A thin layer chromatography method for simultaneously identifying different medicinal ingredients in pediatric anshen bunao medicinal preparation

Abstract

The present application relates to the technical field of medicine quality detection, in particular to a thin layer chromatography method for simultaneously identifying different medicinal ingredients in pediatric tranquilizing and brain-nourishing medicine preparation, the method being as recorded in the specification, and the thin layer chromatography pattern obtained by the impurity removal and purification method and suitable proportion of developing agent has clear spots, good separation degree, strong specificity, no interference background, no interference negative control, good reproducibility, good durability, and is not easily affected by thin layer plate, temperature and humidity.

Description

Technical Field

[0001] This invention relates to the field of drug quality testing technology, specifically to a thin-layer chromatography method for simultaneously identifying different medicinal flavors in pediatric calming and brain-tonifying drug preparations. Background Technology

[0002] Tourette syndrome, also known as tic disorder, is clinically characterized by chronic, fluctuating, multiple rapid tics of motor muscles, accompanied by involuntary vocalizations and speech disturbances. Onset typically occurs between 2 and 12 years of age, with boys being about three times more likely to be affected than girls. The course of the illness is generally long, with tics worsening during periods of stress and disappearing after sleep. Symptoms may spontaneously improve or worsen, and intellectual impairment is not affected. The pathogenesis of Tourette syndrome is not fully understood. Western medicine treatment primarily involves oral chlorpheniramine and other psychotropic drugs, which block dopamine receptors, producing a calming and sedative effect. However, these psychotropic drugs have strong side effects, causing irreversible damage to the child's nervous system. Furthermore, medication cannot be discontinued arbitrarily; stopping medication can worsen symptoms or even trigger an outburst. Therefore, exploring and finding more effective treatments and medications remains a key focus of research on this disease.

[0003] In recent years, extensive and in-depth research on traditional Chinese medicine (TCM) and years of clinical practice by clinicians have confirmed that it not only has good clinical efficacy but also compensates for the shortcomings of Western medicine, such as poor clinical efficacy and significant side effects. Xiaor Anshen Bunao Granules are a TCM preparation developed by pediatric experts based on years of clinical experience in treating childhood tic disorders. The formula consists of Astragalus membranaceus, Citrus reticulata peel, Citrus aurantium, Polygala tenuifolia, Glycyrrhiza uralensis, and other herbs, and has the effects of clearing phlegm, relieving convulsions, calming the mind, and nourishing the brain. It is mainly used for various symptoms of mental disturbance in children, such as Tourette syndrome (TS), ADHD, night crying, sleepwalking, and restless sleep. With the increasing health awareness of the public, there are higher requirements for the quality and safety of TCM products. Currently, there are no standards issued for identifying the effective components in Xiaor Anshen Bunao Granules.

[0004] Thin-layer chromatography (TLC) is a primary method for qualitative identification in the quality standards of traditional Chinese medicine (TCM) compound preparations. The "Drafting Instructions for Quality Standards of TCM Compound Preparations" in the "Technical Requirements for Research on New Traditional Chinese Medicines" requires that "corresponding identification items should be established based on the prescription composition and research data. In principle, all medicinal ingredients in the prescription should be tested, and those selected for inclusion in the standard should be chosen based on the test results." Currently, in the quality standards for TCM compound preparations, including the Chinese Pharmacopoeia, identification items are mostly listed separately for each medicinal ingredient, with one identification item per prescription ingredient. This means one TLC plate is developed once to identify one prescription ingredient. Furthermore, the processing methods for the test solution, the control medicinal material solution, and the reference standard solution differ for each ingredient. If TLC is used to achieve basic coverage of the medicinal ingredients in a TCM compound preparation, multiple solutions need to be processed using different methods, often requiring a large number of tests and consuming significant manpower and resources. Summary of the Invention

[0005] Therefore, the technical problem to be solved by this invention is to overcome the shortcomings of existing thin-layer chromatography methods for identifying the components of pediatric calming and brain-tonifying granules, which require processing multiple solutions using different methods and developing each medicinal ingredient on multiple thin-layer plates for separate identification. This invention provides a thin-layer chromatography method for simultaneously identifying different medicinal ingredients in pediatric calming and brain-tonifying granules. To this end, this invention provides a thin-layer chromatography method for simultaneously identifying different medicinal ingredients in pediatric calming and brain-tonifying granules, comprising the following steps:

[0006] S1: Take the sample to be tested, extract it with an organic solvent, separate the solid and liquid, dry the liquid, dissolve it with an organic solvent, and prepare the test solution.

[0007] S2: Take the reference medicinal materials of dried tangerine peel and immature bitter orange peel, extract them separately with organic solvents, and prepare solutions of each reference medicinal material;

[0008] S3: Take the test sample solution, tangerine peel reference medicinal material solution, and bitter orange peel reference medicinal material solution, and spot them separately on the same thin-layer plate. Develop with the first developing solvent, remove, air dry, spray with the first color developing agent, heat, examine, and identify tangerine peel and bitter orange peel; the first developing solvent is a lower layer solution of chloroform, methanol and water in a volume ratio of 89-93:42-46:10-14.

[0009] Furthermore, the thin-layer chromatography method further includes: taking Notopterygium incisum reference material, extracting it with an organic solvent to obtain Notopterygium incisum reference material solution; taking the test solution and Notopterygium incisum reference material solution, spotting them separately on another thin-layer plate, developing them with a second developing solvent, removing them, drying them, and examining them under ultraviolet light to identify Notopterygium incisum; the second developing solvent is chloroform and methanol in a volume ratio of 7-9:1.5-2.5; optionally, the wavelength of the ultraviolet light is 365 nm.

[0010] Furthermore, the thin-layer chromatography method further includes: taking imperatorin reference standard, adding an organic solvent to prepare an imperatorin reference standard solution, and spotting it, the test solution, and the Notopterygium incisum reference herb solution on the same thin-layer plate before development; optionally, each 1 mL of the reference standard solution contains 0.8 to 1.2 mg of the reference standard; optionally, the organic solvent includes at least one of anhydrous ethanol, methanol, and n-hexane.

[0011] Furthermore, the thin-layer chromatography method further includes: taking Cornus officinalis reference material, extracting it with an organic solvent to obtain a Cornus officinalis reference material solution; taking the test solution and the Cornus officinalis reference material solution, spotting them separately on another thin-layer plate, developing them with a third developing solvent, removing them, drying them, spraying them with a second colorimetric reagent, heating them, examining them, and identifying Cornus officinalis. The third developing solvent is ethyl acetate, ethanol, and glacial acetic acid in a volume ratio of 12-17:3-7:0.3-0.5. Optionally, the second colorimetric reagent includes vanillin sulfuric acid solution.

[0012] Optionally, the concentration of the vanillin sulfuric acid solution is 4-6%.

[0013] Furthermore, the thin-layer chromatography method further includes: taking at least one of hesperidin, neohesperidin, and naringin reference standards, adding an organic solvent to prepare a reference solution; step S3 further includes spotting the reference solution onto the same thin-layer plate used in step S3 before development; optionally, each 1 mL of the reference solution contains 0.8–1.2 mg of each reference standard; optionally, the organic solvent includes at least one of anhydrous ethanol, methanol, and n-hexane.

