preparation Examples 1-2, provide a test solution of Atractylodes macrocephala granules, a test solution of Jiao Atractylodes macrocephala granules and a control medicinal material solution;

[0109] S2: Take 10 μL, 10 μL, and 10 μL of the test solution of Atractylodes macrocephala formula granules, the test solution of Jiao Atractylodes macrocephala formula granules, and the control medicinal material solution, and test them according to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), and spot them on the prefabricated silica gel GF of Qingdao Ocean and Tianjin Silida, respectively. 254 On the plate, develop and inspect according to the TLC conditions described in the TLC identification method established above.

[0110] The results are as follows Figures 6-7 As shown, Figure 6 Qingdao Ocean's prefabricated silica gel GF 254 The thin layer chromatogram obtained from the plate, Figure 7 Tianjin Silida's prefabricated silicone GF 254 Thin layer chromatogram obtained from Figure 6 It can be seen that compared with the Atractylodes macrocephala reference material atlas, the Atractylodes macrocephala formula granules have obvious spots at the ratio transfer values ​​(Rf) of 0.51 and 0.96, while the Jiao Atractylodes macrocephala formula granules have no spots of the same color; Figure 7 It can be seen that the Atractylodes macrocephala formula granules have obvious spots at the Rf values ​​of 0.37 and 0.92, while the Atractylodes macrocephala formula granules have no spots of the same color. The results show that both brands of thin layer plates can meet the expected identification requirements, proving that the method has good durability.

[0111] Example 4

[0112] This embodiment provides a thin layer identification chromatography method for Atractylodes macrocephala formula granules and Atractylodes macrocephala formula granules, and investigates the durability (development temperature), comprising the following steps:

[0113] S1: According to the method of Preparation Examples 1-2, provide a test solution of Atractylodes macrocephala granules, a test solution of Jiao Atractylodes macrocephala granules and a control medicinal material solution;

[0114] S2: Take 10 μL, 10 μL, and 10 μL of the test solution of Atractylodes macrocephala formula granules, the test solution of Jiao Atractylodes macrocephala formula granules, and the control medicinal material solution, and test them according to the thin layer chromatography method (General Rules 0502 of Part I of the 2020 edition of the Chinese Pharmacopoeia), and spot them on the same silica gel GF 254 On the thin layer plate, petroleum ether (60-90°C)-dichloromethane-acetone-formic acid (5:1.5:1:0.1) was used as the developing solvent, and it was developed at low temperature 4°C and room temperature 25°C (humidity was 63RH%) respectively. The plate was taken out, dried, and examined under ultraviolet light (254nm).

[0115] Results Figures 8-9 , Figure 8 This is a thin layer chromatogram obtained at a low temperature of 4°C. Figure 7 This is a thin layer chromatogram obtained at room temperature 25°C. Figure 8 It can be seen that compared with the Atractylodes macrocephala reference material at 4°C, the Atractylodes macrocephala formula granules have the same spots at the Rf values ​​of 0.32 and 0.87, while the Jiao Atractylodes macrocephala formula granules have no spots. Fig. 9 It can be seen that when the temperature is 25°C, the specific migration value (Rf) is 0.32 and 0.88, the Atractylodes macrocephala formula granules have the same spots, while the scorched Atractylodes macrocephala formula granules have no spots. The results show that this method has good durability at different temperatures.

[0116] Example 5

[0117] This embodiment provides a thin layer identification chromatography method for Atractylodes macrocephala formula granules and Atractylodes macrocephala formula granules, and investigates durability (expanding humidity), comprising the following steps:

[0118] S1: According to the method of Preparation Examples 1-2, provide a test solution of Atractylodes macrocephala granules, a test solution of Jiao Atractylodes macrocephala granules and a control medicinal material solution;

[0119] S2: Take 10 μL, 10 μL, and 10 μL of the test solution of the Atractylodes macrocephala formula granules, the test solution of the Jiao Atractylodes macrocephala formula granules, and the control medicinal material solution. 254 On the thin layer plate, petroleum ether (60-90°C)-dichloromethane-acetone-formic acid (5:1.5:1:0.1) was used as the developing solvent, and it was developed in a humidity environment of 32RH% and 63RH% respectively (temperature was 25°C), taken out, dried, and examined under ultraviolet light (254nm).

