Thin-layer chromatography method for simultaneous identification of multiple components in refined Pantao pills and its application

Abstract

The present invention discloses a thin layer chromatography identification method for simultaneously identifying multiple components in a refined flat peach pill preparation and its application, which belongs to the technical field of quality control of traditional Chinese medicine. The thin layer chromatography identification method of the present invention is divided into two parts. The first part adopts methanol ultrasonic extraction and is developed by toluene-ethyl acetate-formic acid-methanol system, which can simultaneously identify the alpinia oxyphylla, wolfberry and fenxinmu in the refined flat peach pill; the second part is based on the methanol ultrasonic extraction, and hydrochloric acid is added to heat and reflux extraction, shaken and evaporated, and then ethanol is redissolved and developed by petroleum ether-ethyl acetate system, which can identify the ophiopogon in the refined flat peach pill. That is, only two sample preparation methods are required, and one thin layer plate can simply, quickly and effectively identify the four medicinal materials in the refined flat peach pill, and the color spots are clear, without tailing, without cross-linking and good specificity.

Description

Technical Field

[0001] The invention relates to a thin layer chromatography identification method for simultaneously identifying multiple components in a refined Pantao pill preparation and application thereof, belonging to the technical field of traditional Chinese medicine quality control. Background Art

[0002] Refined Pantao Pills are a streamlined version of the traditional Chinese medicine Pantao Pills (also known as Qinggong Shoutao Pills, National Medicine Approval No. Z12020286). Traditional Pantao Pills are composed of 13 herbs: ginseng, asparagus cochinchinensis, ophiopogon japonicus, raw rehmannia root, angelica sinensis, wolfberry fruit, alpinia oxyphylla, silkworm excrement, jujube seed, and fenxin wood. Its formula adheres to the longevity theory of the Yellow Emperor's Classic of Internal Medicine, which emphasizes "protecting and supporting yang qi as the foundation" and "yin essence nourishes human longevity." These pills are believed to tonify the kidneys, promote spermatogenesis, and strengthen the vital energy. The refined Pantao Pills described in this invention, however, are formulated with a carefully selected blend of four core herbs: alpinia oxyphylla, fenxin wood, wolfberry fruit, and ophiopogon japonicus.

[0003] Thin layer chromatography, as an important technical means for qualitative identification in the quality standards of traditional Chinese medicine compound preparations, plays a key role in ensuring the quality of drugs. However, there are no relevant research reports on thin layer chromatography identification methods for refined Pantao Pills in the prior art. In the 2020 edition of the Chinese Pharmacopoeia, the thin layer identification method of Psoralea corylifolia was not included, and the identification of the other three medicinal materials still adopted the form of single medicinal flavor identification, that is, different medicinal materials need to be identified independently using different methods. Such existing methods not only cannot meet the overall quality control requirements of refined Pantao Pills, but also require multiple tests in actual operation, which has problems such as time-consuming and consumables.

[0004] In view of this, the present invention aims to establish a thin-layer chromatography method capable of simultaneously identifying multiple components of refined Pantao Pills, achieving good separation and no interference. The establishment of this method is extremely important for the quality control of refined Pantao Pills, effectively improving the drug's quality stability and reliability, and providing strong support for the product's clinical application. Summary of the Invention

[0005] The present invention aims to address the problems of the prior art by providing a thin-layer chromatography identification method for simultaneously identifying multiple components in a refined Pantao Pill preparation. The method utilizes two extraction methods to prepare two test sample solutions, enabling comprehensive identification of the four medicinal ingredients in the refined Pantao Pill formula and simultaneously identifying three of them. While ensuring identification efficiency, the method also exhibits high stability and repeatability.

[0006] At the same time, the present invention provides an application of a thin layer chromatography identification method for simultaneously identifying multiple components in a refined Pantao pill preparation.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0008] A thin layer chromatography identification method for simultaneously identifying multiple components in a refined Pantao pill preparation comprises the following steps:

[0009] (1) Preparation of test solution

[0010] Take the refined Pantao pill preparation, firstly extract it with methanol ultrasonically as the test solution 1;

[0011] Take the test solution 1, add hydrochloric acid to reflux extraction, then shake the filtrate to extract, evaporate the solution to dryness, and re-dissolve the resulting residue in ethanol to prepare the test solution 2;

[0012] (2) Preparation of control medicinal material solution

[0013] Take the control medicinal materials of Alpinia oxyphylla, Fenxinmu and Fructus Lycii, add methanol and ultrasonicate respectively to prepare the control medicinal materials solutions of Alpinia oxyphylla, Fenxinmu and Fructus Lycii;

[0014] Take Ophiopogon japonicus reference medicinal material, add methanol to sonicate, add hydrochloric acid to reflux, filter, shake the filtrate to extract, evaporate the solution, and dissolve the residue in ethanol to prepare Ophiopogon japonicus reference medicinal material solution;

[0015] (3) Thin layer chromatography detection

[0016] The test solution 1 prepared in step (1), the Alpinia oxyphylla control medicinal material solution, the Herba Lycii control medicinal material solution, and the Herba Lycii control medicinal material solution prepared in step (2) are respectively spotted on the same silica gel G thin layer plate, developed with a toluene-ethyl acetate-formic acid-methanol system, taken out, dried, and examined under an ultraviolet lamp. In the chromatogram of the test sample, fluorescent spots of the same color are shown at the positions corresponding to those of the Herba Lycii control medicinal material and the Herba Lycii control medicinal material; and then examined under an ultraviolet lamp of another wavelength, spots of the same color are shown at the positions corresponding to those of the Herba Alpiniae oxyphylla control medicinal material in the chromatogram of the test sample.