[0014] Furthermore, in step S1, the organic solvent used includes at least one of anhydrous ethanol, methanol, and n-hexane;

[0015] And / or, in step S1 or S2, the extraction includes shaking extraction or ultrasonic extraction;

[0016] And / or, step S2 includes solid-liquid separation, drying the liquid, and dissolving it with an organic solvent after extraction with an organic solvent; the organic solvent includes at least one of anhydrous ethanol, methanol, and n-hexane.

[0017] And / or, in step S3, the first colorimetric agent comprises 2-5% aluminum trichloride ethanol solution;

[0018] And / or, in step S3, after spraying the first color developer, the heating temperature is 102-108°C.

[0019] Further, step S1 includes taking 0.5-15g of the sample to be tested, adding 15-60mL of methanol or ethanol, sonicating for 20-40 minutes, filtering, evaporating the filtrate to dryness, and dissolving the residue in 1-3mL of methanol or ethanol to obtain the test solution; preferably, step S1 includes taking 0.5-1.5g of the sample to be tested, adding 40-60mL of methanol or ethanol, sonicating for 20-40 minutes, filtering, evaporating the filtrate to dryness, and dissolving the residue in 1-3mL of methanol or ethanol to obtain the test solution.

[0020] Further, step S2 includes taking 0.05-0.15g of tangerine peel reference material and citrus aurantium reference material, adding 15-60mL of methanol or ethanol to sonicate for 20-40 minutes, filtering, evaporating the filtrate to dryness, and dissolving the residue in 1-3ml of methanol or ethanol to obtain solutions of each reference material.

[0021] Furthermore, the preparation of the Notopterygium incisum reference herb solution includes the steps of solid-liquid separation, drying the liquid, and dissolving it with an organic solvent after extraction with an organic solvent. The organic solvent includes at least one of anhydrous ethanol, methanol, and n-hexane. Preferably, the preparation method of the Notopterygium incisum reference herb solution includes taking 0.05-0.15g of Notopterygium incisum reference herb, adding 15-60mL of methanol or ethanol, sonicating for 20-40 minutes, filtering, evaporating the filtrate to dryness, and dissolving the residue with 1-3ml of methanol or ethanol to obtain the Notopterygium incisum reference herb solution.

[0022] Furthermore, the preparation of the Cornus officinalis reference herb solution includes solid-liquid separation, drying of the liquid, and dissolution with an organic solvent after extraction with an organic solvent. The organic solvent includes at least one of anhydrous ethanol, methanol, and n-hexane. Preferably, the preparation method of the Cornus officinalis reference herb solution includes taking 0.05-0.3g of Cornus officinalis reference herb, adding 15-60mL of methanol or ethanol, sonicating for 20-40 minutes, filtering, evaporating the filtrate to dryness, and dissolving the residue with 1-3ml of methanol or ethanol to obtain the Cornus officinalis reference herb solution.

[0023] In some preferred embodiments, the test solution used for identifying Cornus officinalis includes taking 0.5-15g of the sample to be tested, adding 15-60mL of methanol or ethanol, sonicating for 20-40 minutes, filtering, evaporating the filtrate to dryness, and dissolving the residue in 1-3mL of methanol or ethanol to obtain the test solution; preferably, it includes taking 5-15g of the sample to be tested, adding 15-45mL of methanol or ethanol, sonicating for 20-40 minutes, filtering, evaporating the filtrate to dryness, and dissolving the residue in 1-3mL of methanol or ethanol to obtain the test solution.

[0024] The thin-layer chromatography method further includes: a method for identifying licorice. For the pediatric calming and brain-tonifying drug preparation of the present invention, conventional methods in the art can be used to identify licorice, such as, but not limited to, the following methods.

[0025] The specific steps include: S1: Preparation of the test solution: Take 3g-7g of Xiaor Anshen Bunao Granules, grind them into a fine powder, add 30ml-70ml of methanol, sonicate for 20min-40min, filter, evaporate the filtrate to dryness, add 10ml-30ml of water to dissolve the residue, extract with water-saturated n-butanol 1-3 times, combine the n-butanol solutions, wash with 10ml-30ml of water saturated with n-butanol, discard the washing liquid, evaporate the n-butanol solution to dryness, add 1ml-2ml of methanol to dissolve the residue, and use this as the test solution; S2: Preparation of the reference herb: Take 0.3g-0.7g of licorice reference herb, and prepare the reference herb solution using the same method; S3: Preparation of the reference solution: Take glycyrrhizin reference standard, add methanol to prepare a solution containing 0.5mg-1.5mg per ml, and use this as the reference solution;

[0026] S4: Determination method: Perform the test according to the thin-layer chromatography method (General Rule 0502). Take 1-3 μl of each of the above three solutions and spot them separately on the same silica gel G thin-layer plate. Use ethyl acetate-glacial acetic acid-formic acid-aqueous solution (v / v) of 12-17:1:1:2 as the developing solvent. Develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible. Examine under sunlight and ultraviolet light (365nm). In the chromatogram of the test sample, spots and fluorescent spots of the same color appear at the corresponding positions as in the chromatograms of the reference medicinal material and the reference standard.

[0027] Further, according to the weight parts, the pediatric calming and brain-tonifying drug preparation includes the following raw materials: 220-280 parts of Acorus tatarinowii, 145-185 parts of Arisaema cum Bile, 145-185 parts of Pinellia ternata, 145-185 parts of Poria cocos, 65-105 parts of Citrus reticulata peel, 65-105 parts of Notopterygium incisum, 220-280 parts of chlorite, 220-280 parts of Haliotis diversicolor, 145-185 parts of Polygala tenuifolia, 145-185 parts of Alpinia oxyphylla, 145-185 parts of Astragalus membranaceus, 220-280 parts of Triticum aestivum, 145-185 parts of Cornus officinalis, 145-185 parts of Citrus aurantium, 220-280 parts of Hordeum vulgare, 145-185 parts of Medicated Leaven, 145-185 parts of Crataegus pinnatifida, and 145-185 parts of Glycyrrhiza uralensis.

[0028] In some preferred embodiments, the pediatric calming and brain-tonifying drug preparation comprises the following raw materials by weight: 250 parts of Acorus tatarinowii, 165 parts of Arisaema cum Bile, 165 parts of Pinellia ternata, 165 parts of Poria cocos, 85 parts of Citrus reticulata peel, 85 parts of Notopterygium incisum, 250 parts of chlorite, 250 parts of Haliotis diversicolor, 165 parts of Polygala tenuifolia, 165 parts of Alpinia oxyphylla, 165 parts of Astragalus membranaceus, 250 parts of Triticum aestivum, 165 parts of Cornus officinalis, 165 parts of Citrus aurantium, 250 parts of Hordeum vulgare, 165 parts of Massa fermentata, 165 parts of Crataegus pinnatifida, and 165 parts of Glycyrrhiza uralensis.

[0029] The preferred pediatric calming and brain-boosting drug preparation is at least one of the following: pediatric calming and brain-boosting decoction, pediatric calming and brain-boosting concentrated extract, pediatric calming and brain-boosting freeze-dried powder, and pediatric calming and brain-boosting granules.

