A thin layer identification method for natural myrrh and colloidal myrrh formula particles and its application

Abstract

The present invention provides a thin layer identification method for natural myrrh and colloid myrrh formula particles and its application, including: taking a sample to be tested, solvent dissolving, and obtaining a test solution; the sample to be tested is natural myrrh and colloid myrrh formula particles; taking natural myrrh control medicinal materials and colloid myrrh control medicinal materials, respectively, solvent dissolving, and obtaining a control medicinal material solution; the test solution and the control medicinal material solution are subjected to thin layer chromatography detection, and the thin layer plate is a silica gel G thin layer plate; the developing agent is cyclohexane-ethyl acetate-glacial acetic acid-0.1mol / L hydrochloric acid with a volume ratio of (8-10): (0.8-1.2): (0.8-1.2): (0.3-0.7); under ultraviolet light, in the test sample chromatogram, whether there is a fluorescent spot in Rf, as a distinguishing point, distinguish natural myrrh formula particles and colloid myrrh formula particles. The present invention observes whether there is a spot in the thin layer chromatogram within a specified ratio shift value range as a distinguishing point, so as to quickly and effectively distinguish natural myrrh and colloid myrrh formula particles.

Description

Technical Field

[0001] The present invention relates to the field of identification technology, and in particular to a thin layer identification method for natural myrrh and colloidal myrrh formula particles and application thereof. Background Art

[0002] Myrrh is the dried resin of Commiphora myrrha Engl. or Commiphoramolmol Engl., a plant of the olive family. It is divided into natural myrrh and colloidal myrrh. Its main functions are to dissipate blood stasis, relieve pain, and reduce swelling and promote tissue regeneration. It is used for chest pain, heart pain, stomach pain, dysmenorrhea, amenorrhea, postpartum blood stasis, abdominal pain due to lumps, rheumatic pain, traumatic injuries, carbuncle, ulcers and sores. The two have the same efficacy, but there are literature reports that the main chemical components of natural myrrh include furanoceous diene, curcumene, bicyclo-sound leaf ene, β-cadinene and β-olivene, etc.; the main chemical components of colloidal myrrh include α-trans-bergamotene, α-santalene-α-cis-bergamotene, trans-β-farnesene, trans-β-camphorene, α-bisabolene, β-bisabolene, trans-γ-bisabolene, cis-γ-bisabolene and β-santalaldehyde, etc., indicating that the scope of application of the two medicines is different. In order to ensure the accuracy of clinical medication, it is necessary to establish an effective and feasible identification method for the qualitative distinction between the two.

[0003] There are obvious differences in the properties of natural myrrh and colloidal myrrh, and property identification is an effective method to identify the two. Formula granules are made from medicinal materials through processing, decoction, concentration, drying, total mixing, and granulation, which have lost the original properties of the medicinal materials. Therefore, the property identification method is not suitable for the identification of formula granules. Zhang Wenting et al. established a method for identifying natural myrrh, colloidal myrrh medicinal materials, and decoction pieces through characteristic spectra, but the formula granules are decoction-extracted, and the chemical composition is somewhat different from that of the medicinal materials. Whether this characteristic spectrum method is suitable for the identification of formula granules remains to be verified, and this method requires precise liquid chromatography instruments, the detection cost is too high, and it also has high requirements for the professional skills of the test personnel. Therefore, it is necessary to establish a visual, simple, effective, and reproducible method that can distinguish between natural myrrh and colloidal myrrh formula granules to provide a basis for the quality evaluation of the finished product. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a thin layer identification method for natural myrrh and colloidal myrrh formula particles and its application. The method observes whether there are fluorescent spots in the specified ratio shift value range of the thin layer chromatogram as the identification point, so as to quickly, accurately and effectively identify natural myrrh and colloidal myrrh formula particles.

[0005] The present invention provides a thin layer identification method for natural myrrh and colloidal myrrh formula particles, comprising the following steps:

[0006] A) taking a sample to be tested, dissolving it with a solvent, and obtaining a test solution; the sample to be tested is a natural myrrh formula granule and a colloidal myrrh formula granule;

[0007] B) taking a natural myrrh reference medicinal material and a colloidal myrrh reference medicinal material, dissolving them in solvents respectively, to obtain a reference medicinal material solution;

[0008] C) The test solution and the control medicinal material solution are subjected to thin layer chromatography, the thin layer plate is a silica gel G thin layer plate; the developing solvent is cyclohexane-ethyl acetate-glacial acetic acid-0.1 mol / L hydrochloric acid in a volume ratio of (8-10):(0.8-1.2):(0.8-1.2):(0.3-0.7);

[0009] D) Check under ultraviolet light to see if there are fluorescent spots within the specified ratio shift value Rf in the chromatogram of the test sample, which can be used as a discrimination point to distinguish between natural myrrh formula granules and colloidal myrrh formula granules.