[0017] The test solution 2 prepared in step (1) and the Radix Ophiopogonis control medicinal material solution prepared in step (2) are respectively spotted on the same silica gel G thin layer plate, developed with a petroleum ether-ethyl acetate system, removed, air-dried, and examined under ultraviolet light. In the chromatogram of the test sample, fluorescent spots of the same color appear at positions corresponding to those of the Radix Ophiopogonis control medicinal material. The refined Pantao Pill preparation includes refined Pantao Pill, refined Pantao Pill fluid extract, or a mixture of a refined Pantao Pill aqueous extract and an alcohol extract.

[0018] The refined Pantao pill preparation is composed of 30-55 parts by weight of wolfberry, 5-18 parts by weight of fenxinmu, 5-18 parts by weight of alpinia oxyphylla, and 12-32 parts by weight of ophiopogon japonicus. The preparation method is as follows:

[0019] (1) Preparation of a mixture of water extract and alcohol extract of refined Pantao pills: extract Alpinia oxyphylla with 8-12 times of 50%-90% ethanol for 1-3 times, each time for 0.5-2.5 hours, filter, and set aside the residue and alcohol extract; extract Lycium barbarum, Dioscorea chinensis, Radix Ophiopogonis, and Alpinia oxyphylla with 8-15 times of water for 1-3 times, each time for 1-2.5 hours, filter, and mix the filtrate with the alcohol extract for later use;

[0020] The extraction method is preferably hot reflux extraction;

[0021] (2) Preparation of refined Pantao pills fluid extract: Take the mixture of the water extract and the alcohol extract of the refined Pantao pills in process (1), concentrate under reduced pressure to an extract with a relative density of 1.25-1.30 at 25°C, and set aside;

[0022] (3) Preparation of refined Pantao pills: Take the refined Pantao pills fluid extract prepared in process (2), add the excipients, mix well, and prepare into pills for later use.

[0023] in:

[0024] The volume ratios of methanol during the ultrasonic process and hydrochloric acid during the reflux process in step (1) are 5-30 and 0.1-3, respectively.

[0025] In step (1), the shaking extraction time when preparing the test solution 2 is 0.2-2 h.

[0026] In step (1), the temperature of the evaporated solution when preparing the test solution 2 is 40-90 °C.

[0027] In step (1), when the raw material is refined flat peach pills, the refined flat peach pills must first be crushed into fine powder, and then the test solution 1 or the test solution 2 is prepared.

[0028] In step (1), the mass volume ratio of the refined flat peach pills to methanol is 1.0:4.0-5.0, and the unit is g / mL.

[0029] In step (1), the ultrasonic time is 10-30 min, and the ultrasonic power is 100-200w.

[0030] In step (1), the hydrochloric acid reflux extraction time is 5-10 minutes. In step (2), the volume ratios of methanol during the ultrasonic process and hydrochloric acid during the reflux process are 2-20 and 0.5-2, respectively.

[0031] The mass volume ratio of the Alpinia oxyphylla reference medicinal material and methanol in step (2) is 1.0:5.0, and the unit is g / mL.

[0032] The mass volume ratio of the control medicinal material of the genus Psoralea corylifolia in step (2) to methanol is 1.0:5.0, and the unit is g / mL.

[0033] The mass volume ratio of the wolfberry reference medicinal material and methanol in step (2) is 1.0:5.0, and the unit is g / mL.

[0034] The mass volume ratio of the Ophiopogon japonicus control medicinal material to methanol in step (2) is 1.0:5.0, and the unit is g / mL.

[0035] In step (2), the shaking extraction time for preparing the Radix Ophiopogonis control medicinal material solution is 0.2-2 h.

[0036] In step (2), when preparing the Radix Ophiopogonis control medicinal material solution, the temperature of the evaporated solution is 40-90°C.

[0037] In step (2), the ultrasonic time is 10-30 min and the ultrasonic power is 100-200w;

[0038] In step (2), the hydrochloric acid reflux extraction time is 5-10 min.

[0039] In the toluene-ethyl acetate-formic acid-methanol system of step (3), the volume ratio of toluene, ethyl acetate, formic acid, and methanol is 2-8:2-8:0.1-1:1-3.

[0040] In the petroleum ether-ethyl acetate system of step (3), the volume ratio of petroleum ether to ethyl acetate is 1-5:1-5.

[0041] In step (3), the wavelength of the ultraviolet lamp for the arborvitae and wolfberry is 365 nm, and the wavelength of the ultraviolet lamp for the alpinia oxyphylla is 254 nm.

[0042] In step (3), the wavelength of the ultraviolet lamp for the Ophiopogon japonicus is 365 nm.