[0030] The preferred pediatric calming and brain-boosting medicine preparation is pediatric calming and brain-boosting granules, which are obtained by decocting, concentrating, drying, and granulating a total of eighteen Chinese herbs, including Acorus tatarinowii, Arisaema cum Bile, Pinellia ternata, Poria cocos, Citrus reticulata peel, Notopterygium incisum, Chlorite, Haliotis diversicolor, Polygala tenuifolia, Alpinia oxyphylla, Astragalus membranaceus, Triticum aestivum, Cornus officinalis, Citrus aurantium, Hordeum vulgare, Massa fermentata, Crataegus pinnatifida, and Glycyrrhiza uralensis.

[0031] The pediatric calming and brain-boosting drug preparation of the present invention can be prepared in-house or purchased from a hospital (Approval number: Guangdong Pharmaceutical Preparation Z20070083).

[0032] The technical solution of this invention has the following advantages:

[0033] 1. The present invention provides a thin-layer chromatography method for simultaneously identifying different medicinal flavors in pediatric calming and brain-tonifying drug preparations, comprising the steps of preparing a test solution, preparing a reference medicinal material solution, and detecting by thin-layer chromatography; in preparing the test solution, the sample to be tested is taken, extracted with an organic solvent, separated into solid and liquid, the liquid is dried, dissolved with an organic solvent to obtain the test solution; in preparing the reference medicinal material solution, tangerine peel reference medicinal material and immature bitter orange reference medicinal material are taken, extracted with organic solvents respectively to obtain each reference medicinal material solution; the test solution, tangerine peel reference medicinal material solution, and immature bitter orange reference medicinal material solution are respectively spotted on the same thin-layer plate, developed with a first developing solvent, removed, dried, sprayed with a first colorimetric agent, heated, and examined to identify tangerine peel and immature bitter orange; the first developing solvent is a lower layer solution of chloroform, methanol, and water in a volume ratio of 89-93:42-46:10-14. The thin-layer chromatography (TLC) spectra obtained by the above-described purification method and appropriate ratio of developing solvent of this invention have clear spots, good separation, strong specificity, no background interference, no interference from negative controls, good reproducibility, and good durability. They are not easily affected by the TLC plate, temperature, or humidity. A single identification test can reflect the quality information of multiple medicinal ingredients, making the identification more comprehensive and better reflecting their overall quality characteristics. It also reduces the number of TLC identification tests, reduces reagent consumption, saves time, and improves testing efficiency.

[0034] This invention provides a thin-layer chromatography method for simultaneously identifying different medicinal flavors in pediatric calming and brain-tonifying drug preparations. The same thin-layer plate is used to identify tangerine peel and immature bitter orange peel, reducing the number of tests required for thin-layer identification and saving time and increasing efficiency. Furthermore, only one developing solvent is needed to identify different medicinal flavors (tangerine peel and immature bitter orange peel) in pediatric calming and brain-tonifying drug preparations, reducing reagent usage and making the operation more convenient.

[0035] 2. The present invention provides a thin-layer chromatography method for simultaneously identifying different medicinal flavors in pediatric calming and brain-tonifying drug preparations. The thin-layer chromatography method further includes: taking Notopterygium incisum reference material, extracting it with an organic solvent to obtain a Notopterygium incisum reference material solution; taking the test solution and the Notopterygium incisum reference material solution, spotting them separately on another thin-layer plate, developing them with a second developing solvent, removing them, drying them, and examining them under ultraviolet light to identify Notopterygium incisum; the second developing solvent is chloroform and methanol in a volume ratio of 7-9:1.5-2.5. Using the same test solution as tangerine peel and immature bitter orange peel under the second developing solvent can simultaneously identify Notopterygium incisum. The spots are clear, the separation is good, the specificity is strong, there is no background interference, there is no interference from the negative control, the reproducibility is good, and the durability is good. It is not easily affected by the thin-layer plate, temperature, and humidity, reducing experimental steps and saving time and manpower.

[0036] 3. The present invention provides a thin-layer chromatography method for simultaneously identifying different medicinal flavors in pediatric calming and brain-tonifying drug preparations. A reference herb, *Cornus officinalis*, is extracted with an organic solvent to obtain a *Cornus officinalis* reference herb solution. The test solution and the *Cornus officinalis* reference herb solution are spotted separately onto another thin-layer plate, developed with a third developing solvent, removed, dried, sprayed with a second colorimetric reagent, heated, and examined to identify *Cornus officinalis*. The third developing solvent is ethyl acetate, ethanol, and glacial acetic acid in a volume ratio of 12–17:3–7:0.3–0.5. Using the same test solution as *Citrus reticulata* and *Citrus aurantium* under the third developing solvent allows for simultaneous identification of *Cornus officinalis*. The method produces clear spots, good separation, strong specificity, no background interference, no interference from the negative control, good reproducibility, and good durability. It is not easily affected by the thin-layer plate, temperature, or humidity, reducing experimental steps and saving time and manpower.

[0037] 4. The thin-layer chromatography method provided by the present invention for simultaneously identifying different medicinal flavors in pediatric calming and brain-tonifying drug preparations includes using at least one of hesperidin, neohesperidin, and naringin as indicator components or using purslane as indicator components, to assist in the identification of different medicinal flavors in pediatric calming and brain-tonifying drug preparations, making the identification more accurate.

[0038] In this invention, only one sample solution is needed to identify different medicinal flavors, without the need to treat the sample solution separately for each medicinal flavor, which helps to improve the testing efficiency. When preparing the sample solution, the reference medicinal material solution, and the reference solution, only solvents such as methanol and ethanol are used, and no easily toxic or explosive reagents are used throughout the process, making it environmentally friendly. Attached Figure Description

[0039] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0040] Figure 1 These are thin-layer identification spectra of dried tangerine peel and immature bitter orange obtained in Example 1 of this invention;

[0041] Figure 2 This is the thin-layer identification spectrum of Notopterygium incisum obtained in Example 1 of the present invention;

[0042] Figure 3 This is a thin-layer identification spectrum of Cornus officinalis obtained in Example 2 of the present invention;

[0043] Figure 4 These are the thin-layer identification spectra of dried tangerine peel and immature bitter orange obtained in Example 3 of this invention;

[0044] Figure 5 This is a thin-layer identification spectrum of dried tangerine peel and immature bitter orange obtained in Comparative Example 1 of this invention;

[0045] Figure 6 This is the thin-layer identification spectrum of Notopterygium incisum obtained in Comparative Example 2 of this invention;

[0046] Figure 7 This is the thin-layer identification spectrum of Cornus officinalis obtained in Comparative Example 3 of this invention;

[0047] Figure 8 This is the result of the investigation of thin-layer plates from different manufacturers in the methodological verification of tangerine peel and bitter orange peel in Experiment Example 1 of this invention;

[0048] Figure 9 This is the result of the investigation at different temperatures in the methodological verification of tangerine peel and bitter orange peel in Experiment Example 1 of this invention;

[0049] Figure 10 This is the result of the investigation of different humidity levels in the methodological verification of tangerine peel and bitter orange peel in Experiment Example 1 of this invention;

[0050] Figure 11 This is the result of the investigation of thin-layer plates from different manufacturers in the methodological verification of Notopterygium incisum in Experiment Example 2 of this invention;

[0051] Figure 12 This is the result of the investigation at different temperatures in the methodological verification of Notopterygium incisum in Experiment Example 2 of this invention;

[0052] Figure 13 This is the result of the investigation of different humidity levels in the methodological verification of Notopterygium incisum in Experiment Example 2 of this invention;

[0053] Figure 14 This is the result of the investigation of thin-layer plates from different manufacturers in the methodological verification of Cornus officinalis in Experimental Example 3 of this invention;

[0054] Figure 15 This is the result of the investigation at different temperatures in the methodological verification of Cornus officinalis in Experiment Example 3 of this invention;

[0055] Figure 16 This is the result of the investigation of different humidity levels in the methodological verification of Cornus officinalis in Experiment Example 3 of this invention. Detailed Implementation

[0056] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0057] Unless otherwise specified, the reagents used in the following examples and comparative examples are conventional reagents and can be purchased from conventional reagent manufacturers and distributors. The proportions of each reagent in the developing solvent are volume ratios.

