Thin-layer chromatography identification method for simultaneously identifying multiple components in refined flat peach pill preparation and application of thin-layer chromatography identification method

Abstract

The invention discloses a thin-layer chromatography identification method for simultaneously identifying multiple components in a refined flat peach pill preparation and application of the thin-layer chromatography identification method, and belongs to the technical field of quality control of traditional Chinese medicines. The thin-layer chromatography identification method is divided into two parts, in the first part, methanol is adopted for ultrasonic extraction, a methylbenzene-ethyl acetate-formic acid-methanol system is adopted for expansion, and then fructus alpiniae oxyphyllae, fructus lycii and diaphragma juglandis in the refined flat peach pills can be identified at the same time; and adding hydrochloric acid into the second part on the basis of methanol ultrasonic treatment, heating, refluxing and extracting, shaking, evaporating to dryness, redissolving with ethanol, and expanding by using a petroleum ether-ethyl acetate system, so as to identify the radix ophiopogonis in the refined flat peach pill. The four medicinal materials in the refined flat peach pill can be simply, quickly and effectively identified only by two sample preparation methods and one thin-layer plate in sequence, and the method has the advantages of clear color spots, no trailing, no crossing 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 quality control of traditional Chinese medicines. Background Art

[0002] Refined Pantao Pills are simplified from the traditional medicine Pantao Pills (also known as Qinggong Shoutao Pills, national medicine standard Z12020286). Traditional Pantao Pills are composed of 13 medicinal materials, including ginseng, asparagus, ophiopogon, raw rehmannia, angelica, wolfberry, alpinia oxyphylla, silkworm excrement, spiny date seed, and fenxinmu. Its formula follows the theory of longevity in the Yellow Emperor's Internal Classic, such as "protecting yang qi as the basis" and "yin essence is the life of the person", and has the effects of nourishing the kidney, producing essence, and strengthening the body. The refined Pantao Pills described in the present invention are made of four core medicinal materials, namely alpinia oxyphylla, fenxinmu, wolfberry and ophiopogon.

[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 the thin layer chromatography identification method for refined Pantao Pills in the prior art. In the 2020 edition of the "Chinese Pharmacopoeia", the thin layer identification method of Fenxinmu is not included, and the identification of the other three medicinal materials still adopts the form of single medicinal flavor identification, that is, different medicinal materials need to be identified independently by 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 detection method that can simultaneously identify multiple components of refined Pantao Pills to achieve good separation without interference. The establishment of this method is of great significance for the quality control of refined Pantao Pills, and can effectively improve the quality stability and reliability of the drug, providing a strong guarantee for the clinical application of the product. Summary of the invention

[0005] The purpose of the present invention is to solve the problems existing in the prior art and provide a thin layer chromatography identification method for simultaneously identifying multiple components in a refined Pantao Pill preparation. The identification method uses two extraction methods to prepare two test sample solutions, which can achieve comprehensive identification of the four medicinal materials in the refined Pantao Pill, and has the ability to simultaneously identify three of the medicinal materials. On the basis of ensuring the identification efficiency, it also has 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: A thin-layer chromatography identification method for simultaneously identifying multiple components in refined Pantao Pills preparation, which consists of the following steps: (1) Preparation of test solution Take the refined Pantao Pills preparation, first extract it with methanol by ultrasonic wave to obtain test solution 1; Take test solution 1, reflux and extract it with hydrochloric acid, then shake and extract the filtrate, evaporate the solution to dryness, and dissolve the obtained residue in ethanol to obtain test solution 2; (2) Preparation of reference medicinal material solution Take Alpiniae Oxyphyllae Fructus reference medicinal material, Semen Juglandis reference medicinal material and Lycii Fructus reference medicinal material, and extract them with methanol by ultrasonic wave respectively to prepare Alpiniae Oxyphyllae Fructus reference medicinal material solution, Semen Juglandis reference medicinal material solution and Lycii Fructus reference medicinal material solution; Take Ophiopogonis Radix reference medicinal material, extract it with methanol by ultrasonic wave, reflux and extract it with hydrochloric acid, filter, shake and extract the filtrate, evaporate the solution to dryness, and dissolve the residue in ethanol to prepare Ophiopogonis Radix reference medicinal material solution; (3) Detection by thin-layer chromatography Absorb test solution 1 prepared in step (1), Alpiniae Oxyphyllae Fructus reference medicinal material solution, Semen Juglandis reference medicinal material solution and Lycii Fructus reference medicinal material solution prepared in step (2), and spot them on the same silica gel G thin-layer plate respectively. Develop with toluene-ethyl acetate-formic acid-methanol system, take out, dry in air, and examine under ultraviolet lamp. In the test solution chromatogram, at the positions corresponding to Semen Juglandis reference medicinal material and Lycii Fructus reference medicinal material, there are fluorescent spots of the same color; then examine under ultraviolet lamp at another wavelength. In the test solution chromatogram, at the position corresponding to Alpiniae Oxyphyllae Fructus reference medicinal material, there are spots of the same color; Absorb test solution 2 prepared in step (1) and Ophiopogonis Radix reference medicinal material solution prepared in step (2), and spot them on the same silica gel G thin-layer plate respectively. Develop with petroleum ether-ethyl acetate system, take out, dry in air, and examine under ultraviolet lamp. In the test solution chromatogram, at the position corresponding to Ophiopogonis Radix reference medicinal material, there are fluorescent spots of the same color. The refined Pantao Pills preparation includes refined Pantao Pills, refined Pantao Pills liquid extract or a mixture of water extract and alcohol extract of refined Pantao Pills.