[0010] The thin layer chromatography method for identifying natural myrrh and colloidal myrrh formula particles established by the present invention uses fluorescent spots as identification points by observing whether there are fluorescent spots in a specified ratio shift value range in a thin layer chromatography spectrum to quickly, accurately and effectively identify natural myrrh and colloidal myrrh formula particles.

[0011] The present invention takes a sample to be tested, dissolves it in a solvent, and obtains a test solution; the sample to be tested is a natural myrrh formula granule and a colloidal myrrh formula granule. In the present invention, the step A) comprises:

[0012] Take natural myrrh formula granules and colloidal myrrh formula granules, add water and hydrochloric acid respectively, heat and reflux, filter, evaporate the filtrate to dryness, add water to dissolve the residue, add ethyl acetate to extract, evaporate the obtained ethyl acetate solution to dryness, add ethanol to the residue, and obtain natural myrrh formula granule solution and colloidal myrrh formula granule solution respectively;

[0013] The mass ratio of the natural myrrh formula granules to water and hydrochloric acid is 2.0 g: (18-23) mL: (1.8-2.3) mL;

[0014] The mass ratio of the colloidal myrrh formula particles to the volume ratio of water and hydrochloric acid is 2.0 g: (18-23) mL: (1.8-2.3) mL;

[0015] The mass ratio of the natural myrrh formula particles to the volume ratio of ethyl acetate is 2.0 g: (18-23) mL;

[0016] The mass ratio of the colloidal myrrh formula particles to the volume ratio of ethyl acetate is 2.0 g: (18-23) mL.

[0017] The heating reflux time is 50 to 70 min, preferably 55 to 65 min.

[0018] In a specific embodiment, the test solution is prepared according to the following method: take 2g of natural myrrh formula granules and colloidal myrrh formula granules, grind them, add 20ml of water, then add 2ml of hydrochloric acid, heat and reflux for 1h, filter, evaporate the filtrate to dryness, add 20ml of water to dissolve the residue, add 20ml of ethyl acetate to extract twice, combine the ethyl acetate solution, evaporate to dryness, add 2ml of ethanol to dissolve the residue, and use it as the test solution.

[0019] The present invention takes natural myrrh reference medicinal material and colloidal myrrh reference medicinal material, dissolves them in solvents respectively, and obtains reference medicinal material solutions. The step B) of the present invention comprises:

[0020] Take natural myrrh reference medicinal material and colloidal myrrh reference medicinal material and mix them with cyclohexane respectively, heat to slightly boil, and take cyclohexane solution after standing to obtain natural myrrh reference medicinal material solution and colloidal myrrh reference medicinal material solution;

[0021] The mass ratio of natural myrrh reference medicinal material to cyclohexane is 1 g: (1.8-2.3) mL.

[0022] The present invention prepares the control medicinal material solution according to the volatile oil determination method (Chinese Pharmacopoeia 2020 General Rules 2204 B method). The micro-boiling time is 2 to 3 hours, preferably 2.5 hours.

[0023] In a specific embodiment, the control medicinal material solution is prepared according to the following method:

[0024] Take 1g of natural myrrh reference medicinal material, add 2ml of cyclohexane according to the volatile oil determination method (Chinese Pharmacopoeia 2020 General Rules 2204 B method), slowly heat to boiling, and keep it slightly boiling for about 2.5 hours. After standing, take the cyclohexane liquid to make a natural myrrh reference medicinal material solution. Take 1g of colloidal myrrh reference medicinal material and make a colloidal myrrh reference medicinal material solution in the same way.