[0043] In the 2020 edition of the Chinese Pharmacopoeia, the thin layer chromatography identification method of the wood is not included, and for the identification of the other three medicinal materials, the form of single medicinal flavor identification is still adopted, that is, different medicinal materials need to be independently identified using different methods. Such existing methods not only cannot meet the overall quality control requirements of the refined flat peach pills, but also require multiple tests in actual operation, which has problems such as time-consuming and consumables. The thin layer chromatography identification method of the present invention is divided into two parts. The first part uses methanol ultrasonic extraction and toluene-ethyl acetate-formic acid-methanol system for development, which can simultaneously identify the Alpinia oxyphylla, wolfberry and wood in the refined flat peach pills; the second part is based on methanol ultrasonic extraction, hydrochloric acid is added to heat reflux extraction, and then the solution is shaken, evaporated and redissolved in ethanol, and developed with petroleum ether-ethyl acetate system to identify the Ophiopogon japonicus in the refined flat peach pills. Only two thin-layer plates are needed to simply and effectively identify the four medicinal ingredients in refined Pantao Pills. The color spots are clear, without tailing or cross-talk, and have good specificity. They can meet the daily work of identifying the active ingredients in refined Pantao Pills and achieve quality control of refined Pantao Pills.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] The present invention provides a thin-layer chromatography identification method for simultaneously identifying multiple components in refined Pantao Pills. This method utilizes two sequential extraction methods to prepare two test solutions, enabling comprehensive identification of all four medicinal ingredients in the refined Pantao Pills formula. This method not only ensures efficient identification but also exhibits high stability and repeatability. It effectively addresses the existing challenges of requiring the preparation of multiple test solutions and cumbersome testing procedures, providing an important reference for quality control of refined Pantao Pills and possessing significant significance.

[0046] The present invention belongs to the technical field of quality control of traditional Chinese medicine, and specifically relates to a thin-layer chromatography identification method for simultaneously identifying multiple ingredients in refined Pantao Pills. The thin-layer chromatography identification method of the present invention is divided into two parts. The first part uses methanol ultrasonic extraction and development with a toluene-ethyl acetate-formic acid-methanol system to simultaneously identify Alpinia oxyphylla, Lycium barbarum, and Dioscorea chinensis in refined Pantao Pills. The second part, based on the methanol ultrasonic extraction, adds hydrochloric acid and heats under reflux for extraction, shakes and evaporates to dryness, then redissolves in ethanol and develops with a petroleum ether-ethyl acetate system to identify Radix Ophiopogonis in refined Pantao Pills. That is, only two sample preparation methods are required, and one thin-layer plate can simply, quickly, and effectively identify the four medicinal materials in refined Pantao Pills, and the color spots are clear, without tailing, cross-linking, and good specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1The TLC spectrum of the refined Pantao Pills of Alpinia oxyphylla, Fenxinmu, and Lycium barbarum under 365 nm ultraviolet light (including the specificity investigation spectrum) in Example 1 is as follows: 1 is the test solution; 2 is the Alpinia oxyphylla control medicinal material; 3 is the Alpinia oxyphylla negative control; 4 is the Fenxinmu control medicinal material; 5 is the Fenxinmu negative control; 6 is the Lycium barbarum control medicinal material; 7 is the Lycium barbarum negative control;

[0048] Figure 2 The TLC spectrum of the refined Pantao Pills of Alpinia oxyphylla, Fenxinmu, and Lycium barbarum at 254 nm (including the specificity investigation spectrum) in Example 1 is shown. The numbers in the figure are as follows: 1 is test solution 1; 2 is Alpinia oxyphylla control medicinal material; 3 is Alpinia oxyphylla negative control; 4 is Fenxinmu control medicinal material; 5 is Fenxinmu negative control; 6 is Lycium barbarum control medicinal material; 7 is Lycium barbarum negative control;

[0049] Figure 3 This is the TLC spectrum of the refined Pantao Pills and Radix Ophiopogonis at 365 nm (including the specificity analysis spectrum) in Example 1. The labels in the figure are as follows: 1 is the test solution 2; 2 is the Radix Ophiopogonis control medicinal material; 3 is the Radix Ophiopogonis negative control;

[0050] Figure 4 This is the TLC spectrum of the refined Pantao Pills of Example 2, Alpinia oxyphylla, Fenxinmu, and Lycium barbarum, under a 365 nm UV lamp; the labels in the figure are as follows: 1, 2, and 3 are test solution 1; 4 is Alpinia oxyphylla control medicinal material; 5 is Fenxinmu control medicinal material; 6 is Lycium barbarum control medicinal material;

[0051] Figure 5 This is the TLC spectrum of the refined Pantao Pills of Alpinia oxyphylla, Fenxinmu, and Lycium barbarum at 254 nm in Example 2; the labels in the figure are as follows: 1, 2, and 3 are test solution 1; 4 is Alpinia oxyphylla control medicinal material; 5 is Fenxinmu control medicinal material; 6 is Lycium barbarum control medicinal material;

[0052] Figure 6 This is the TLC spectrum of the refined Pantao Pills and Radix Ophiopogonis identified in Example 2 at 365 nm; the labels in the figure are as follows: 1, 2, and 3 are test solution 2; 4 is Radix Ophiopogonis control medicinal material;

[0053] Figure 7 This is the TLC spectrum of the refined Pantao Pills of Example 3, Alpinia oxyphylla, Fenxinmu, and Lycium barbarum, under a 365 nm UV lamp; the labels in the figure are as follows: 1, 2, and 3 are test solution 1; 4 is Alpinia oxyphylla control medicinal material; 5 is Fenxinmu control medicinal material; 6 is Lycium barbarum control medicinal material;

[0054] Figure 8This is the TLC spectrum of the refined Pantao Pills containing Alpinia oxyphylla, Fenxinmu, and Lycium barbarum at 254 nm in Example 3; the labels in the figure are as follows: 1, 2, and 3 are test solution 1; 4 is Alpinia oxyphylla control medicinal material; 5 is Fenxinmu control medicinal material; 6 is Lycium barbarum control medicinal material;

[0055] Figure 9 This is the TLC spectrum of the refined Pantao Pills and Radix Ophiopogonis identified in Example 3 at 365 nm; the labels in the figure are as follows: 1.2.3 are test solution 2; 4 is Radix Ophiopogonis control medicinal material;