[0008] The refined Pantao Pills preparation is composed of 30-55 parts by weight of Lycii Fructus, 5-18 parts by weight of Semen Juglandis, 5-18 parts by weight of Alpiniae Oxyphyllae Fructus, and 12-32 parts by weight of Ophiopogonis Radix; the preparation method is as follows: (1) Preparation of a mixture of water extract and alcohol extract of refined Pantao Pills: Extract Alpiniae Oxyphyllae Fructus with 8-12 times of 50%-90% ethanol for 1-3 times, 0.5-2.5 h each time, filter, and reserve the residue and alcohol extract; add 8-15 times of water to the residue of Lycii Fructus, Semen Juglandis, Ophiopogonis Radix and Alpiniae Oxyphyllae Fructus and extract for 1-3 times, 1-2.5 h each time, filter, and mix the filtrate with the alcohol extract for reserve; The extraction method is preferably heat reflux extraction; (2)Preparation of refined flat peach pill liquid extract: Take the mixture of water extract and alcohol extract of refined flat peach pills in process (1), and concentrate it under reduced pressure to an extract with a relative density of 1.25 - 1.30 at 25 °C for standby; (3)Preparation of refined flat peach pills: Take the refined flat peach pill liquid extract in process (2), add auxiliary materials, mix evenly, and prepare into pills for standby.

[0009] Among them: In the ultrasonic process in step (1), the volume ratios of methanol and hydrochloric acid in the reflux process are 5 - 30 and 0.1 - 3 respectively.

[0010] In step (1), when preparing the test solution 2, the shaking extraction time is 0.2 - 2 h.

[0011] In step (1), when preparing the test solution 2, the temperature for evaporating the solution to dryness is 40 - 90 °C.

[0012] In step (1), when the raw material is refined flat peach pills, the refined flat peach pills need to be first crushed into fine powder, and then the test solution 1 or test solution 2 is prepared.

[0013] In step (1), the mass - volume ratio of refined flat peach pills to methanol is 1.0:4.0 - 5.0, with the unit of g / mL.

[0014] In step (1), the ultrasonic time is 10 - 30 min, and the ultrasonic power is 100 - 200 w.

[0015] In step (1), the time for hydrochloric acid reflux extraction is 5 - 10 min. In the ultrasonic process in step (2), the volume ratios of methanol and hydrochloric acid in the reflux process are 2 - 20 and 0.5 - 2 respectively.

[0016] In step (2), the mass - volume ratio of the control medicinal material of Alpinia oxyphylla to methanol is 1.0:5.0, with the unit of g / mL.

[0017] In step (2), the mass - volume ratio of the control medicinal material of Semen Juglandis to methanol is 1.0:5.0, with the unit of g / mL.

[0018] In step (2), the mass - volume ratio of the control medicinal material of Fructus Lycii to methanol is 1.0:5.0, with the unit of g / mL.

[0019] In step (2), the mass - volume ratio of the control medicinal material of Radix Ophiopogonis to methanol is 1.0:5.0, with the unit of g / mL.

[0020] In step (2), when preparing the solution of the control medicinal material of Radix Ophiopogonis, the shaking extraction time is 0.2 - 2 h.

[0021] In step (2), when preparing the control crude drug solution of Ophiopogon japonicus, the temperature for evaporating the solution to dryness is 40 - 90 °C.

[0022] In step (2), the ultrasonic time is 10 - 30 min, and the ultrasonic power is 100 - 200 w; In step (2), the time for reflux extraction with hydrochloric acid is 5 - 10 min.

[0023] In the toluene - ethyl acetate - formic acid - methanol system described in step (3), the volume ratio of toluene, ethyl acetate, formic acid, and methanol is 2 - 8 : 2 - 8 : 0.1 - 1 : 1 - 3.

[0024] In the petroleum ether - ethyl acetate system described in step (3), the volume ratio of petroleum ether and ethyl acetate is 1 - 5 : 1 - 5.

[0025] In step (3), the ultraviolet lamp wavelengths for the Semen Juglandis and Fructus Lycii are 365 nm, and the ultraviolet lamp wavelength for Fructus Alpiniae Oxyphyllae is 254 nm.

[0026] In step (3), the ultraviolet lamp wavelength for the Radix Ophiopogonis is 365 nm.