[0025] The present invention performs thin layer chromatography on the test solution and the control medicinal material solution, and the thin layer plate is a silica gel G thin layer plate; the developing agent is cyclohexane-ethyl acetate-glacial acetic acid-0.1 mol / L hydrochloric acid with a volume ratio of (8-10): (0.8-1.2): (0.8-1.2): (0.3-0.7); preferably, cyclohexane-ethyl acetate-glacial acetic acid-0.1 mol / L hydrochloric acid with a volume ratio of (9-10): (0.9-1.1): (0.9-1.1): (0.4-0.6), and more preferably, cyclohexane-ethyl acetate-glacial acetic acid-0.1 mol / L hydrochloric acid with a volume ratio of (9.5-10): (0.95-1.05): (0.95-1.05): (0.45-0.55). In a specific embodiment, the developing agent is cyclohexane-ethyl acetate-glacial acetic acid-0.1 mol / L hydrochloric acid with a volume ratio of 10:1:1:0.5.

[0026] In a specific embodiment of the present invention, according to the thin layer chromatography method (Appendix VIB of Part I of the Chinese Pharmacopoeia 2020 Edition), the test sample solution and the control medicinal material solution were respectively spotted on the same silica gel G thin layer plate, and cyclohexane-ethyl acetate-glacial acetic acid-0.1 mol / L hydrochloric acid was used as the developing agent. The plate was developed, taken out, dried, and inspected under ultraviolet light.

[0027] In the present invention, during the thin layer chromatography test, the sample volume of the test solution and the control medicinal material solution is 5 to 15 μL. In a specific embodiment, the sample volume is 5 μL, 10 μL, or 15 μL.

[0028] The thin layer plate used in the present invention is a silica gel G thin layer plate; the present invention preferably uses a cuttable thin layer chromatography plate of Tianjin Silida Technology Co., Ltd., Merck, and a silica gel plate of Qingdao Yumingyuan Silica Gel Reagent Co., Ltd. The results show that the pink fluorescent spot ratio shift values ​​(Rf) of the three thin layer plates at 7.3 to 9.9 cm from the origin are 0.522, 0.511, and 0.519, respectively, which can meet the identification requirements. And at the ratio shift values ​​of 0.522, 0.511, and 0.519, the natural myrrh formula particles show pink fluorescent spots, while the colloidal myrrh formula particles do not have such spots.

[0029] The wavelength examined in the present invention is 365 nm.

[0030] The inspection temperature in the present invention is 4-23° C. The method has good adaptability to different temperatures, and the natural myrrh formula particles show pink fluorescent spots at the ratio shift values ​​of 0.538 and 0.516, while the colloidal myrrh formula particles do not have such spots.

[0031] The humidity examined in the present invention is 31% rh to 60% rh. The method provided by the present invention has good adaptability to different humidity, and the natural myrrh formula particles show pink fluorescent spots at the ratio shift values ​​of 0.532 and 0.529, while the colloidal myrrh formula particles do not have such spots.

[0032] Inspect under ultraviolet light, and whether there are fluorescent spots within the specified ratio shift value Rf in the chromatogram of the test sample is used as a distinguishing point to distinguish natural myrrh formula particles from colloidal myrrh formula particles. Preferably, when placed under ultraviolet light, if there are spots at the Rf value of 0.469 to 0.573, it is a natural myrrh formula particle; if there are no spots at the Rf value of 0.469 to 0.573, it is a colloidal myrrh formula particle. In the present invention, the Rf value is preferably 0.511 to 0.538; specifically, it can be 0.511, 0.513, 0.514, 0.516, 0.519, 0.522, 0.529, 0.532, 0.538.

[0033] The identification method provided by the present invention is also applicable to the identification of natural myrrh water extract.

[0034] The present application also provides an application of the identification method described in the above technical solution in distinguishing natural myrrh from colloidal myrrh.

[0035] The invention provides a thin layer identification method for natural myrrh and colloidal myrrh formula particles, comprising the following steps: A) taking a sample to be tested, dissolving it with a solvent, and obtaining a test solution; the sample to be tested is natural myrrh formula particles and colloidal myrrh formula particles; B) taking a natural myrrh control medicinal material and a colloidal myrrh control medicinal material, dissolving them with solvents respectively, and obtaining a control medicinal material solution; C) performing thin layer chromatography on the test solution and the control medicinal material solution, wherein the thin layer plate is a silica gel G thin layer plate; the developing agent is cyclohexane-ethyl acetate-glacial acetic acid-0.1 mol / L hydrochloric acid in a volume ratio of (8-10):(0.8-1.2):(0.8-1.2):(0.3-0.7); D) inspecting under ultraviolet light whether there are fluorescent spots in the specified ratio shift value Rf in the chromatogram of the test sample as an identification point to distinguish the natural myrrh formula particles and the colloidal myrrh formula particles. The thin layer chromatography identification method for natural myrrh and colloidal myrrh formula particles established by the present invention uses the observation of whether spots exist in the thin layer chromatography spectrum within a specified ratio shift value range as identification points to quickly and effectively identify the natural myrrh formula particles and the colloidal myrrh formula particles.