[0058] Pediatric calming and brain-nourishing drug preparations are made from raw materials such as Acorus tatarinowii, Arisaema cum Bile, Pinellia ternata, Poria cocos, Citrus reticulata peel, Notopterygium incisum, Chlorite, Haliotis diversicolor, Polygala tenuifolia, Alpinia oxyphylla, Astragalus membranaceus, Triticum aestivum, Cornus officinalis, Citrus aurantium, Hordeum vulgare, Massa fermentata, Crataegus pinnatifida, and Glycyrrhiza uralensis. Conventional excipients are added, and the preparations are manufactured according to conventional processes to produce clinically acceptable tablets, capsules, pills, granules, honey-refined pills, sustained-release preparations, immediate-release preparations, controlled-release preparations, oral liquid preparations, or injection preparations.

[0059] The preparation methods of various pediatric calming and brain-nourishing drug formulations in the following examples and experimental cases are as follows.

[0060] The prescription for Xiaor Anshen Bunao Granules (Children's Calming and Brain-Nourishing Granules) is shown in the table below:

[0061] 250g of Acorus tatarinowii 165g of Arisaema cum Bile 165g of Pinellia ternata Poria cocos 165g 85g of dried tangerine peel 85g of Notopterygium root 250g of Qingmeng Stone 250g of abalone shell Polygala tenuifolia 165g 165g of Alpinia oxyphylla Astragalus membranaceus 165g 250g of floating wheat 165g of Cornus officinalis 165g of Citrus aurantium 250g of malt Divine Comedy 165g 165g hawthorn 165g of licorice

[0062] Preparation process: The above eighteen ingredients are decocted twice with water, 2 hours each time. The decoctions are combined, filtered, and the filtrate is concentrated into a clear paste with a relative density of 1.25-1.30 (80℃). After cooling, 1500ml of ethanol is added, stirred evenly, and allowed to stand for 24 hours. The supernatant is collected, the ethanol is recovered, and the paste is concentrated into a clear paste with a relative density of 1.25-1.30 (80℃). Sucrose powder and dextrin are added in appropriate amounts, mixed evenly, granulated, and dried at 70℃ to obtain 1000g of the product. Using the same batch of medicinal materials, different batches of pediatric calming and brain-nourishing granules were prepared according to the above prescription and process, with batch numbers 20220804, 20220822, 20220824, and 20220829, respectively.

[0063] The granules lacking the tangerine peel and bitter orange peel, the granules lacking the notopterygium root and the granules lacking the cornus fruit were prepared using the same method as the pediatric calming and brain-nourishing granules. The only difference in the preparation method was the absence of the corresponding medicinal materials.

[0064] Example 1

[0065] This embodiment provides a thin-layer chromatography method for identifying pediatric calming and brain-nourishing granules, specifically including:

[0066] (I) Identification of dried tangerine peel and immature bitter orange peel

[0067] Preparation of test solution: Four batches of Xiao'er Anshen Bunao Granules were used as test samples. 1g of the test sample was taken, ground into a fine powder, 50ml of methanol was added, and the mixture was sonicated for 30 minutes. The mixture was filtered, the filtrate was evaporated to dryness, and the residue was dissolved in 2ml of methanol to obtain the test solution.

[0068] Preparation of reference herbal solutions: Take 0.1g each of dried tangerine peel and immature bitter orange peel as reference herbs, grind them into a fine powder, add 50ml of methanol to each, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 2ml of methanol to dissolve the residue, and obtain the dried tangerine peel reference herbal solution and immature bitter orange peel reference herbal solution.

[0069] Preparation of reference solutions: Take hesperidin, neohesperidin and naringin reference standards separately, and add methanol to prepare solutions containing 1 mg of reference standard per 1 ml, thus obtaining the reference solution.

[0070] Preparation of negative control solution: Take 1g of granules lacking both tangerine peel and bitter orange peel, grind them into a fine powder, add 50ml of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 2ml of methanol to dissolve the residue, and use it as the negative control solution lacking both tangerine peel and bitter orange peel.

[0071] Identification: Perform thin-layer chromatography (General Rule 0502). Apply 5 μl of each of the above solutions to the same silica gel G thin-layer plate. Then, apply 5 μl each of the test solution (batch number: 20220804), hesperidin reference solution, neohesperidin reference solution, and naringin reference solution sequentially to the same location on the plate. Develop using the lower layer of a chloroform-methanol-water (91:44:12) solution as the developing solvent. Remove the plate, air dry, spray with 3% aluminum trichloride ethanol solution, heat at 105℃ for 5 minutes, and examine under ultraviolet light (365 nm). Results are shown below. Figure 1 .

[0072] Figure 1 Among them, 1. A double negative solution for tangerine peel and immature bitter orange peel; 2. Tangerine peel reference material solution; 3. Immature bitter orange peel reference material solution; 4. Hesperidin reference solution; 5. Neohesperidin reference solution; 6. Naringin reference solution; 7. Test solution (batch number: 20220804); 8. Test solution (batch number: 20220822); 9. Test solution (batch number: 20220824); 10. Test solution (batch number: 20220829); 11. Test solution (batch number: 20220804) + hesperidin reference solution + neohesperidin reference solution + naringin reference solution.

[0073] As can be seen from the images, in the chromatogram of the test sample, fluorescent main spots of the same color appear at the corresponding positions as in the chromatograms of hesperidin, neohesperidin, naringin reference standards, tangerine peel reference material, and bitter orange peel reference material, and there is no interference from the negative control. Validated with four batches of samples, this method demonstrates good specificity and reproducibility in identifying tangerine peel and bitter orange peel in the preparation, with no interference from the negative control.

[0074] (II) Identification of Notopterygium incisum

[0075] Preparation of the reference herb solution: Take 0.1g of Notopterygium incisum reference herb, grind it into a fine powder, add 50ml of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 2ml of methanol to dissolve the residue, and obtain the Notopterygium incisum reference herb solution.

[0076] Preparation of reference solution: Take the imperatorin reference standard and add methanol to prepare a solution containing 1 mg per 1 ml, which is used as the reference solution.

[0077] Preparation of negative control solution: Take 1g of notopterygium glutamate negative control granules, grind them into a fine powder, add 50ml of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 2ml of methanol to dissolve the residue, and use it as notopterygium glutamate negative control solution.

[0078] Identification: Perform thin-layer chromatography (General Rule 0502). Apply 5 μl each of the test solution, the Notopterygium incisum reference material solution, the Imperatorin reference solution, and the Notopterygium incisum-deficient negative solution to the same silica gel G thin-layer plate. Then, apply 5 μl each of the test solution (batch number: 20220804) and the Imperatorin reference solution sequentially to the same location on the same thin-layer plate in a stacked manner. Develop using chloroform-methanol (8:2) as the developing solvent. Remove the plate, air-dry, and examine under ultraviolet light (365 nm). Results are shown below. Figure 2 .