[0027] In the 2020 edition of the Chinese Pharmacopoeia, the thin - layer chromatography identification method for Semen Juglandis is not included, and for the identification of the other three crude drugs, the form of separate identification for each single crude drug is still adopted, that is, different crude drugs need to be identified independently by different methods. Such existing methods not only cannot meet the overall quality control requirements of the refined Pantao Pills, but also require multiple tests in actual operation, resulting in problems such as time - consuming and consumable materials. The thin - layer chromatography identification method of the present invention is divided into two parts. The first part uses methanol ultrasonic extraction and develops with the toluene - ethyl acetate - formic acid - methanol system, and thus can simultaneously identify Fructus Alpiniae Oxyphyllae, Fructus Lycii, and Semen Juglandis in the refined Pantao Pills; the second part, on the basis of methanol ultrasonic extraction, adds hydrochloric acid for heating and reflux extraction, then shakes the solution, evaporates the solution to dryness and redissolves it with ethanol, and develops with the petroleum ether - ethyl acetate system, and thus can identify the Radix Ophiopogonis in the refined Pantao Pills. Only two thin - layer plates are needed to simply and effectively identify the four crude drugs in the refined Pantao Pills, and the developed color spots are clear, do not tail, have no cross - over, and have good specificity, which can meet the daily work of identifying the active ingredients in the refined Pantao Pills and realize the quality control of the refined Pantao Pills.

[0028] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a thin-layer chromatography identification method, which can be used to simultaneously identify multiple components in refined Pantao Pills. This method uses two extraction methods successively to prepare two test solution samples, and can comprehensively identify all the medicinal herbs in the four herbs of the whole formula of refined Pantao Pills. This method not only ensures the identification efficiency, but also has high stability and repeatability. It effectively solves the problems in the prior art of needing to prepare multiple test solution samples and having a cumbersome detection procedure, provides an important reference basis for the quality control of refined Pantao Pills, and has great significance.

[0029] The present invention belongs to the technical field of traditional Chinese medicine quality control, and specifically relates to a thin-layer chromatography identification method for simultaneously identifying multiple components in refined Pantao Pills. The thin-layer chromatography identification method of the present invention is divided into two parts. In the first part, ultrasonic extraction with methanol is used, and development is carried out with a toluene-ethyl acetate-formic acid-methanol system, and thus the semen coicis, wolfberry fruit and cortex juglandis in refined Pantao Pills can be simultaneously identified; in the second part, on the basis of ultrasonic extraction with methanol, hydrochloric acid is added for heating under reflux extraction, shaken and evaporated to dryness, and then redissolved with ethanol, and development is carried out with a petroleum ether-ethyl acetate system, and thus the radix ophiopogonis in refined Pantao Pills can be identified. That is, only two sample preparation methods are needed successively, and one thin-layer plate can simply, quickly and effectively identify the four medicinal herbs in refined Pantao Pills, and the developed color spots are clear, do not tail, have no cross-interference, and have good specificity. Description of the Drawings