[0056] Figure 10 The TLC spectrum of the refined Pantao Pills of Alpinia oxyphylla, Fenxinmu, and Lycium barbarum under 365 nm ultraviolet light (including the specificity investigation spectrum) in Example 4 is shown in the figure. The labels in the figure are as follows: 1 is the test solution; 2 is the Alpinia oxyphylla control medicinal material; 3 is the Alpinia oxyphylla negative control; 4 is the Fenxinmu control medicinal material; 5 is the Fenxinmu negative control; 6 is the Lycium barbarum control medicinal material; 7 is the Lycium barbarum negative control;

[0057] Figure 11 The TLC spectrum of the refined Pantao Pills of Alpinia oxyphylla, Fenxinmu, and Lycium barbarum at 254 nm (including the specificity investigation spectrum) in Example 4 is shown. The labels in the figure are as follows: 1 is test solution 1; 2 is Alpinia oxyphylla control medicinal material; 3 is Alpinia oxyphylla negative control; 4 is Fenxinmu control medicinal material; 5 is Fenxinmu negative control; 6 is Lycium barbarum control medicinal material; 7 is Lycium barbarum negative control;

[0058] Figure 12 This is the TLC spectrum of the refined Pantao Pills and Radix Ophiopogonis at 365 nm (including the specificity analysis spectrum) in Example 4. The labels in the figure are as follows: 1 is the test solution 2; 2 is the Radix Ophiopogonis control medicinal material; 3 is the Radix Ophiopogonis negative control;

[0059] Figure 13 The TLC spectrum of the refined Pantao Pills of Alpinia oxyphylla, Fenxinmu, and Lycium barbarum under 365 nm ultraviolet light (including the specificity investigation spectrum) in Example 5 is shown in the figure. The labels in the figure are as follows: 1 is the test solution; 2 is the Alpinia oxyphylla control medicinal material; 3 is the Alpinia oxyphylla negative control; 4 is the Fenxinmu control medicinal material; 5 is the Fenxinmu negative control; 6 is the Lycium barbarum control medicinal material; 7 is the Lycium barbarum negative control;

[0060] Figure 14 The TLC spectrum of the refined Pantao Pills of Alpinia oxyphylla, Fenxinmu, and Lycium barbarum at 254 nm (including the specificity investigation spectrum) in Example 5 is shown in the figure. The labels in the figure are as follows: 1 is test solution 1; 2 is Alpinia oxyphylla control medicinal material; 3 is Alpinia oxyphylla negative control; 4 is Fenxinmu control medicinal material; 5 is Fenxinmu negative control; 6 is Lycium barbarum control medicinal material; 7 is Lycium barbarum negative control;

[0061] Figure 15 This is the TLC spectrum at 365 nm (including the specificity investigation spectrum) for the thin layer identification of the refined Pantao Pills and Ophiopogon japonicus in Example 5. The labels in the figure are as follows: 1 is the test solution 2; 2 is the Ophiopogon japonicus control medicinal material; 3 is the Ophiopogon japonicus negative control. DETAILED DESCRIPTION

[0062] The present invention will be further described in detail below with reference to the accompanying drawings and specific examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Example 1

[0063] In Examples 1, 4, and 5, negative samples were prepared. The purpose of preparing negative samples is to verify the specificity of the thin-layer chromatography analysis method. By performing thin-layer chromatography on a negative test sample (a sample without the target medicinal material), a control medicinal material solution, and a test sample solution, and comparing the resulting chromatograms, it is possible to determine whether the target identifying component is interfered with by other components. If the negative test sample chromatogram shows no interfering spots of the same color at positions corresponding to the control medicinal material chromatogram and the test sample chromatogram, this indicates that the negative sample has no interference and the method is highly specific.

[0064] A thin layer chromatography identification method for simultaneously identifying multiple components in a refined Pan Tao Wan preparation was developed, using the refined Pan Tao Wan as the test sample. The preparation methods for the refined Pan Tao Wan and the negative sample were as follows:

[0065] Refined Pantao Pills: Take 10 parts by weight of Alpinia oxyphylla kernel, grind into coarse particles (pass through a 20-mesh sieve), add 8 times the volume of 70% ethanol, soak for 0.5 h, and reflux extract twice, each time for 1.5 h. Reserve the ethanol extract and the Alpinia oxyphylla kernel residue.

[0066] Take 12 parts by weight of fenxin wood, 30 parts by weight of wolfberry, and 12 parts by weight of ophiopogon, and grind them separately (pass the fenxin wood through a 20-mesh sieve, and grind the wolfberry and ophiopogon appropriately. Grind the wolfberry and ophiopogon after grinding, as they are sticky and cannot pass through the sieve, so they can be ground appropriately). Add 10 parts by weight of Alpinia oxyphylla residue (Alpinia oxyphylla can be extracted multiple times to obtain 10 parts by weight of Alpinia oxyphylla residue). Add 10 times the amount of water, and reflux extract twice, each for 1 hour. The aqueous extract is set aside. The aqueous and alcohol extracts are combined and concentrated to a relative density of 1.30 (25°C) to obtain a refined Pantao Pill fluid extract. This refined Pantao Pill fluid extract is then added to starch and microcrystalline cellulose, and pellets are formed using a plasticizing method. After drying, the refined Pantao Pills are obtained.

[0067] Alpinia oxyphylla negative sample: weigh the other three medicinal ingredients except Alpinia oxyphylla. The composition, content and preparation method are the same as those of refined Pantao pills.