[0079] Figure 2 Among them, 1. Notopterygium incisum negative control solution; 2. Notopterygium incisum reference medicinal material solution; 3. Imperatorin reference solution; 4. Test solution (batch number: 20220804); 5. Test solution (batch number: 20220822); 6. Test solution (batch number: 20220824); 7. Test solution (batch number: 20220829); 8. Test solution (batch number: 20220804) + Imperatorin reference solution.

[0080] As can be seen from the images, the test sample chromatogram shows a fluorescent main spot of the same color at the corresponding position as the reference standard for purslane and the reference herb for Notopterygium root, and there is no interference from the negative control. Validation with four batches of samples shows that this method has good specificity and reproducibility in identifying Notopterygium root in the preparation, and there is no interference from the negative control.

[0081] Example 2

[0082] This embodiment provides a thin-layer chromatography method for identifying pediatric calming and brain-nourishing granules, comprising the following steps:

[0083] For the preparation of the test solution, different batches of Xiao'er Anshen Bunao Granules were used as test samples. 10g of the test sample was taken, ground into a fine powder, 30ml of ethanol was added, and the mixture was sonicated for 30min. The mixture was filtered, the supernatant was evaporated to dryness, and the residue was dissolved in 2ml of ethanol to obtain the test solution.

[0084] Preparation of the reference herb solution: Take 0.2g of Cornus officinalis reference herb, grind it into a fine powder, add 30ml of ethanol, sonicate for 30min, filter, evaporate the supernatant to dryness, add 2ml of ethanol to dissolve the residue, and use it as the reference herb solution.

[0085] Preparation of negative solution: Take 10g of Cornus officinalis negative granules, grind them into a fine powder, add 30ml of ethanol, sonicate for 30min, filter, evaporate the supernatant to dryness, add 2ml of ethanol to dissolve the residue, and use it as Cornus officinalis negative solution.

[0086] Identification: Perform thin-layer chromatography (General Rule 0502). Apply 5 μl of each of the above solutions to the same silica gel G thin-layer plate. Also, apply 5 μl each of the test solution (batch number: 20220804) and the Cornus officinalis reference material solution sequentially to the same location on the same thin-layer plate in a stacked manner. Develop with ethyl acetate-ethanol-glacial acetic acid (15:5:0.4). Remove the plate, air dry, spray with 5% vanillin-sulfuric acid solution, and heat at 105℃ until the spots are clearly visible. Examine under sunlight. Results are shown below. Figure 3 .

[0087] Figure 3 Among them, 1. Cornus officinalis negative solution; 2. Cornus officinalis reference material solution; 3. Test solution (batch number: 20220804); 4. Test solution (batch number: 20220822); 5. Test solution (batch number: 20220824); 6. Test solution (batch number: 20220829); 7. Test solution (batch number: 20220804) + Cornus officinalis reference material solution.

[0088] As can be seen from the images, the test sample chromatogram shows a fluorescent main spot of the same color at the corresponding position as the Cornus officinalis reference material chromatogram, and there is no interference from the negative control. Validation with four batches of samples shows that this method has good specificity and reproducibility in identifying Notopterygium incisum in preparations, and there is no interference from the negative control.

[0089] Example 3

[0090] This embodiment provides a thin-layer chromatography method for identifying pediatric calming and brain-nourishing granules, comprising the following steps:

[0091] Preparation of the test solution: It is basically the same as in Example 1, except that the amount of test sample is increased from 1g to 10g.

[0092] Preparation of the control herbal solution: It is basically the same as in Example 1, except that the amount of the control herbal material is increased from 0.1g to 0.5g.

[0093] Preparation of reference solutions: Take hesperidin and naringin reference standards separately, and add methanol to prepare solutions containing 1 mg of reference standard per 1 ml, thus obtaining the reference solution.

[0094] Preparation of negative control solution: Take 10g of granules lacking both tangerine peel and bitter orange peel, grind them into a fine powder, add 50ml of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 2ml of methanol to dissolve the residue, and use it as the negative control solution lacking both tangerine peel and bitter orange peel.

[0095] Identification: Perform thin-layer chromatography (General Rule 0502). Apply 2 μl of each of the above solutions to the same silica gel G thin-layer plate. Develop using the lower layer of a chloroform-methanol-water (91:44:12) solution as the developing solvent. Remove the plate, air dry, spray with 3% aluminum trichloride ethanol solution, heat at 105°C for 5 minutes, and examine under ultraviolet light (365 nm). Results are shown below. Figure 4 .

[0096] Figure 4 Among them, 1. Double negative solution for tangerine peel and immature bitter orange peel; 2. Tangerine peel reference material solution (2μl); 3. Immature bitter orange peel reference material solution; 4. Hesperidin reference standard; 5. Naringin reference standard solution; 6. Test solution (batch number: 20220804).

[0097] As can be seen from the images, in the chromatogram of the test sample, there are fluorescent main spots of the same color at the corresponding positions as those of the reference standards of hesperidin, naringin, tangerine peel, and bitter orange peel. The negative sample has no interference, but there are defects such as tailing and inconsistent Rf values ​​of thin-layer spots.

[0098] Compared with Example 1, it can be seen that when identifying tangerine peel and bitter orange peel, it is preferable to control the sample amount of the test sample to be 0.5-1.5g.

[0099] Comparative Example 1

[0100] This comparative example provides a thin-layer chromatography method for identifying pediatric calming and brain-nourishing granules, comprising the following steps:

[0101] Preparation of test solutions: Using Xiao'er Anshen Bunao Granules as the test sample, take 10g of the sample, grind it into a fine powder, add 50ml of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 2ml of methanol to obtain test solution A; Using Xiao'er Anshen Bunao Granules as the test sample, take 10g of the sample, add 50ml of methanol, heat under reflux for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 25ml of water, extract with 10ml of ether, discard the ether, then extract three times with 20ml of ethyl acetate each time, combine the ethyl acetate extracts, wash twice with 20ml of water each time, discard the washings, evaporate the ethyl acetate extract to dryness, dissolve the residue in 1ml of methanol to obtain test solution B.

[0102] Preparation of reference herbal solutions: Take 0.5g each of dried tangerine peel and immature bitter orange peel as reference herbs, grind them into a fine powder, add 50ml of methanol to each, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 2ml of methanol to dissolve the residue, and obtain the dried tangerine peel reference herbal solution and immature bitter orange peel reference herbal solution.

[0103] Preparation of reference solution: Separately prepare a solution containing 1 mg of reference standard per 1 ml by adding hesperidin to methanol.

[0104] Preparation of negative control solution: Take 10g of granules lacking tangerine peel and bitter orange peel, grind them into a fine powder, add 50ml of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 2ml of methanol to dissolve the residue, and use it as the negative control solution.

[0105] Identification: Following thin-layer chromatography (General Rule 0502), apply 2 μl of the tangerine peel reference solution, 2 μl of the bitter orange peel reference solution, 5 μl of the negative control solution, 10 μl of the reference solution, 5 μl of the test solution A, and 5 μl of the test solution B to the same silica gel G thin-layer plate. Develop the plate using ethyl acetate-methanol-water (100:17:10) solution. Remove the plate, air dry, spray with 3% aluminum trichloride ethanol solution, heat at 105℃ for 5 minutes, and examine under ultraviolet light (365 nm). Results are shown below. Figure 5 .