[0030] Figure 1 It is the TLC chromatogram (including the chromatogram for specificity investigation) of the thin-layer identification of semen coicis, cortex juglandis and wolfberry fruit in refined Pantao Pills in Example 1 under a 365 nm ultraviolet lamp. The labels in the drawing are described as follows: 1 is the test solution sample; 2 is the control medicinal material of semen coicis; 3 is the negative control of semen coicis; 4 is the control medicinal material of cortex juglandis; 5 is the negative control of cortex juglandis; 6 is the control medicinal material of wolfberry fruit; 7 is the negative control of wolfberry fruit; Figure 2 It is the TLC chromatogram (including the chromatogram for specificity investigation) of the thin-layer identification of semen coicis, cortex juglandis and wolfberry fruit in refined Pantao Pills in Example 1 under 254 nm. The labels in the drawing are described as follows: 1 is the test solution sample 1; 2 is the control medicinal material of semen coicis; 3 is the negative control of semen coicis; 4 is the control medicinal material of cortex juglandis; 5 is the negative control of cortex juglandis; 6 is the control medicinal material of wolfberry fruit; 7 is the negative control of wolfberry fruit; Figure 3 It is the TLC chromatogram (including the chromatogram for specificity investigation) of the thin-layer identification of radix ophiopogonis in refined Pantao Pills in Example 1 under 365 nm. The labels in the drawing are described as follows: 1 is the test solution sample 2; 2 is the control medicinal material of radix ophiopogonis; 3 is the negative control of radix ophiopogonis; Figure 4TLC chromatogram of refined Pantao Pills in Example 2 for the thin-layer identification of Alpiniae Oxyphyllae Fructus, Semen Juglandis, and Lycii Fructus under a 365 nm ultraviolet lamp; the labels in the attached figure are described as follows: 1, 2, and 3 are the test solution 1; 4 is the control crude drug of Alpiniae Oxyphyllae Fructus; 5 is the control crude drug of Semen Juglandis; 6 is the control crude drug of Lycii Fructus; Figure 5 TLC chromatogram of refined Pantao Pills in Example 2 for the thin-layer identification of Alpiniae Oxyphyllae Fructus, Semen Juglandis, and Lycii Fructus under 254 nm; the labels in the attached figure are described as follows: 1, 2, and 3 are the test solution 1; 4 is the control crude drug of Alpiniae Oxyphyllae Fructus; 5 is the control crude drug of Semen Juglandis; 6 is the control crude drug of Lycii Fructus; Figure 6 TLC chromatogram of refined Pantao Pills in Example 2 for the thin-layer identification of Ophiopogonis Radix under 365 nm; the labels in the attached figure are described as follows: 1, 2, and 3 are the test solution 2; 4 is the control crude drug of Ophiopogonis Radix; Figure 7 TLC chromatogram of refined Pantao Pills in Example 3 for the thin-layer identification of Alpiniae Oxyphyllae Fructus, Semen Juglandis, and Lycii Fructus under a 365 nm ultraviolet lamp; the labels in the attached figure are described as follows: 1, 2, and 3 are the test solution 1; 4 is the control crude drug of Alpiniae Oxyphyllae Fructus; 5 is the control crude drug of Semen Juglandis; 6 is the control crude drug of Lycii Fructus; Figure 8 TLC chromatogram of refined Pantao Pills in Example 3 for the thin-layer identification of Alpiniae Oxyphyllae Fructus, Semen Juglandis, and Lycii Fructus under 254 nm; the labels in the attached figure are described as follows: 1, 2, and 3 are the test solution 1; 4 is the control crude drug of Alpiniae Oxyphyllae Fructus; 5 is the control crude drug of Semen Juglandis; 6 is the control crude drug of Lycii Fructus; Figure 9 TLC chromatogram of refined Pantao Pills in Example 3 for the thin-layer identification of Ophiopogonis Radix under 365 nm; the labels in the attached figure are described as follows: 1, 2, and 3 are the test solution 2; 4 is the control crude drug of Ophiopogonis Radix; Figure 10 TLC chromatogram of refined Pantao Pills in Example 4 for the thin-layer identification of Alpiniae Oxyphyllae Fructus, Semen Juglandis, and Lycii Fructus under a 365 nm ultraviolet lamp (including the specificity investigation chromatogram), the labels in the attached figure are described as follows: 1 is the test solution; 2 is the control crude drug of Alpiniae Oxyphyllae Fructus; 3 is the negative control of Alpiniae Oxyphyllae Fructus; 4 is the control crude drug of Semen Juglandis; 5 is the negative control of Semen Juglandis; 6 is the control crude drug of Lycii Fructus; 7 is the negative control of Lycii Fructus; Figure 11 TLC chromatogram of refined Pantao Pills in Example 4 for the thin-layer identification of Alpiniae Oxyphyllae Fructus, Semen Juglandis, and Lycii Fructus under 254 nm (including the specificity investigation chromatogram), the labels in the attached figure are described as follows: 1 is the test solution 1; 2 is the control crude drug of Alpiniae Oxyphyllae Fructus; 3 is the negative control of Alpiniae Oxyphyllae Fructus; 4 is the control crude drug of Semen Juglandis; 5 is the negative control of Semen Juglandis; 6 is the control crude drug of Lycii Fructus; 7 is the negative control of Lycii Fructus; Figure 12TLC chromatogram (including the chromatogram for specificity study) of refined flat peach pills with Ophiopogon japonicus identification in Example 4 under 365 nm. The markings in the attached figure are described as follows: 1 is the test solution 2; 2 is the reference crude drug of Ophiopogon japonicus; 3 is the negative control of Ophiopogon japonicus. Figure 13 TLC chromatogram (including the chromatogram for specificity study) of refined flat peach pills with Alpinia oxyphylla, Semen Juglandis, and Lycium barbarum identification in Example 5 under 365 nm ultraviolet lamp. The markings in the attached figure are described as follows: 1 is the test solution; 2 is the reference crude drug of Alpinia oxyphylla; 3 is the negative control of Alpinia oxyphylla; 4 is the reference crude drug of Semen Juglandis; 5 is the negative control of Semen Juglandis; 6 is the reference crude drug of Lycium barbarum; 7 is the negative control of Lycium barbarum. Figure 14 TLC chromatogram (including the chromatogram for specificity study) of refined flat peach pills with Alpinia oxyphylla, Semen Juglandis, and Lycium barbarum identification in Example 5 under 254 nm. The markings in the attached figure are described as follows: 1 is the test solution 1; 2 is the reference crude drug of Alpinia oxyphylla; 3 is the negative control of Alpinia oxyphylla; 4 is the reference crude drug of Semen Juglandis; 5 is the negative control of Semen Juglandis; 6 is the reference crude drug of Lycium barbarum; 7 is the negative control of Lycium barbarum. Figure 15 TLC chromatogram (including the chromatogram for specificity study) of refined flat peach pills with Ophiopogon japonicus identification in Example 5 under 365 nm. The markings in the attached figure are described as follows: 1 is the test solution 2; 2 is the reference crude drug of Ophiopogon japonicus; 3 is the negative control of Ophiopogon japonicus. Detailed implementation manners

[0031] The present invention will be further described in detail below with reference to the attached drawings and specific embodiments. The following embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Example 1

[0032] 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 analysis on the negative test solution (sample without the target crude drug), the reference crude drug solution, and the test solution together, and comparing their chromatograms, it can be confirmed whether the target identification components are interfered by other components. If there are no spots of the same color interfering at the corresponding positions in the chromatogram of the negative test solution as in the chromatograms of the reference crude drug and the test solution, it indicates that the negative sample has no interference and the specificity of the method is good.

[0033] At the same time, for the thin-layer chromatography identification method for simultaneously identifying multiple components in the refined flat peach pill preparation, the refined flat peach pill is used as the test sample. The preparation methods of the refined flat peach pill and the negative sample are as follows: Refined flat peach pill: Take 10 parts by weight of Alpinia oxyphylla, crush it into coarse grains (pass through a 20-mesh sieve), add 8 times the amount of ethanol solution (70%), soak for 0.5 h, and reflux extract twice, each time for 1.5 h. The ethanol extract and the residue of Alpinia oxyphylla are reserved.