[0068] Negative sample of the Chinese prickly ash: weigh the other three medicinal ingredients except the Chinese prickly ash. The composition, content and preparation method are the same as those of the refined Pantao pills.

[0069] Lycium barbarum negative sample: weigh the other three medicinal ingredients except wolfberry, and the composition, content and preparation method are the same as the preparation method of refined Pantao pills.

[0070] Ophiopogon japonicus negative sample: weigh the other three medicinal ingredients except Ophiopogon japonicus. The composition, content and preparation method are the same as those of refined Pantao pills.

[0071] The thin layer chromatography identification method for simultaneously identifying multiple components of the refined Pantao Pills preparation described in Example 1, i.e., the thin layer chromatography identification method for simultaneously identifying the four medicinal ingredients of Alpinia oxyphylla, Fenxinmu, Fructus Lycii, and Radix Ophiopogonis in the refined Pantao Pills, comprises the following steps:

[0072] (1) Preparation of test solution

[0073] Take an appropriate amount of refined Pantao pills (about 5 g), add 20 mL of methanol, ultrasonicate for 30 min (power 100 w), filter, and use as test solution 1.

[0074] Take an appropriate amount of test solution 1 (approximately 10 mL), add 0.5 mL of hydrochloric acid, and heat under reflux in a water bath for 10 minutes. Cool and filter. Shake the filtrate for 1 hour, evaporate to dryness, and dissolve in 2 mL of ethanol to prepare test solution 2.

[0075] (2) Preparation of negative test solutions of Alpinia oxyphylla, Fenxinmu, and Lycium barbarum

[0076] Weigh out the same amount of the negative sample of Angelica sinensis and Alpinia oxyphylla, the negative sample of Dioscorea chinensis, and the negative sample of Lycium barbarum in step (1), and prepare the negative sample solution of Alpinia oxyphylla, the negative sample solution of Dioscorea chinensis, and the negative sample solution of Lycium barbarum in accordance with the method of the sample solution 1 in step (1).

[0077] (3) Preparation of Ophiopogon japonicus negative test solution

[0078] Weigh out an equal amount of Ophiopogon japonicus negative sample as in step (1) and prepare an Ophiopogon japonicus negative test solution according to the method of test solution 2 in step (1).

[0079] (4) Preparation of control medicinal material solution

[0080] Take 1 g of Alpinia oxyphylla, 1 g of Fenxinmu, and 1 g of Fructus Lycii. Follow the method of test solution 1 in step (1) to prepare Alpinia oxyphylla control medicinal material solution, Fenxinmu control medicinal material solution, and Fructus Lycii control medicinal material solution, respectively. The volume of methanol added is 5 mL.

[0081] Take 1 g of Radix Ophiopogonis control medicinal material and prepare Radix Ophiopogonis control medicinal material solution according to the method of test solution 2 in step (1). That is, take 1 g of Radix Ophiopogonis control medicinal material, add 5 mL of methanol, ultrasonicate for 30 min (power 100w), filter, and use as test solution 1. Take an appropriate amount of test solution 1 (about 5 mL), add 0.5 mL of hydrochloric acid, place in a water bath, heat under reflux for 10 min, and cool. Shake for 1 h, evaporate the solution to dryness, and dissolve it in 1 mL of ethanol to use as test solution 2.

[0082] (5) Thin layer chromatography analysis 1

[0083] According to the thin layer chromatography method (General Rules 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia), 1 μL of the test solution prepared in step (1), the negative test solution of Alpinia oxyphylla, the negative test solution of Fructus Lycii, and the negative test solution of Alpinia oxyphylla, the negative test solution of Fructus Lycii prepared in step (2), and 5 μL of the control medicinal material solution of Alpinia oxyphylla, the control medicinal material solution of Fructus Lycii prepared in step (4) were respectively spotted on the same silica gel G thin layer plate, and developed with toluene:ethyl acetate:methanol:formic acid (the volume ratio of toluene:ethyl acetate:methanol:formic acid was 5:4:1:1). The plate was taken out, dried, and examined under ultraviolet light (365 nm). The results are shown in FIG. Figure 1 .

[0084] from Figure 1 As can be seen in the figure, under 365 nm inspection, in the chromatogram of the test sample, at the corresponding position of the control medicinal materials of Fenxinmu and Lycium barbarum, the same color fluorescent spots appear. There is no interference from the negative test samples of Lycium barbarum and Fenxinmu, and the specificity is good. Then put it under ultraviolet light (254 nm) for inspection, the results are shown. Figure 2 In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the chromatogram of the reference medicinal material Alpinia oxyphylla. The negative test sample of Alpinia oxyphylla had no interference and had good specificity.

[0085] (6) Thin layer chromatography analysis 2

[0086] According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 edition, Part IV, General Rules 0502), 2 μL of the test solution prepared in step (1) and 5 μL of the negative test solution of Radix Ophiopogonis prepared in step (3) and 5 μL of the control medicinal material solution of Radix Ophiopogonis prepared in step (4) were taken and spotted on the same silica gel G thin layer plate and the same silica gel GF254 thin layer plate, respectively. The plates were developed with petroleum ether (60-90°C): ethyl acetate (the volume ratio of petroleum ether: ethyl acetate was 1:1), taken out, dried, and examined under ultraviolet light (365 nm). The results are shown in the figure. Figure 3 .