[0036] The invention observes whether the thin layer chromatography of the test sample and the thin layer chromatography of the reference sample and the reference medicinal material show spots of the same color, and serves as a thin layer identification method for natural myrrh and colloidal myrrh formula granules. The thin layer chromatography method established by the invention can quickly and effectively identify natural myrrh and colloidal myrrh formula granules. The method is simple to operate, highly sensitive, and stable, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 To investigate the amount of samples;

[0038] Figure 2 For exclusive investigation;

[0039] Figure 3 Investigation of different thin layer plates - Tianjin Silida;

[0040] Figure 4 For different thin layer plates to investigate - Merck;

[0041] Figure 5 For different thin layer plates inspection-Qingdao Yumingyuan;

[0042] Figure 6 For investigations at different temperatures -23°C;

[0043] Figure 7 For investigations at different temperatures -4°C;

[0044] Figure 8For investigation of different humidity -60%;

[0045] Fig. 9 For the investigation of different humidity -31%;

[0046] Fig.10 This is a multi-batch verification graph;

[0047] Fig.11 It is the thin layer identification diagram of comparative example 1;

[0048] Fig.12 It is the thin layer identification diagram of comparative example 2;

[0049] Fig.13 It is the thin layer identification diagram of comparative example 3;

[0050] Fig.14 This is a thin layer identification diagram of Comparative Example 4 using different proportions of developing agents;

[0051] Fig.15 This is the thin layer identification image of Comparative Example 5 using different developing agents. DETAILED DESCRIPTION

[0052] In order to further illustrate the present invention, a thin layer identification method for natural myrrh and colloidal myrrh formula particles and applications thereof provided by the present invention are described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0053] Example 1

[0054] 1.1 Instruments and reagents

[0055] 1.1.1 Instruments

[0056] Heating plate, mortar, thin layer imaging system: CAMAG TLC Visualizer, silica gel G thin layer plate (Qingdao Yumingyuan Silica Gel Reagent Co., Ltd., batch number: 20230612, Tianjin Silida Technology Co., Ltd., batch number: 221030, Merck, batch number: HX32019153);

[0057] 1.1.2 Reagents

[0058] Cyclohexane, ether, xylene, ethanol, ethyl acetate, and anhydrous ethanol were all of analytical grade, and water was ultrapure water (prepared in the laboratory);

[0059] 1.1.3 Drug testing

[0060] Natural myrrh reference medicinal materials (China Food and Drug Inspection Institute, batch number: 120967-201706), colloidal myrrh reference medicinal materials (China Food and Drug Inspection Institute, batch number: 121250-201806), natural myrrh formula granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch number: 2309060; 2309061; 2309062), colloidal myrrh formula granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch number: 2309063; 2309064; 2309065).

[0061] 1.2 Thin layer method establishment

[0062] 1.2.1 Preparation of test solution

[0063] Take 2g of this product, grind it into powder, add 20ml of water, then add 2ml of hydrochloric acid, heat and reflux for 1h, filter, evaporate the filtrate to dryness, dissolve the residue in 20ml of water, extract twice with 20ml of ethyl acetate, combine the ethyl acetate liquid, evaporate to dryness, dissolve the residue in 2ml of ethanol, and use it as the test solution.

[0064] 1.2.2 Preparation of reference medicinal material solution

[0065] Take 1g of natural myrrh reference medicinal material, add 2ml of cyclohexane according to the volatile oil determination method (Chinese Pharmacopoeia 2020 General Rules 2204 B method), slowly heat to boiling, and keep it slightly boiling for about 2.5 hours. After standing, take the cyclohexane liquid to make a natural myrrh reference medicinal material solution. Take 1g of colloidal myrrh reference medicinal material and make a colloidal myrrh reference medicinal material solution in the same way.

[0066] 1.3.3 Determination method

[0067] According to the thin layer chromatography method (Appendix VIB of Part I of the Chinese Pharmacopoeia 2020 Edition), 10 μl of the test sample solution and 10 μl of the control drug solution were respectively spotted on the same silica gel G thin layer plate, and cyclohexane: ethyl acetate: glacial acetic acid: 0.1 mol / L hydrochloric acid (10:1:1:0.5) was used as the developing solvent. The plate was developed, taken out, dried, and examined under ultraviolet light (365 nm).