[0034] Take 12 parts by weight of semen juglandis, 30 parts by weight of fructus lycii, and 12 parts by weight of radix ophiopogonis, and pulverize them separately (semen juglandis passes through a 20-mesh sieve, and fructus lycii and radix ophiopogonis are appropriately pulverized. After pulverization, fructus lycii and radix ophiopogonis are sticky and cannot pass through the sieve, so just appropriately pulverize them). Add 10 parts by weight of the residue of semen alpinaekatsumadai (10 parts by weight of the residue of semen alpinaekatsumadai can be obtained by extracting semen alpinaekatsumadai multiple times), add 10 times the amount of water, reflux and extract 2 times, each time for 1 h, and reserve the water extract. Mix the water extract and the ethanol extract, concentrate to a relative density of 1.30 (25 °C) to obtain the refined flat peach pill fluid extract. Add starch and microcrystalline cellulose to the refined flat peach pill fluid extract, and make pills by the plasticizing method. After drying, the refined flat peach pills are obtained.

[0035] Negative sample of semen alpinaekatsumadai: Weigh the other 3 medicinal flavors except semen alpinaekatsumadai, and the composition content and preparation method are the same as those of the preparation method of the refined flat peach pills.

[0036] Negative sample of semen juglandis: Weigh the other 3 medicinal flavors except semen juglandis, and the composition content and preparation method are the same as those of the preparation method of the refined flat peach pills.

[0037] Negative sample of fructus lycii: Weigh the other 3 medicinal flavors except fructus lycii, and the composition content and preparation method are the same as those of the preparation method of the refined flat peach pills.

[0038] Negative sample of radix ophiopogonis: Weigh the other 3 medicinal flavors except radix ophiopogonis, and the composition content and preparation method are the same as those of the preparation method of the refined flat peach pills.

[0039] The thin-layer chromatography identification method for simultaneously identifying multiple components of the refined flat peach pill preparation described in Example 1 of the present invention, that is, the thin-layer identification method for simultaneously identifying 4 medicinal materials of semen alpinaekatsumadai, semen juglandis, fructus lycii, and radix ophiopogonis in the refined flat peach pills, consists of the following steps: (1) Preparation of the test solution Take an appropriate amount of refined flat peach pills (about 5 g), add 20 mL of methanol, ultrasonicate for 30 min (power 100 w), filter, and use it as the test solution 1.

[0040] Take an appropriate amount of test solution 1 (about 10 mL), add 0.5 mL of hydrochloric acid, place it in a water bath and heat under reflux for 10 min, cool, and filter. Shake the filtrate for 1 h, evaporate the solution to dryness, add 2 mL of ethanol to dissolve, and use it as the test solution 2.

[0041] (2) Preparation of the negative test solutions of semen alpinaekatsumadai, semen juglandis, and fructus lycii Weigh the negative samples of semen alpinaekatsumadai, semen juglandis, and fructus lycii equivalent to the same amount in step (1) respectively, and prepare the negative test solutions of semen alpinaekatsumadai, semen juglandis, and fructus lycii according to the method of the test solution 1 in step (1) respectively.

[0042] (3) Preparation of Ophiopogon japonicus negative test solution 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 for test solution 2 in step (1).

[0043] (4) Preparation of reference medicinal material solution Take 1 g of Alpinia oxyphylla, 1 g of Fenxinmu and 1 g of Fructus Lycii and prepare Alpinia oxyphylla, Fenxinmu and Fructus Lycii control medicinal materials solutions according to the method of test solution 1 in step (1). The volume of methanol added is 5 mL.

[0044] Take 1 g of Ophiopogon japonicus control medicinal material and prepare Ophiopogon japonicus control medicinal material solution according to the method of test solution 2 in step (1). That is, take 1 g of Ophiopogon japonicus 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, heat and reflux in a water bath for 10 min, and cool. Shake for 1 h, evaporate the solution, add 1 mL of ethanol to dissolve, and use as test solution 2.

[0045] (5) Thin layer chromatography analysis 1 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 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), taken out, dried, and examined under ultraviolet light (365 nm). The results are shown in Table 1. Figure 1 .

[0046] from Figure 1 It can be seen that 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. The negative test samples of Lycium barbarum and Fenxinmu have no interference and good specificity. Then put it under ultraviolet light (254 nm) for inspection, the results are shown in 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.