[0087] from Figure 3It can be seen that in the chromatogram of the test sample, spots of the same color appear at the corresponding positions in the chromatogram of the reference medicinal material Ophiopogon japonicus. The negative reference Ophiopogon japonicus has no interference and has good specificity. Example 2

[0088] A thin layer chromatography identification method for simultaneously identifying multiple components in a refined Pantao Pill preparation is provided, using a refined Pantao Pill fluid extract as the test sample. The preparation method of the refined Pantao Pill fluid extract is as follows:

[0089] Refined Pantao Pills fluid extract: Take 18 parts by weight of Alpinia oxyphylla kernel, grind into coarse particles (pass through a 20-mesh sieve), add 12 times the volume of 50% ethanol, soak for 0.5 h, and reflux extract three times, each for 2.5 h. Reserve the ethanol extract and Alpinia oxyphylla kernel residue.

[0090] Grind 18 parts by weight of fenxin wood, 55 parts by weight of wolfberry, and 32 parts by weight of ophiopogon japonicus separately (pass the fenxin wood through a 20-mesh sieve, and grind the wolfberry and ophiopogon japonicus appropriately. Grind the wolfberry and ophiopogon japonicus appropriately, as they are sticky after grinding and cannot pass through a sieve, so they can be ground appropriately). Add 18 parts by weight of Alpinia oxyphylla residue (Alpinia oxyphylla can be extracted multiple times to obtain 20 parts by weight of Alpinia oxyphylla residue). Add 15-fold the amount of water, and reflux extract three times for 2.5 hours each time. Set aside the aqueous extract. Combine the aqueous and alcohol extracts and concentrate to a relative density of 1.25 (25°C) to obtain the refined Pantao Pill fluid extract.

[0091] The thin layer chromatography identification method for simultaneously identifying multiple components of the refined Pantao Pills preparation described in Example 2, i.e., the thin layer chromatography identification method for simultaneously identifying four medicinal materials, namely, Alpinia oxyphylla, Fructus sylvestris, Fructus Lycii, and Radix Ophiopogonis in the refined Pantao Pills fluid extract, comprises the following steps:

[0092] (1) Preparation of test solution

[0093] Take an appropriate amount of refined Pantao Pills fluid extract (about 4 g), add 20 mL of methanol, ultrasonicate for 10 min (power 200 w), filter, and use as test solution 1.

[0094] Take an appropriate amount of test solution 1 (approximately 5 mL), add 0.1 mL of hydrochloric acid, and heat under reflux in a water bath for 5 minutes. Cool and filter. Shake the filtrate for 0.2 hours, evaporate to dryness at 40°C, and dissolve in 2 mL of ethanol to prepare test solution 2.

[0095] (2) Preparation of control medicinal material solution

[0096] Take 1 g of Alpinia oxyphylla, 1 g of Fenxinmu, and 1 g of Fructus Lycii. Follow the method of test solution 1 in step (1) to prepare Alpinia oxyphylla control medicinal material solution, Fenxinmu control medicinal material solution, and Fructus Lycii control medicinal material solution, respectively. The volume of methanol added is 5 mL.

[0097] Take 10 g of Radix Ophiopogonis control medicinal material and prepare Radix Ophiopogonis control medicinal material solution according to the method of test solution 2 in step (1). That is, take 10 g of Radix Ophiopogonis control medicinal material, add 50 mL of methanol, ultrasonicate for 10 min (power 200w), filter, and use as test solution 1. Take an appropriate amount of test solution 1 (about 20 mL), add 2 mL of hydrochloric acid, heat under reflux in a water bath for 5 min, and cool. Shake for 0.2 h, evaporate the solution to dryness, and dissolve it in 5 mL of ethanol to use as test solution 2.

[0098] (3) Thin layer chromatography detection

[0099] like Figure 4 As shown, 1 μL of the test solution prepared in step (1), 5 μL of the control medicinal material solution of Alpinia oxyphylla, the control medicinal material solution of Fenxinmu, and the control medicinal material solution of Lycium barbarum were taken and spotted on the same silica gel G thin layer plate, respectively. The plate was developed with a toluene-ethyl acetate-formic acid-methanol = 2:2:0.3:3 system. The plate was taken out, dried, and examined under a UV lamp (365 nm). The results are shown in FIG. Figure 4 In the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions of the control medicinal materials of Fenxinmu and Lycium barbarum. When examined under ultraviolet light (254 nm), the results were shown. Figure 5 In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the control medicinal material Alpinia oxyphylla.

[0100] Take 2 μL of the test solution prepared in step (1) and 5 μL of the Radix Ophiopogonis control solution prepared in step (2), and spot them on the same silica gel G thin layer plate, develop them with petroleum ether-ethyl acetate = 2:5 system, take them out, dry them, and inspect them under ultraviolet light (365nm). Figure 6 In the chromatogram of the test sample, a fluorescent spot of the same color appeared at the corresponding position of the reference herbal medicine Ophiopogon japonicus. Example 3

[0101] like Figure 7-Figure 9 As shown, a thin layer chromatography identification method for simultaneously identifying multiple components in a refined Pan Tao Wan preparation is used, wherein a mixture of a water extract and an alcohol extract of the refined Pan Tao Wan is used as a test sample, wherein the preparation method of the mixture of a water extract and an alcohol extract of the refined Pan Tao Wan is as follows:

[0102] Refined Pantao Pills: Mixture of aqueous and ethanol extracts: Grind 5 parts by weight of Alpinia oxyphylla kernel into coarse particles (pass through a 20-mesh sieve). Add 10 times the volume of 90% ethanol solution and soak for 0.5 h. Reflux extraction is repeated once, each time for 0.5 h. Reserve the ethanol extract and Alpinia oxyphylla kernel residue.