[0068] 1.3 Methodological considerations

[0069] 1.3.1 Sampling quantity inspection

[0070] Under the experimental conditions proposed above, 5μl, 10μl, and 15μl of natural myrrh control medicinal material solution, 5μl, 10μl, and 15μl of natural myrrh formula granule solution, 5μl, 10μl, and 15μl of colloidal myrrh control medicinal material solution, and 5μl, 10μl, and 15μl of colloidal myrrh formula granule solution were taken and spotted on the same silica gel G thin layer plate. The plate was developed and examined according to the proposed thin layer chromatography conditions. The results are shown in Figure 1(Among them, 1 to 3 are natural myrrh control medicinal material solutions, 4 to 6 are natural myrrh formula granule solutions, 7 to 9 are natural myrrh formula granule solutions, and 10 to 12 are colloidal myrrh formula granule solutions). Figure 1 It can be seen that when 5-15μl of the reference solution, reference medicinal material solution, and test solution are spotted, the spots are clearly colored and have good separation, and the spot volume is determined to be 5-15μl. In addition, at the ratio shift value of 0.513, the natural myrrh formula particles show pink fluorescent spots, while the colloidal myrrh formula particles do not have such spots. The fluorescent spots can be used to distinguish the natural myrrh formula particles from the colloidal myrrh formula particles. Whether there is a pink fluorescent spot at the ratio shift value of 0.513 can be used as a distinguishing point.

[0071] 1.3.2 Exclusivity inspection

[0072] According to the above test sample preparation method, negative solution, natural myrrh reference medicinal material solution, natural myrrh formula granule solution, colloidal myrrh reference medicinal material solution, and colloidal myrrh formula granule solution were prepared respectively, and thin layer identification experiments were carried out. The results are shown in Figure 2 , wherein 1 is a negative solution, 2 is a natural myrrh reference medicinal material solution, 3 is a natural myrrh formula granule solution, 4 is a colloidal myrrh reference medicinal material solution, and 5 is a colloidal myrrh formula granule solution; Figure 2 It can be seen that the negative sample has no interference with the natural myrrh and colloidal myrrh formula granules, and the method has good specificity. At the ratio shift value of 0.519, the natural myrrh formula granules show pink fluorescent spots, while the colloidal myrrh formula granules do not have such spots.

[0073] 1.3.3 Durability test

[0074] 1.3.3.1 Comparison of different thin layer plates

[0075] The cuttable thin layer chromatography plates of Tianjin Silida Technology Co., Ltd., Merck, and the silica gel plates of Qingdao Yumingyuan Silica Gel Reagent Co., Ltd. were selected and tested according to the proposed test methods. Figures 3 to 5 , where 1 is a natural myrrh reference medicinal material solution, 2 is a natural myrrh formula granule solution, 3 is a colloidal myrrh reference medicinal material solution, and 4 is a colloidal myrrh formula granule solution; the results show that the pink fluorescent spot ratio shift values ​​(Rf) of the three thin layer plates at 7.3 to 9.9 cm from the origin are 0.522, 0.511, and 0.519, respectively, which can meet the identification requirements. And at the ratio shift values ​​of 0.522, 0.511, and 0.519, the natural myrrh formula granules show pink fluorescent spots, while the colloidal myrrh formula granules do not have such spots.

[0076] 1.3.3.2 Comparison of different temperatures

[0077] Take the spotted thin layer plate and develop it at a low temperature of 4°C and a normal temperature of 23°C. Figures 6-7 It can be seen that this method has good adaptability to different temperatures, and the natural myrrh formula particles show pink fluorescent spots at the ratio shift values ​​of 0.538 and 0.516, while the colloidal myrrh formula particles do not have such spots.

[0078] 1.3.3.3 Comparison of different humidity levels

[0079] The thin layer plate after spotting was developed in a humidity environment of 31% RH and 60% RH, respectively. Figures 8-9 ; Among them, 1 is a natural myrrh reference medicinal material solution, 2 is a natural myrrh formula granule solution, 3 is a colloidal myrrh reference medicinal material solution, and 4 is a colloidal myrrh formula granule solution. Figures 8-9 It can be seen that this method has good adaptability to different humidity levels, and the natural myrrh formula particles show pink fluorescent spots at the ratio shift values ​​of 0.532 and 0.529, while the colloidal myrrh formula particles do not have such spots.