[0047] (6) Thin layer chromatography analysis 2 Test according to the thin-layer chromatography method (General Principles 0502, Volume IV, Chinese Pharmacopoeia 2020 Edition). Pipette 5 μL of the test solution 2 prepared in step (1), 5 μL of the negative Ophiopogon japonicus test solution prepared in step (3), and 5 μL of the reference crude drug solution of Ophiopogon japonicus prepared in step (4) onto the same silica gel G thin-layer plate and the same silica gel GF254 thin-layer plate respectively. Develop with petroleum ether (60 - 90 °C): ethyl acetate (the volume ratio of petroleum ether to ethyl acetate is 1:1). Take out, dry in air, and examine under an ultraviolet lamp (365 nm). The results are shown in Figure 3 。

[0048] From Figure 3 it can be seen that in the chromatogram of the test solution, at the position corresponding to the chromatogram of the reference crude drug of Ophiopogon japonicus, spots of the same color appear, and there is no interference from the negative control of Ophiopogon japonicus, indicating good specificity. Example 2

[0049] A thin-layer chromatography identification method for simultaneously identifying multiple components in the refined flat peach pill preparation. The refined flat peach pill extract is used as the test sample, and the preparation method of the refined flat peach pill extract is as follows: Refined flat peach pill extract: Take 18 parts by weight of Alpinia oxyphylla, crush it into coarse particles (pass through a 20-mesh sieve), add 12 times the amount of ethanol solution (50%), soak for 0.5 h, and reflux extract 3 times, each time for 2.5 h. The alcohol extract and the residue of Alpinia oxyphylla are reserved for later use.

[0050] Take 18 parts by weight of Semen Juglandis, 55 parts by weight of Lycium barbarum, and 32 parts by weight of Ophiopogon japonicus, and crush them respectively (Semen Juglandis passes through a 20-mesh sieve, and Lycium barbarum and Ophiopogon japonicus are appropriately crushed. After crushing, Lycium barbarum and Ophiopogon japonicus are sticky and cannot pass through the sieve, so appropriate crushing is sufficient). Add 18 parts by weight of the residue of Alpinia oxyphylla (20 parts by weight of the residue of Alpinia oxyphylla can be obtained by multiple extractions of Alpinia oxyphylla), add 15 times the amount of water, reflux extract 3 times, each time for 2.5 h, and reserve the water extract for later use. Mix the water extract and the alcohol extract, and concentrate to a relative density of 1.25 (25 °C) to obtain the refined flat peach pill extract.

[0051] The thin-layer chromatography identification method for simultaneously identifying multiple components in the refined flat peach pill preparation described in this Example 2, that is, the thin-layer identification method for simultaneously identifying 4 medicinal materials of Alpinia oxyphylla, Semen Juglandis, Lycium barbarum, and Ophiopogon japonicus in the refined flat peach pill extract, consists of the following steps: (1) Preparation of the test solution Take an appropriate amount (about 4 g) of the refined flat peach pill extract, add 20 mL of methanol, ultrasonicate for 10 min (power 200 w), filter, and use it as the test solution 1.

[0052] Take an appropriate amount (about 5 mL) of the test solution 1, add 0.1 mL of hydrochloric acid, heat under reflux in a water bath for 5 min, cool, and filter. Shake the filtrate for 0.2 h, evaporate the solution to dryness at 40 °C, dissolve with 2 mL of ethanol to obtain the test solution 2.

[0053] (2)Preparation of the control crude drug solution Take 1 g of the control crude drug of Alpiniae Oxyphyllae Fructus, 1 g of the control crude drug of Semen Juglandis, and 1 g of the control crude drug of Lycii Fructus, and prepare the control crude drug solution of Alpiniae Oxyphyllae Fructus, the control crude drug solution of Semen Juglandis, and the control crude drug solution of Lycii Fructus respectively according to the method of the test solution 1 in step (1). The volume of methanol added is 5 mL for each.

[0054] Take 10 g of the control crude drug of Radix Ophiopogonis, and prepare the control crude drug solution of Radix Ophiopogonis according to the method of the test solution 2 in step (1). That is: take 10 g of the control crude drug of Radix Ophiopogonis, add 50 mL of methanol, ultrasonicate for 10 min (power 200 w), filter to obtain the test solution 1. Take an appropriate amount (about 20 mL) of the test solution 1, add 2 mL of hydrochloric acid, heat under reflux in a water bath for 5 min, cool. Shake for 0.2 h, evaporate the solution to dryness, dissolve with 5 mL of ethanol to obtain the test solution 2.

[0055] (3)Detection by thin layer chromatography As Figure 4 shown, pipette 5 μL of the test solution 1 prepared in step (1), the control crude drug solution of Alpiniae Oxyphyllae Fructus, the control crude drug solution of Semen Juglandis, and the control crude drug solution of Lycii Fructus prepared in step (2) respectively, and spot them on the same silica gel G thin layer plate. Develop with the system of toluene - ethyl acetate - formic acid - methanol = 2:2:0.3:3, take out, air dry, and examine under an ultraviolet lamp (365 nm). The results are shown in Figure 4 , in the test sample chromatogram, at the positions corresponding to the control crude drug of Semen Juglandis and the control crude drug of Lycii Fructus, fluorescent spots of the same color appear; then examine under an ultraviolet lamp (254 nm), and the results are shown in Figure 5 , in the test sample chromatogram, at the position corresponding to the control crude drug of Alpiniae Oxyphyllae Fructus, spots of the same color appear.