[0103] Take 5 parts by weight of fenxin wood, 30 parts by weight of wolfberry, and 15 parts by weight of ophiopogon, and grind them separately (pass the fenxin wood through a 20-mesh sieve, and grind the wolfberry and ophiopogon appropriately. Grind the wolfberry and ophiopogon after grinding, as they are sticky after grinding and cannot pass through a sieve, so they can be ground appropriately). Add 5 parts by weight of Alpinia oxyphylla residue (Alpinia oxyphylla can be extracted multiple times to obtain 5 parts of Alpinia oxyphylla residue), add 8 times the amount of water, and reflux extract once for 1 hour each time. The aqueous extract is set aside. The aqueous extract and the alcohol extract are mixed to obtain a mixture of the aqueous extract and the alcohol extract of refined Pantao Pills.

[0104] The thin-layer chromatography identification method for simultaneously identifying multiple components of the refined Pantao Pills preparation described in Example 2, i.e., the thin-layer chromatography identification method for simultaneously identifying four medicinal materials, namely, Alpinia oxyphylla, Fructus sylvestris, Fructus Lycii, and Radix Ophiopogonis, in a mixture of the aqueous extract and the alcohol extract of the refined Pantao Pills, comprises the following steps:

[0105] (1) Preparation of test solution

[0106] Take an appropriate amount of the mixture of the water extract and the alcohol extract of the refined Pantao Pills (about 100 mL), concentrate it to 20 mL, add methanol to make up to 40 mL, and ultrasonicate for 30 min to prepare the test solution 1.

[0107] Take an appropriate amount of test solution 1 (approximately 30 mL), add 3 mL of hydrochloric acid, and heat under reflux in a water bath for 5 minutes. Cool and filter. Shake the filtrate for 2 hours, evaporate to dryness at 90°C, and dissolve in 10 mL of ethanol to prepare test solution 2.

[0108] (2) Preparation of control medicinal material solution

[0109] Take 1 g of Alpinia oxyphylla, 1 g of Fenxinmu, and 1 g of Fructus Lycii. Follow the method of test solution 1 in step (1) to prepare Alpinia oxyphylla control medicinal material solution, Fenxinmu control medicinal material solution, and Fructus Lycii control medicinal material solution, respectively. The volume of methanol added is 5 mL.

[0110] Take 1 g of Radix Ophiopogonis control medicinal material and prepare Radix Ophiopogonis control medicinal material solution according to the method of test solution 2 in step (1). That is, take 1 g of Radix Ophiopogonis control medicinal material, add 5 mL of methanol, sonicate for 7 minutes (power 100w), filter, and use as test solution 1. Take an appropriate amount of test solution 1 (about 2 mL), add 0.5 mL of hydrochloric acid, place in a water bath, heat under reflux for 5 minutes, and cool. Shake for 2 hours, evaporate the solution to dryness, and dissolve it in 1 mL of ethanol to use as test solution 2.

[0111] (3) Thin layer chromatography detection

[0112] 1 μL of the test solution prepared in step (1) and 5 μL of the Alpinia oxyphylla control medicinal material solution, the Herba Lycii control medicinal material solution, and the Herba Lycii control medicinal material solution prepared in step (2) were respectively spotted on the same silica gel G thin layer plate, and developed with a toluene-ethyl acetate-formic acid-methanol = 8:8:0.5:2 system. The plate was taken out, dried, and examined under an ultraviolet lamp (365 nm). In the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions of the Herba Lycii control medicinal material and the Herba Lycii control medicinal material. Then, in the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the Herba Alpiniae oxyphylla control medicinal material.

[0113] 2 μL of the test solution prepared in step (1) and 5 μL of the Ophiopogon japonicus control solution prepared in step (2) were applied to the same silica gel G thin layer plate. The plate was developed with a 5:3 ratio of petroleum ether to ethyl acetate. The plate was removed, air-dried, and examined under a UV lamp (365 nm). In the chromatogram of the test sample, a fluorescent spot of the same color appeared at the corresponding position of the Ophiopogon japonicus control. Example 4

[0114] like Figure 10-12 As shown, the difference between this embodiment and embodiment 1 is only that:

[0115] Toluene-ethyl acetate-formic acid-methanol = 5:5:0.3:3;

[0116] Petroleum ether-ethyl acetate = 5:1. Example 5

[0117] like Figure 13-15 As shown, the difference between this embodiment and embodiment 1 is only that:

[0118] Toluene-ethyl acetate-formic acid-methanol = 2:8:1:3;

[0119] Petroleum ether-ethyl acetate = 1:5.

[0120] It should be understood that in order to streamline the present disclosure and aid understanding of one or more of the various inventive aspects, in the above description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together into a single embodiment, figure, or description thereof. However, this method of disclosure should not be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as reflected in the claims, inventive aspects lie in less than all the features of the foregoing disclosed embodiments. Accordingly, the claims that follow the detailed description are hereby expressly incorporated into this detailed description, with each claim standing on its own as a separate embodiment of the invention.

[0121] Although the present invention has been described with respect to a limited number of embodiments, it will be apparent to those skilled in the art, having benefit of the foregoing description, that other embodiments are contemplated within the scope of the invention thus described. Furthermore, it should be noted that the language used in this specification has been selected primarily for readability and didactic purposes, and not for the purpose of explaining or limiting the subject matter of the invention. Accordingly, many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the appended claims. The disclosure of the present invention is intended to be illustrative and not restrictive of the scope of the invention, which is defined by the appended claims.