[0080] 1.3.4 Verification

[0081] Take the natural myrrh reference medicinal material solution, the natural myrrh formula granule solution, the colloidal myrrh reference medicinal material solution, and the colloidal myrrh formula granule solution respectively, spot them on the same silica gel G thin layer plate, and analyze them according to the determined method. The experimental results are shown in Fig.12 ; Among them, 1 is a natural myrrh control medicinal material solution, 2-4 are natural myrrh formula granule solutions, 5 is a colloidal myrrh control medicinal material solution, and 6-8 are colloidal myrrh formula granule solutions. The results show that the natural myrrh formula granules have pink fluorescent spots at a ratio transfer value (Rf) of 0.514, while the colloidal myrrh formula granules do not have such spots, and the fluorescent spots can be used to distinguish the two.

[0082] 1.4 Determination of the identification point Rf of natural myrrh formula granules and colloidal myrrh formula granules

[0083] The data of various ratio shift values ​​in the thin layer chromatography methodology investigation are summarized, and the results are shown in Table 1. According to the summary results, it is determined that the ratio shift value (Rf) of the identification spot of natural myrrh and colloidal myrrh formula particles should be within the range of 10%, and the specified value is 0.469-0.573.

[0084] Table 1 Summary of methodological investigation ratios

[0085]

[0086] Comparative Example 1

[0087] Take 20g of natural myrrh and colloid myrrh (remove impurities) and measure according to the volatile oil determination method (General Rule 2204 Method B). Take an appropriate amount of volatile oil and add cyclohexane to make a solution containing 10mg of natural myrrh and 50mg of colloid myrrh per 1ml, as the test solution. Take 2g of natural myrrh control medicinal materials and colloid myrrh control medicinal materials, add 2ml of cyclohexane according to the volatile oil determination method (General Rule 2204 Method B), slowly heat to boiling, and keep it slightly boiling for about 2.5 hours. After standing, take the cyclohexane solution as the control medicinal material solution. According to the thin layer chromatography method (General Rule 0502), take 4μl of each of the above two solutions and spot them on the same silica gel G thin layer plate, use cyclohexane-ether (4:1) as the developing solvent, develop, take out, dry, and immediately spray with 10% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly colored. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions in the chromatogram of the control medicinal material, such as Fig.11 Among them, 1-2 is a natural myrrh reference medicinal material solution; 3-4 is a natural myrrh formula granule solution; 5-6 is a colloidal myrrh reference medicinal material solution; 7-8 is a colloidal myrrh formula granule solution.

[0088] Fig.11 The results showed that using the above-mentioned thin layer identification method, there were no obvious differences in the natural myrrh and colloidal myrrh formula particles, and the two could not be identified.

[0089] Comparative Example 2

[0090] Chromatographic conditions: Chromatographic plate: Silica gel G was wet-plated, activated at 105°C for 1 h, and placed in a desiccator for use; developing agent: cyclohexane; color developer: 5% vanillin hydrochloric acid solution.

[0091] Preparation of test solution: Take 0.5g of the test sample and control medicinal material powder, add 5ml of ether respectively, plug and soak for 1h, filter, and use the filtrate as the test solution. Take appropriate amount of the test sample and control medicinal material powder, extract the volatile oil by distillation, add anhydrous sodium sulfate to dehydrate, and add ether to dilute to a 10% solution as the test solution.

[0092] Chromatographic method and results: Take 5ul of each of the above test solutions, spot them on the same silica gel G thin layer plate, develop about 11cm with cyclohexane, take out and dry, then spray with 5% vanillin hydrochloric acid solution, and observe under sunlight. The results showed that the ether extract of natural myrrh showed 1 blue spot, and the volatile oil showed 2 blue spots; while the two extracts of colloid showed no spots at the corresponding positions. Fig.12 Among them, 1-2 is a natural myrrh reference medicinal material solution; 3-4 is a natural myrrh formula granule solution; 5-6 is a colloidal myrrh reference medicinal material solution; 7-8 is a colloidal myrrh formula granule solution.

[0093] Fig.12The results showed that using the above-mentioned thin layer identification method, there were no obvious differences in the granules of natural myrrh and colloidal myrrh formula, and the two could not be distinguished.