[0106] Figure 5 Among them, 1. Tangerine peel reference solution; 2. Bitter orange peel reference solution; 3. Negative test solution; 4. Hesperidin reference solution; 5. Test solution B; 6. Test solution A.

[0107] As can be seen from the images, the separation of the main spots in the chromatograms of both test samples was poor, and the negative solution had fluorescent interference spots.

[0108] Comparative Example 2

[0109] This comparative example provides a thin-layer chromatography method for identifying pediatric calming and brain-nourishing granules, comprising the following steps:

[0110] Preparation of test solution: Using Xiao'er Anshen Bunao Granules as the test sample, take 10g of the product, grind it into a fine powder, add 50ml of water to dissolve it, centrifuge, take the supernatant, extract it twice with 30ml of ether each time, combine the ether extracts, concentrate it to about 1ml, and use it as the test solution.

[0111] Preparation of the reference herb solution: Take 0.5g of Notopterygium incisum reference herb, add 50ml of water, decoct for 20 minutes, cool, centrifuge, take the supernatant, extract twice with 20ml of ether each time, combine the ether liquids, evaporate to about 1ml, and use as the reference herb solution.

[0112] Preparation of negative control solution: Take 10g of Qianghuo negative granules, grind them into a fine powder, add 50ml of water to dissolve them, centrifuge, take the supernatant, extract with ether twice, 30ml each time, combine the ether extracts, concentrate to about 1ml, and use as negative control solution.

[0113] Identification: Perform thin-layer chromatography (General Rule 0502). Apply 10 μl of the test solution, 20 μl of the test solution, 10 μl of the reference herb solution, and 10 μl of the negative control solution separately to the same silica gel G thin-layer plate. Develop with cyclohexane-ethyl acetate (4:1) solution as the developing solvent. Spray with 10% sulfuric acid ethanol solution and heat at 85℃ for about 5 minutes. Examine under ultraviolet light (365 nm). The results are shown in the figure. Figure 6 .

[0114] Figure 6 In the sample, 1. Notopterygium incisum reference solution; 2. Negative control solution; 3. Test solution (10 μl); 4. Test solution (20 μl).

[0115] As can be seen from the image, the negative sample in the chromatogram of the test sample is interfered with, and there are no characteristic spots that can be used to identify this preparation.

[0116] Comparative Example 3

[0117] This comparative example provides a thin-layer chromatography method for identifying pediatric calming and brain-nourishing granules, comprising the following steps:

[0118] Preparation of the test solution: Using Xiaor Anshen Bunao Granules as the test sample, take 10g of the product, place it in a stoppered conical flask, add 50ml of diethyl ether, sonicate for 30 minutes, centrifuge, and evaporate the supernatant to dryness. Wash the residue three times with 15ml of petroleum ether (30-60℃), and discard the petroleum ether solution. Dissolve the residue in 1ml of anhydrous ethanol-chloroform (3:2) mixed solution to obtain the test solution.

[0119] Preparation of the reference herb solution: Take 0.5g of Cornus officinalis reference herb, add 10ml of ethyl acetate, sonicate for 15 minutes, filter, evaporate the filtrate to dryness, add 1ml of anhydrous ethanol-chloroform (3:2) mixed solution to dissolve the residue, and use it as the reference herb solution.

[0120] Preparation of reference solution: Separately prepare a solution containing 1 mg of reference standard per 1 ml by adding loganin, ursolic acid and monoglucoside reference standards to methanol.

[0121] Preparation of negative control solution: Take 10g of Cornus officinalis negative control granules, place them in a stoppered conical flask, add 50ml of diethyl ether, sonicate for 30 minutes, centrifuge, and evaporate the supernatant to dryness. Wash the residue three times with 15ml of petroleum ether (30-60℃), and discard the petroleum ether solution. Dissolve the residue in 1ml of anhydrous ethanol-chloroform (3:2) mixed solution to obtain the negative control solution.

[0122] Identification: Perform thin-layer chromatography (General Rule 0502). Apply 20 μl of the test solution, 10 μl of the reference herb solution, 20 μl of the negative control solution, 5 μl of loganin reference solution, 5 μl of ursolic acid reference solution, and 5 μl of monosodium glutamate reference solution separately to the same silica gel G thin-layer plate. Use toluene-ethyl acetate-glacial acetic acid (12:2:0.5) as the developing solvent, spray with 10% sulfuric acid ethanol solution, heat at 85℃ for about 5 minutes, and examine under ultraviolet light (365 nm). The results are shown in the figure. Figure 7 .

[0123] Figure 7 Among them, 1. Cornus officinalis reference solution; 2. Loganin; 3. Monoglycoside; 4. Ursolic acid; 5. Negative control solution; 6. Test solution 20220824.

[0124] As can be seen from the image, the negative sample in the chromatogram of the test sample is interfered with, and there are no characteristic spots that can be used to identify this preparation.

[0125] Methodological verification of tangerine peel and immature bitter orange peel in Experiment Example 1

[0126] 1. Comparison of thin-layer boards from different manufacturers

[0127] Following the preparation method given in Example 1, four batches of pediatric calming and brain-nourishing granules test solution, double negative solution lacking tangerine peel and bitter orange peel, tangerine peel and bitter orange peel reference medicinal material solution, and hesperidin, neohesperidin and naringin reference standard solution were prepared respectively.

[0128] Identification: Apply 5 μl of each of the above solutions to the same silica gel G thin-layer plate. Then, apply 5 μl each of the test solution (batch number: 20220804), hesperidin reference solution, neohesperidin reference solution, and naringin reference solution sequentially to the same location on the same thin-layer plate in a stacked manner. Develop using the lower layer of chloroform-methanol-water (91:44:12) as the developing solvent. Remove the plate, air dry, spray with 3% aluminum trichloride ethanol solution, heat at 105℃ for 5 minutes, and examine under ultraviolet light (365 nm). The development of thin-layer plates from different brands (Yantai plate, Merck plate) was investigated; the results are shown below. Figure 8 .

[0129] Figure 8Among them, 1. A double negative solution for tangerine peel and immature bitter orange peel; 2. Tangerine peel reference material solution; 3. Immature bitter orange peel reference material solution; 4. Hesperidin reference solution; 5. Neohesperidin reference solution; 6. Naringin reference solution; 7. Test solution (batch number: 20220804); 8. Test solution (batch number: 20220822); 9. Test solution (batch number: 20220824); 10. Test solution (batch number: 20220829); 11. Test solution (batch number: 20220804) + hesperidin reference solution + neohesperidin reference solution + naringin reference solution.

[0130] The left side shows a Merck thin-layer plate, and the right side shows a Yantai thin-layer plate. T (temperature): 18.2℃; RH (relative humidity): 36%.

[0131] As can be seen from the attached figures, the unfolding effects of the two methods are basically the same, and there is no significant difference in the separation effect and spot display, indicating that the method has good durability on different thin-layer plates.

[0132] 2. Investigation at different temperatures

[0133] Following the preparation method given in Example 1, four batches of pediatric calming and brain-nourishing granules test solution, double negative solution lacking tangerine peel and bitter orange peel, tangerine peel and bitter orange peel reference medicinal material solution, and hesperidin, neohesperidin and naringin reference standard solution were prepared respectively.