[0122] 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 principles 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 chromatography identification method for simultaneously identifying multiple components in a refined Pantao pill preparation, characterized in that: It consists of the following steps: (1) Preparation of test solution: Take the refined Pantao Pill preparation, first use methanol ultrasonic extraction as the test solution 1, or take the extract of the refined Pantao Pill preparation and directly use it as the test solution 1; Take the test solution 1, add hydrochloric acid to reflux extraction, filter after extraction, then shake the filtrate to extract, evaporate the solution to dryness, and re-dissolve the resulting residue in ethanol to prepare the test solution 2; (2) Preparation of control medicinal material solution: Take the control medicinal materials of Alpinia oxyphylla, Fenxinmu and Fructus Lycii, add methanol and ultrasonicate respectively to prepare the control medicinal materials solutions of Alpinia oxyphylla, Fenxinmu and Fructus Lycii; Take Ophiopogon japonicus reference medicinal material, add methanol to sonicate, add hydrochloric acid to reflux, filter, shake the filtrate to extract, evaporate the solution, and dissolve the residue in ethanol to prepare Ophiopogon japonicus reference medicinal material solution; (3) Thin layer chromatography detection The test solution 1 prepared in step (1), the Alpinia oxyphylla control medicinal material solution, the Herba Lycii control medicinal material solution, and the Herba Lycii control medicinal material solution prepared in step (2) were respectively spotted on the same silica gel G thin layer plate, developed with a toluene-ethyl acetate-formic acid-methanol system, taken out, dried, and examined under a 365 nm ultraviolet lamp. In the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions of the Herba Lycii control medicinal material and the Herba Lycii control medicinal material; and then examined under a 254 nm wavelength ultraviolet lamp. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the Herba Alpiniae oxyphylla control medicinal material. The test solution 2 prepared in step (1) and the Ophiopogon japonicus control medicinal material solution prepared in step (2) were respectively spotted on the same silica gel G thin layer plate, developed with petroleum ether-ethyl acetate system, taken out, dried, and examined under a 365 nm ultraviolet lamp. In the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions of the Ophiopogon japonicus control medicinal material. In step (3), in the toluene-ethyl acetate-formic acid-methanol system, the volume ratio of toluene, ethyl acetate, formic acid, and methanol is 2-8:2-8:0.3-1:1-3.

2. The method according to claim 1, characterized in that The refined flat peach pill preparation includes refined flat peach pills, refined flat peach pill fluid extract or a mixture of refined flat peach pill water extract and alcohol extract.

3. The method according to claim 2, characterized in that The refined Pantao pill preparation is composed of 30-55 parts by weight of wolfberry, 5-18 parts by weight of fenxinmu, 5-18 parts by weight of alpinia oxyphylla, and 12-32 parts by weight of ophiopogon japonicus. The preparation method is as follows: Preparation of a mixture of water extract and alcohol extract of refined Pantao pills: Extract the Alpinia oxyphylla with 8-12 times the amount of 50%-90% ethanol for 1-3 times, each time for 0.5-2.5 hours, filter, and set aside the residue and alcohol extract; extract the wolfberry fruit, fenxinmu, ophiopogon japonicus, and Alpinia oxyphylla with 8-15 times the amount of water for 1-3 times, each time for 1-2.5 hours, filter, and mix the filtrate with the alcohol extract for later use; Preparation of refined Pantao pills fluid extract: Take the mixture of the water extract and the alcohol extract of the refined Pantao pills in process (1), concentrate under reduced pressure to an extract with a relative density of 1.25-1.30 at 25°C, and set aside; Preparation of refined Pantao pills: Take the refined Pantao pills fluid extract prepared in process (2), add the excipients, mix well, and prepare into pills for later use.

4. The method according to claim 1, wherein When preparing test solution 2, the shaking extraction time is 0.2-2 h; When preparing test solution 2, the temperature of the evaporated solution should be 40-90°C; The mass volume ratio of refined Pantao Pills or refined Pantao Pills fluid extract to methanol is 1.0:4.0-5.0, and the unit is g / mL.

5. The method according to claim 4, characterized in that The ultrasonic time is 10-30 min, the ultrasonic power is 100-200w; the hydrochloric acid reflux extraction time is 5-10 min.

6. The method according to claim 1, characterized in that The mass volume ratio of Alpinia oxyphylla control medicinal material to methanol is 1.0:5.0, and the unit is g / mL; The mass volume ratio of the control medicinal material of Fenxiong chinensis to methanol was 1.0:5.0, and the unit is g / mL; The mass-to-volume ratio of wolfberry control medicinal material to methanol was 1.0:5.0, and the unit is g / mL; The mass volume ratio of Radix Ophiopogonis control medicinal material to methanol was 1.0:5.0, and the unit is g / mL; The shaking extraction time for preparing the control herbal solution of Ophiopogon japonicus was 0.2-2 h; When preparing the Radix Ophiopogonis control medicinal material solution, the temperature of the evaporated solution was 40-90°C.

7. The method according to claim 6, characterized in that The ultrasonic time is 10-30 min, the ultrasonic power is 100-200w; the hydrochloric acid reflux extraction time is 5-10 min.

8. The method according to claim 1, characterized in that In step (3), in the petroleum ether-ethyl acetate system, the volume ratio of petroleum ether to ethyl acetate is 1-5:1-5.

9. Application of the method according to any one of claims 1 to 8 in the quality control of refined Pantao Pill preparations.

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