[0094] Comparative Example 3

[0095] Take an appropriate amount of this product, grind it into powder, take 0.2g, add 10ml of methanol, ultrasonically treat for 20 minutes, filter, evaporate the filtrate to dryness below 60°C, add 1ml of methanol to the residue to dissolve it, and use it as the test solution. Take another 0.2g of myrrh (natural myrrh) as a control medicinal material, and prepare a control medicinal solution in the same way. According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), take 5ul of each of the above two solutions, and spot them on the same silica gel G thin layer plate, using xylene-ethyl acetate (19:1) as the developing agent, develop, take out, dry, and immediately spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and examine them under sunlight and ultraviolet light (365nm) respectively. In the chromatogram of the test product, spots of the same color or fluorescent spots appear at the corresponding positions of the chromatogram of the control medicinal material. If Fig.13 , among which, 1 is a natural myrrh control medicinal material solution; 2 is a natural myrrh formula granule solution; 3 is a colloidal myrrh control medicinal material solution; and 4 is a colloidal myrrh formula granule solution.

[0096] Fig.13 The results showed that using the above-mentioned thin layer identification method, there were no obvious differences in the granules of natural myrrh and colloidal myrrh formula, and the two could not be distinguished.

[0097] Comparative Example 4

[0098] Take 2g of this product, grind it, add 20ml of water, then add 2ml of hydrochloric acid, heat and reflux for 1h, filter, evaporate the filtrate, dissolve the residue in 20ml of water, extract twice with 20ml of ethyl acetate, combine the ethyl acetate solution, evaporate to dryness, and dissolve the residue in 2ml of ethanol as the test solution. Take 1g of natural myrrh reference medicinal material, add 2ml of cyclohexane according to the volatile oil determination method (Chinese Pharmacopoeia 2020 General Rules 2204 B method), slowly heat to boiling, and keep it slightly boiling for about 2.5 hours. After standing, take the cyclohexane solution to make a natural myrrh reference medicinal material solution. Take 1g of colloidal myrrh reference medicinal material and make a colloidal myrrh reference medicinal material solution in the same way. According to the thin layer chromatography method (Appendix VIB of Part I of the Chinese Pharmacopoeia 2020 edition), 5 μl of the test sample solution and 5 μl of the control drug solution were respectively spotted on the same silica gel G thin layer plate, and cyclohexane: ethyl acetate: glacial acetic acid: 0.1 mol / L hydrochloric acid (15:1:1:0.5) was used as the developing solvent. The plate was developed, taken out, dried, and examined under ultraviolet light (365 nm).

[0099] Test results such as Fig.14, wherein 1 is a natural myrrh reference medicinal material solution; 2 is a natural myrrh formula granule solution; 3 is a colloidal myrrh reference medicinal material solution; 4 is a colloidal myrrh formula granule solution; Fig.14 The results showed that using the above-mentioned thin layer identification method, there were no obvious differences in the granules of natural myrrh and colloidal myrrh formula, and the two could not be distinguished.

[0100] Comparative Example 5

[0101] Take 2g of this product, grind it, add 20ml of water, then add 2ml of hydrochloric acid, heat and reflux for 1h, filter, evaporate the filtrate, dissolve the residue in 20ml of water, extract twice with 20ml of ethyl acetate, combine the ethyl acetate solution, evaporate to dryness, and dissolve the residue in 2ml of ethanol as the test solution. Take 1g of natural myrrh reference medicinal material, add 2ml of cyclohexane according to the volatile oil determination method (Chinese Pharmacopoeia 2020 General Rules 2204 B method), slowly heat to boiling, and keep it slightly boiling for about 2.5 hours. After standing, take the cyclohexane solution to make a natural myrrh reference medicinal material solution. Take 1g of colloidal myrrh reference medicinal material and make a colloidal myrrh reference medicinal material solution in the same way. According to the thin layer chromatography method (Appendix VIB of Part I of the Chinese Pharmacopoeia 2020 edition), 5 μl of the test sample solution and 5 μl of the control drug solution were respectively spotted on the same silica gel G thin layer plate, and cyclohexane: ethyl acetate: glacial acetic acid: 0.1 mol / L hydrochloric acid (5:1:1:0.5) was used as the developing solvent. The plate was developed, taken out, dried, and examined under ultraviolet light (365 nm).

[0102] Test results such as Fig.15 , wherein 1 is a natural myrrh reference medicinal material solution; 2 is a natural myrrh formula granule solution; 3 is a colloidal myrrh reference medicinal material solution; 4 is a colloidal myrrh formula granule solution; Fig.15 The results showed that using the above-mentioned thin layer identification method, there were no obvious differences in the granules of natural myrrh and colloidal myrrh formula, and the two could not be distinguished.