[0056] Pipette 5 μL of the test solution 2 prepared in step (1) and the control crude drug solution of Radix Ophiopogonis prepared in step (2) respectively, and spot them on the same silica gel G thin layer plate. Develop with the system of petroleum ether - ethyl acetate = 2:5, take out, air dry, and examine under an ultraviolet lamp (365 nm). The results are shown in Figure 6 . In the test sample chromatogram, at the position corresponding to the control crude drug of Radix Ophiopogonis, fluorescent spots of the same color appear. Example 3

[0057] As Figures 7 - 9As shown, a thin-layer chromatography identification method for simultaneously identifying multiple components in the refined flat peach pill preparation uses a mixture of the water extract and alcohol extract of the refined flat peach pill as the test sample. The preparation method of the mixture of the water extract and alcohol extract of the refined flat peach pill is as follows: Mixture of the water extract and alcohol extract of the refined flat peach pill: Take 5 parts by weight of Alpinia oxyphylla, crush it into coarse grains (pass through a 20-mesh sieve), add 10 times the amount of ethanol solution (90%), soak for 0.5 h, and reflux extract once for 0.5 h each time. Keep the alcohol extract and the residue of Alpinia oxyphylla for later use.

[0058] Take 5 parts by weight of Semen Juglandis, 30 parts by weight of Lycium barbarum, and 15 parts by weight of Ophiopogon japonicus, and crush them respectively (Semen Juglandis passes through a 20-mesh sieve, Lycium barbarum and Ophiopogon japonicus are appropriately crushed. After crushing, Lycium barbarum and Ophiopogon japonicus are sticky and cannot pass through the sieve, so just appropriately crush them). Add 5 parts by weight of the residue of Alpinia oxyphylla (the residue of Alpinia oxyphylla can be obtained by extracting Alpinia oxyphylla multiple times), add 8 times the amount of water, reflux extract once for 1 h each time, and keep the water extract for later use. Mix the water extract and the alcohol extract to obtain the mixture of the water extract and alcohol extract of the refined flat peach pill.

[0059] The thin-layer chromatography identification method for simultaneously identifying multiple components of the refined flat peach pill preparation described in Example 2, that is, the thin-layer identification method for simultaneously identifying 4 medicinal materials of Alpinia oxyphylla, Semen Juglandis, Lycium barbarum, and Ophiopogon japonicus in the mixture of the water extract and alcohol extract of the refined flat peach pill, consists of the following steps: (1) Preparation of the test solution Take an appropriate amount (about 100 mL) of the mixture of the water extract and alcohol extract of the refined flat peach pill, concentrate it to 20 mL, add methanol to make the volume up to 40 mL, and ultrasonicate for 30 min to obtain Test Solution 1.

[0060] Take an appropriate amount (about 30 mL) of Test Solution 1, add 3 mL of hydrochloric acid, place it in a water bath and heat under reflux for 5 min, let it cool, and filter. Shake the filtrate for 2 h, evaporate the solution to dryness at 90 °C, and dissolve it with 10 mL of ethanol to obtain Test Solution 2.

[0061] (2) Preparation of the reference medicinal material solution Take 1 g of the reference medicinal material of Alpinia oxyphylla, 1 g of the reference medicinal material of Semen Juglandis, and 1 g of the reference medicinal material of Lycium barbarum, and prepare the reference medicinal material solution of Alpinia oxyphylla, the reference medicinal material solution of Semen Juglandis, and the reference medicinal material solution of Lycium barbarum respectively according to the method of Test Solution 1 in step (1). The volume of methanol added is 5 mL in each case.

[0062] Take 1 g of Ophiopogon japonicus control medicinal material and prepare Ophiopogon japonicus control medicinal material solution according to the method of test solution 2 in step (1). That is, take 1 g of Ophiopogon japonicus control medicinal material, add 5 mL of methanol, ultrasonicate for 7 min (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, heat and reflux in a water bath for 5 min, and cool. Shake for 2 h, evaporate the solution, add 1 mL of ethanol to dissolve, and use as test solution 2.

[0063] (3) Thin layer chromatography detection Pipette 1 μL of the test solution prepared in step (1) and 5 μL of the Alpinia oxyphylla control medicinal material solution, the Fructus sylvestris control medicinal material solution, and the Fructus lycii control medicinal material solution prepared in step (2), respectively, and spot them on the same silica gel G thin layer plate, develop with toluene-ethyl acetate-formic acid-methanol = 8:8:0.5:2 system, take out, dry, and examine under ultraviolet light (365 nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions of the Fructus sylvestris control medicinal material and the Fructus lycii control medicinal material; then examine under ultraviolet light (254 nm). In the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the Alpinia oxyphylla control medicinal material.

[0064] Take 2 μL of the test solution prepared in step (1) and 5 μL of the Ophiopogon japonicus control solution prepared in step (2), spot them on the same silica gel G thin layer plate, develop them with a petroleum ether-ethyl acetate = 5:3 system, take them out, dry them, and examine them under ultraviolet light (365 nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions of the Ophiopogon japonicus control. Example 4

[0065] like Figures 10 - 12 As shown, the difference between this embodiment and embodiment 1 is only that: Toluene-ethyl acetate-formic acid-methanol = 5:5:0.3:3; Petroleum ether-ethyl acetate = 5:1. Example 5

[0066] like Figures 13 - 15 As shown, the difference between this embodiment and embodiment 1 is only that: Toluene-ethyl acetate-formic acid-methanol = 2:8:1:3; Petroleum ether-ethyl acetate = 1:5.