[0134] Identification: Apply 5 μl of each of the above solutions to the same silica gel G thin-layer plate. Then, apply 5 μl each of the test solution (batch number: 20220804), hesperidin reference solution, neohesperidin reference solution, and naringin reference solution sequentially to the same location on the same thin-layer plate in a stacked manner. Develop using the lower layer of chloroform-methanol-water (91:44:12) as the developing solvent. Remove the plate, air dry, spray with 3% aluminum trichloride ethanol solution, heat at 105℃ for 5 minutes, and examine under ultraviolet light (365 nm). The development at different temperatures (5.5℃, 25.5℃) was investigated, and the results are shown in the table below. Figure 9 .

[0135] Figure 9The following solutions were prepared: 1. Double negative solution for tangerine peel and immature bitter orange peel; 2. Tangerine peel reference solution; 3. Immature bitter orange peel reference solution; 4. Hesperidin reference solution; 5. Neohesperidin reference solution; 6. Naringin reference solution; 7. Test solution (batch number: 20220804); 8. Test solution (batch number: 20220822); 9. Test solution (batch number: 20220824); 10. Test solution (batch number: 20220829); 11. Test solution (batch number: 20220804) + hesperidin reference solution + neohesperidin reference solution + naringin reference solution. The temperature for thin-layer chromatography on the left was 5.5℃, and on the right it was 25.5℃. The relative humidity was 36% for both.

[0136] As can be seen from the attached figures, the test between 5.5 and 25.5℃ has no significant effect on identification, and there is no significant difference in separation effect and spot display, indicating that this method has good durability at different temperatures.

[0137] 3. Investigation of different humidity levels

[0138] Following the preparation method given in Example 1, four batches of pediatric calming and brain-nourishing granules test solution, double negative solution lacking tangerine peel and bitter orange peel, tangerine peel and bitter orange peel reference medicinal material solution, and hesperidin, neohesperidin and naringin reference standard solution were prepared respectively.

[0139] Identification: Apply 5 μl of each of the above solutions to the same silica gel G thin-layer plate. Then, apply 5 μl each of the test solution (batch number: 20220804), hesperidin reference solution, neohesperidin reference solution, and naringin reference solution sequentially to the same location on the same thin-layer plate in a stacked manner. Develop using the lower layer of chloroform-methanol-water (91:44:12) as the developing solvent. Remove the plate, air dry, spray with 3% aluminum trichloride ethanol solution, heat at 105℃ for 5 minutes, and examine under ultraviolet light (365 nm). The development under different humidity levels (32% and 88% relative humidity) was investigated, and the results are shown in the table below. Figure 10 .

[0140] Figure 10The following solutions were prepared: 1. Double negative solution for tangerine peel and immature bitter orange peel; 2. Tangerine peel reference solution; 3. Immature bitter orange peel reference solution; 4. Hesperidin reference solution; 5. Neohesperidin reference solution; 6. Naringin reference solution; 7. Test solution (batch number: 20220804); 8. Test solution (batch number: 20220822); 9. Test solution (batch number: 20220824); 10. Test solution (batch number: 20220829); 11. Test solution (batch number: 20220804) + hesperidin reference solution + neohesperidin reference solution + naringin reference solution. The humidity (RH) for the thin-layer chromatography detection on the left was 32%, and on the right it was 88%, with a temperature of 18.2℃ for both.

[0141] As can be seen from the attached figures, the test was conducted with relative humidity between 32% and 88%, which had no significant impact on identification. There was no significant difference in the separation effect and spot display, indicating that the method is durable under different humidity conditions.

[0142] Experimental Example 2: Methodological Verification of Notopterygium incisum

[0143] 1. Comparison of thin-layer boards from different manufacturers

[0144] Following the preparation method given in Example 1, four batches of Xiao'er Anshen Bunao Granules test solution, Qianghuo-deficient negative solution, Qianghuo reference herb solution, and Zihuaqianhu glycoside reference solution were prepared respectively.

[0145] Identification: Perform thin-layer chromatography (General Rule 0502). Apply 5 μl of each of the above solutions to the same silica gel G thin-layer plate. Also, apply 5 μl each of the test solution (batch number: 20220804) and the imperatorin reference solution sequentially to the same location on the same thin-layer plate in a stacked manner. Develop using chloroform-methanol (8:2) as the developing solvent. Remove the plate, air-dry, and examine under ultraviolet light (365 nm). The development of different brands of thin-layer plates (Yantai plate, Merck plate) was investigated. The results are shown in [Figure number missing]. Figure 11 .

[0146] Figure 11 Among them, 1. Notopterygium incisum negative control solution; 2. Notopterygium incisum reference medicinal material solution; 3. Imperatorin reference solution; 4. Test solution (batch number: 20220804); 5. Test solution (batch number: 20220822); 6. Test solution (batch number: 20220824); 7. Test solution (batch number: 20220829); 8. Test solution (batch number: 20220804) + Imperatorin reference solution.

[0147] The left side shows a Merck thin-layer plate, and the right side shows a Yantai thin-layer plate. T (temperature): 17.9℃; RH (relative humidity): 51%.

[0148] As can be seen from the attached figures, the unfolding effects of the two methods are basically the same, and there is no significant difference in the separation effect and spot display, indicating that the method has good durability on different thin-layer plates.

[0149] 2. Investigation at different temperatures

[0150] Following the preparation method given in Example 1, four batches of Xiao'er Anshen Bunao Granules test solution, Qianghuo-deficient negative solution, Qianghuo reference herb solution, and Zihuaqianhu glycoside reference solution were prepared respectively.

[0151] Identification: Perform thin-layer chromatography (General Rule 0502). Apply 5 μl of each of the above solutions to the same silica gel G thin-layer plate. Also, apply 5 μl each of the test solution (batch number: 20220804) and the imperatorin reference solution sequentially to the same location on the same thin-layer plate in a stacked manner. Develop using chloroform-methanol (8:2) as the developing solvent. Remove the plate, air dry, and examine under ultraviolet light (365 nm). Investigate the development at different temperatures (5.5℃, 25.5℃). Results are shown in [Figure number missing]. Figure 12 .

[0152] Figure 12 Among them, 1. Notopterygium incisum negative control solution; 2. Notopterygium incisum reference medicinal material solution; 3. Imperatorin reference solution; 4. Test solution (batch number: 20220804); 5. Test solution (batch number: 20220822); 6. Test solution (batch number: 20220824); 7. Test solution (batch number: 20220829); 8. Test solution (batch number: 20220804) + Imperatorin reference solution.

[0153] The temperature for thin-layer chromatography on the left was 5.5℃, and on the right it was 25.5℃. The relative humidity for both was 51%.

[0154] As can be seen from the attached figures, the test between 5.5 and 25.5℃ has no significant effect on identification, and there is no significant difference in separation effect and spot display, indicating that this method has good durability at different temperatures.

[0155] 3. Investigation of different humidity levels

[0156] Following the preparation method given in Example 1, four batches of Xiao'er Anshen Bunao Granules test solution, Qianghuo-deficient negative solution, Qianghuo reference herb solution, and Zihuaqianhu glycoside reference solution were prepared respectively.