[0103] It can be seen from the above embodiments that the present invention provides a thin layer identification method for natural myrrh and colloidal myrrh formula particles, comprising the following steps: A) taking a sample to be tested, dissolving it with a solvent, and obtaining a test solution; the sample to be tested is natural myrrh formula particles and colloidal myrrh formula particles; B) taking a natural myrrh control medicinal material and a colloidal myrrh control medicinal material, dissolving them with solvents respectively, to obtain a control medicinal material solution; C) subjecting the test solution and the control medicinal material solution to thin layer chromatography detection, the thin layer plate is a silica gel G thin layer plate; the developing agent is cyclohexane-ethyl acetate-glacial acetic acid-0.1 mol / L hydrochloric acid in a volume ratio of (8-10):(0.8-1.2):(0.8-1.2):(0.3-0.7); D) inspecting under ultraviolet light whether there are fluorescent spots in the specified ratio shift value Rf in the test sample chromatogram as an identification point to distinguish the natural myrrh formula particles and the colloidal myrrh formula particles. The thin layer chromatography identification method for natural myrrh and colloidal myrrh formula particles established by the present invention uses the observation of whether spots exist in the thin layer chromatography spectrum within a specified ratio shift value range as identification points to quickly and effectively identify the natural myrrh formula particles and the colloidal myrrh formula particles.

[0104] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A thin layer identification method for natural myrrh and colloidal myrrh formula particles, comprising the following steps: A) Take natural myrrh formula granules and colloidal myrrh formula granules, add water and hydrochloric acid respectively, heat and reflux, filter, evaporate the filtrate to dryness, dissolve the residue in water, and then extract with ethyl acetate, evaporate the obtained ethyl acetate solution to dryness, add ethanol to the residue, and obtain natural myrrh formula granule solution and colloidal myrrh formula granule solution respectively as test solution; The mass of the natural myrrh formula particles and the volume ratio of water and hydrochloric acid is 2.0 g: (18-23) mL: (1.8-2.3) mL; The mass ratio of the colloidal myrrh formula particles to water and hydrochloric acid is 2.0 g: (18-23) mL: (1.8-2.3) mL; The mass ratio of the natural myrrh formula particles to the volume ratio of ethyl acetate is 2.0 g: (18-23) mL; The mass ratio of the colloidal myrrh formula particles to the volume ratio of ethyl acetate is 2.0 g: (18-23) mL; B) taking a natural myrrh reference medicinal material and a colloidal myrrh reference medicinal material and mixing them with cyclohexane respectively, heating them to a slight boil, and taking out the cyclohexane solution after standing to obtain a natural myrrh reference medicinal material solution and a colloidal myrrh reference medicinal material solution; The mass ratio of natural myrrh reference medicinal material to the volume ratio of cyclohexane is 1 g: (1.8-2.3) mL; C) The test solution and the control medicinal material solution are subjected to thin layer chromatography, the thin layer plate is a silica gel G thin layer plate; the developing solvent is cyclohexane-ethyl acetate-glacial acetic acid-0.1 mol / L hydrochloric acid in a volume ratio of (8-10):(0.8-1.2):(0.8-1.2):(0.3-0.7); D) Check under ultraviolet light to see if there are fluorescent spots within the specified shift value Rf in the chromatogram of the test sample, which can be used as the identification point to distinguish the natural myrrh formula granules from the colloidal myrrh formula granules.

2. The thin layer identification method according to claim 1, characterized in that: If there are spots at the Rf value of 0.469-0.573, it is a natural myrrh formula granule; If there are no spots at the Rf value of 0.469-0.573, it is a colloidal myrrh formula granule.

3. The identification method according to claim 1, characterized in that: In the thin layer chromatography detection in step C), the spotting volume of the test solution and the control medicinal material solution is 5-15 μL.

4. The identification method according to claim 1, characterized in that: The wavelength examined in step D) is 365 nm.

5. The identification method according to claim 1, characterized in that: The temperature examined in step D) is 4-23°C.

6. The identification method according to claim 1, characterized in that: The humidity checked in step D) is 31% rh~60% rh.

7. The identification method according to claim 1, characterized in that: In the step D), the Rf value is 0.511-0.

538.

8. Use of the identification method according to any one of claims 1 to 7 in distinguishing natural myrrh from colloidal myrrh.

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