[0067] It should be understood that, in order to streamline the present disclosure and assist in understanding one or more of the various inventive aspects, in the foregoing description of the exemplary embodiments of the present invention, various features of the present invention are sometimes grouped together into a single embodiment, figure, or description thereof. However, the disclosed method should not be construed as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as reflected by the claims, the inventive aspects lie in less than all the features of the foregoing disclosed embodiments. Thus, the claims following the detailed description hereby expressly incorporate the detailed description, where each claim itself serves as a separate embodiment of the present invention.

[0068] Although the present invention has been described in terms of a limited number of embodiments, those skilled in the art, having the benefit of the foregoing description, will appreciate that other embodiments can be contemplated within the scope of the invention as thus described. Additionally, it should be noted that the language used in this specification has been principally selected for readability and instructional 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 of ordinary skill in the art without departing from the scope and spirit of the appended claims. For the scope of the present invention, the disclosure of the present invention is illustrative, not restrictive, and the scope of the present invention is defined by the appended claims.

[0069] The foregoing are only the preferred embodiments of the present invention, and it should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A thin-layer chromatography identification method for simultaneously identifying multiple components in refined flat peach pill preparations, characterized in that, It consists of the following steps: (1) Preparation of test solution: Take the refined Pantao Pill preparation, firstly use methanol ultrasonic extraction to take the refined Pantao Pill preparation as the test solution 1 or take the extract of the refined Pantao Pill preparation and directly take it as the test solution 1; Take the test solution 1, add hydrochloric acid to reflux and extract, filter after extraction, then shake the filtrate to extract, evaporate the solution, and re-dissolve the residue in ethanol to prepare the test solution 2; (2) Preparation of control medicinal material solution: Take Alpinia oxyphylla, Fenxinmu and Fructus Lycii control medicinal materials, add methanol and ultrasonicate to prepare Alpinia oxyphylla control medicinal material solution, Fenxinmu control medicinal material solution and Fructus Lycii control medicinal material solution; Take Ophiopogon japonicus reference medicinal material, add methanol to sonicate, add hydrochloric acid to reflux, filter, shake the filtrate to extract, evaporate the solution, add ethanol to dissolve the residue, and 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 Fructus sylvestris control medicinal material solution, and the Fructus 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, air-dried, and examined under a 365 nm 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 Fructus sylvestris control medicinal material and the Fructus 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 are shown at the positions corresponding to those of the Alpinia oxyphylla control medicinal material. The test sample solution 2 prepared in step (1) and the Ophiopogon japonicus control medicinal material solution prepared in step (2) are 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 appear at the corresponding positions of the Ophiopogon japonicus control medicinal material.

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 fenxin wood, 5-18 parts by weight of alpinia oxyphylla, and 12-32 parts by weight of ophiopogon japonicus; the preparation method is as follows: (1) Preparation of a mixture of refined Pantao pills water extract and alcohol extract: extract Alpinia oxyphylla with 8-12 times 50%-90% ethanol for 1-3 times, each time for 0.5-2.5 h, filter, and set aside the residue and alcohol extract; extract Lycium barbarum, Fructus schizonepetae, Radix Ophiopogonis, and Alpinia oxyphylla with 8-15 times water for 1-3 times, each time for 1-2.5 h, filter, and mix the filtrate with the alcohol extract for later use; (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), and concentrate under reduced pressure to an extract having a relative density of 1.25-1.30 at 25°C, and set aside; (3) Preparation of refined Pantao Pills: Take the refined Pantao Pills fluid extract prepared in process (2), add the excipients, mix well, prepare into pills, and set aside.

4. The method according to claim 1, wherein In step (1), the volume ratios of methanol during ultrasonication and hydrochloric acid during reflux are 5-30 and 0.1-3 respectively; When preparing the test solution 2, the shaking extraction time is 0.2-2 h; The temperature for evaporating the solution to dryness when preparing the test solution 2 is 40 - 90 °C; The mass - volume ratio of refined Pantao Pills or refined Pantao Pills extractive to methanol is 1.0:4.0 - 5.0, with the unit of g / mL.

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

6. The method according to claim 1, characterized in that, In step (2), during the ultrasonic process, the volume ratio of methanol and during the reflux process, the volume ratio of hydrochloric acid are 2 - 20 and 0.5 - 2 respectively; The mass - volume ratio of the control medicinal material of Alpinia oxyphylla to methanol is 1.0:5.0, with the unit of g / mL; The mass - volume ratio of the control medicinal material of Semen Juglandis to methanol is 1.0:5.0, with the unit of g / mL; The mass - volume ratio of the control medicinal material of Fructus Lycii to methanol is 1.0:5.0, with the unit of g / mL; The mass - volume ratio of the control medicinal material of Radix Ophiopogonis to methanol is 1.0:5.0, with the unit of g / mL; When preparing the control medicinal material solution of Radix Ophiopogonis, the shaking extraction time is 0.2 - 2 h; The temperature for evaporating the solution to dryness when preparing the control medicinal material solution of Radix Ophiopogonis is 40 - 90 °C.

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

8. The method according to claim 1, characterized in that 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.

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

10. The application of the method according to any one of claims 1 - 9 in the quality control of refined Pantao Pills preparation.

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