[0120] Results Figures 10-11 , Fig.10 is the thin layer chromatogram obtained at 63 RH%, Fig.11 The thin layer chromatogram obtained at 32 RH% is shown in FIG. Fig.10It can be seen that compared with the Atractylodes macrocephala reference material at a humidity of 63%, the Atractylodes macrocephala formula granules have the same spots at the Rf values ​​of 0.32 and 0.88, while the Jiao Atractylodes macrocephala formula granules have no spots. Fig.11 It can be seen that when the humidity is 32%, the specific migration value (Rf) is 0.32 and 0.89, the Atractylodes macrocephala formula granules have the same spots, while the scorched Atractylodes macrocephala formula granules have no spots. The results show that the method has good durability at different humidity.

[0121] verify

[0122] In order to verify the above-mentioned thin-layer identification method, the present invention used 3 batches of Atractylodes macrocephala formula granules and 3 batches of scorched Atractylodes macrocephala formula granules, and carried out and inspected them according to the above-mentioned established thin-layer identification method.

[0123] Results Fig.12 It can be seen that compared with the Atractylodes macrocephala reference material at the Rf of 0.33 and 0.89, different batches of Atractylodes macrocephala formula granules have the same spots, while different batches of Jiao Atractylodes macrocephala formula granules have no spots. Therefore, the presence or absence of spots at the Rf of 0.33 and 0.89 can be used as a distinguishing point.

[0124] Determination of the Rf value of the identification point of the formula granules of Atractylodes macrocephala and Atractylodes macrocephala

[0125] The data of various ratio transfer values ​​in the thin layer chromatography methodology investigation were summarized, and the results are shown in Table 1. Based on the summary results, the ratio transfer value (Rf 1 ) should be within the range of 0.32 to 0.51; the ratio shift value (Rf 2 ) should be within 10% and the specified value is 0.9.

[0126] Table 1

[0127]

[0128] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A thin layer chromatography identification method for Atractylodes macrocephala formula granules and burnt Atractylodes macrocephala formula granules, characterized in that: The following steps are involved: The test solution was applied to a silica gel thin layer plate, developed according to the thin layer chromatography conditions, and inspected after the development; in the inspected thin layer chromatography, the Atractylodes macrocephala formula granules had spots in the range of Rf1 of 0.32-0.51 and Rf2 of 0.88-0.95, while the Jiao Atractylodes macrocephala formula granules had no spots; The test solution was prepared according to the following method: After mixing the test sample with water, extract it with ethyl acetate, evaporate the ethyl acetate to dryness, and add methanol to the residue to obtain the test sample solution; The developing solvent in the thin layer chromatography conditions is petroleum ether-dichloromethane-acetone-formic acid, and the volume ratio of the petroleum ether-dichloromethane-acetone-formic acid is (4-6):(1.2-1.8):(0.8-1.2):(0.08-0.12).

2. The thin layer chromatography identification method according to claim 1, characterized in that: The volume ratio of petroleum ether-dichloromethane-acetone-formic acid is (5-6):(1.3-1.5):1:(0.08-0.1); The sample volume of the test solution is 5~15 μL.

3. The thin layer chromatography identification method according to claim 1 or 2, characterized in that: The silica gel thin layer plate is silica gel GF 254 Thin layer board; The distillation range of the petroleum ether in the developing agent is 60-90°C.

4. The thin layer chromatography identification method according to claim 1 or 2, characterized in that: The ambient temperature of the development is 4-25° C., and the ambient humidity is 32-63 RH%.

5. The thin layer chromatography identification method according to claim 1, characterized in that: The inspection is carried out under ultraviolet light; The wavelength of the ultraviolet lamp is 254 nm.

6. The thin layer chromatography identification method according to claim 1, characterized in that: The number of times of ethyl acetate extraction is 1 to 3 times; The mass volume ratio of the test sample to ethyl acetate in a single ethyl acetate extraction is 1:(15~25) mL.

7. The thin layer chromatography identification method according to claim 1, characterized in that: The method also includes the step of spot coating the control medicinal material solution and the negative solution on the silica gel thin layer plate.

8. The thin layer chromatography identification method according to claim 7, characterized in that: The control medicinal material solution is a control medicinal material solution of Atractylodes macrocephala; The negative solution is a maltodextrin solution; The spotting volume of the control medicinal material solution is 5-15 μL; The spotting volume of the negative solution is 5-15 μL.

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