[0157] Identification: Perform thin-layer chromatography (General Rule 0502). Apply 5 μl of each of the above solutions to the same silica gel G thin-layer plate. Also, apply 5 μl each of the test solution (batch number: 20220804) and the imperatorin reference solution sequentially to the same location on the plate. Develop using chloroform-methanol (8:2) as the developing solvent. Remove the plate, air dry, and examine under ultraviolet light (365 nm). Investigate the development under different humidity levels (32% and 88% relative humidity). Results are shown in [Figure number missing]. Figure 13 .

[0158] Figure 13 Among them, 1. Notopterygium incisum negative control solution; 2. Notopterygium incisum reference medicinal material solution; 3. Imperatorin reference solution; 4. Test solution (batch number: 20220804); 5. Test solution (batch number: 20220822); 6. Test solution (batch number: 20220824); 7. Test solution (batch number: 20220829); 8. Test solution (batch number: 20220804) + Imperatorin reference solution.

[0159] The humidity detected by thin-layer chromatography on the left is RH: 32%, and on the right it is 88%, with a temperature of 17.9℃ for both.

[0160] As can be seen from the attached figures, the test was conducted with relative humidity between 32% and 88%, which had no significant impact on identification. There was no significant difference in the separation effect and spot display, indicating that the method is durable under different humidity conditions.

[0161] Experimental Example 3: Methodological Verification of Cornus officinalis

[0162] 1. Comparison of thin-layer boards from different manufacturers

[0163] Following the preparation method given in Example 2, four batches of pediatric calming and brain-nourishing granules test solution, Cornus officinalis-deficient negative solution, and Cornus officinalis-reference medicinal material solution were prepared respectively.

[0164] Identification: Perform thin-layer chromatography (General Rule 0502). Apply 5 μl of each of the above solutions to the same silica gel G thin-layer plate. Also, apply 5 μl each of the test solution (batch number: 20220804) and the Cornus officinalis reference material solution sequentially to the same location on the same thin-layer plate in a stacked manner. Develop with ethyl acetate-ethanol-glacial acetic acid (15:5:0.4). Remove the plate, air dry, spray with 5% vanillin-sulfuric acid solution, and heat at 105℃ until the spots are clearly visible. Examine under sunlight. The development of different brands of thin-layer plates (Yantai plate, Merck plate) was investigated; the results are shown below. Figure 14 .

[0165] Figure 14Among them, 1. Cornus officinalis negative solution; 2. Cornus officinalis reference material solution; 3. Test solution (batch number: 20220804); 4. Test solution (batch number: 20220822); 5. Test solution (batch number: 20220824); 6. Test solution (batch number: 20220829); 7. Test solution (batch number: 20220804) + Cornus officinalis reference material solution.

[0166] The left side shows a Merck thin-layer plate, and the right side shows a Yantai thin-layer plate. T (temperature): 17.7℃; RH (relative humidity): 53%.

[0167] As can be seen from the attached figures, the unfolding effects of the two methods are basically the same, and there is no significant difference in the separation effect and spot display, indicating that the method has good durability on different thin-layer plates.

[0168] 2. Investigation at different temperatures

[0169] Following the preparation method given in Example 2, four batches of pediatric calming and brain-nourishing granules test solution, Cornus officinalis-deficient negative solution, and Cornus officinalis-reference medicinal material solution were prepared respectively.

[0170] Identification: Perform thin-layer chromatography (General Rule 0502). Apply 5 μl of each of the above solutions to the same silica gel G thin-layer plate. Also, apply 5 μl each of the test solution (batch number: 20220804) and the Cornus officinalis reference material solution sequentially to the same location on the plate. Develop with ethyl acetate-ethanol-glacial acetic acid (15:5:0.4). Remove the plate, air dry, spray with 5% vanillin-sulfuric acid solution, and heat at 105℃ until the spots are clearly visible. Examine under sunlight. The development at different temperatures (5.5℃, 25.5℃) was investigated, and the results are shown below. Figure 15 .

[0171] Figure 15 Among them, 1. Cornus officinalis negative solution; 2. Cornus officinalis reference material solution; 3. Test solution (batch number: 20220804); 4. Test solution (batch number: 20220822); 5. Test solution (batch number: 20220824); 6. Test solution (batch number: 20220829); 7. Test solution (batch number: 20220804) + Cornus officinalis reference material solution.

[0172] The temperature for thin-layer chromatography on the left was 5.5℃, and on the right it was 25.5℃. The relative humidity for both was 53%.

[0173] As can be seen from the attached figures, the test between 5.5 and 25.5℃ has no significant effect on identification, and there is no significant difference in separation effect and spot display, indicating that this method has good durability at different temperatures.

[0174] 3. Investigation of different humidity levels

[0175] Following the preparation method given in Example 2, four batches of pediatric calming and brain-nourishing granules test solution, Cornus officinalis-deficient negative solution, and Cornus officinalis-reference medicinal material solution were prepared respectively.

[0176] Identification: Perform thin-layer chromatography (General Rule 0502). Apply 5 μl of each of the above solutions to the same silica gel G thin-layer plate. Also, apply 5 μl each of the test solution (batch number: 20220804) and the Cornus officinalis reference material solution sequentially to the same location on the plate. Develop with ethyl acetate-ethanol-glacial acetic acid (15:5:0.4) as the developing solvent. Remove the plate, air dry, spray with 5% vanillin-sulfuric acid solution, and heat at 105℃ until the spots are clearly visible. Examine under sunlight. The development under different humidity levels (32% and 88% relative humidity) was investigated. Results are shown in the figure. Figure 16 .

[0177] Figure 16 Among them, 1. Cornus officinalis negative solution; 2. Cornus officinalis reference material solution; 3. Test solution (batch number: 20220804); 4. Test solution (batch number: 20220822); 5. Test solution (batch number: 20220824); 6. Test solution (batch number: 20220829); 7. Test solution (batch number: 20220804) + Cornus officinalis reference material solution.

[0178] The humidity detected by thin-layer chromatography on the left is RH: 32%, and on the right it is 88%, with a temperature of 17.7℃ for both.

[0179] As can be seen from the attached figures, the test was conducted with relative humidity between 32% and 88%, which had no significant impact on identification. There was no significant difference in the separation effect and spot display, indicating that the method is durable under different humidity conditions.

[0180] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A thin-layer chromatography method for simultaneously identifying different medicinal flavors in a pediatric calming and brain-tonifying drug preparation, characterized in that, Includes the following steps: S1: Take the sample to be tested, extract it with an organic solvent, separate the solid and liquid, dry the liquid, dissolve it with an organic solvent to obtain the test solution; the organic solvent used in step S1 is methanol; S2: Take the reference medicinal materials of dried tangerine peel and immature bitter orange peel, extract them separately with organic solvents, and prepare solutions of each reference medicinal material; S3: Take the test sample solution, tangerine peel reference medicinal material solution, and bitter orange peel reference medicinal material solution, and spot them separately on the same thin-layer plate. Develop with the first developing solvent, remove, air dry, spray with the first color developing agent, heat, examine, and identify tangerine peel and bitter orange peel; the first developing solvent is a lower layer solution of chloroform, methanol, and water in a volume ratio of 89~93:42~46:10~14; the first color developing agent includes 2~5% aluminum trichloride ethanol solution.

2. The thin-layer chromatography method for simultaneously identifying different medicinal flavors in a pediatric calming and brain-tonifying drug preparation according to claim 1, characterized in that, The thin-layer chromatography method further includes: taking Notopterygium incisum reference material, extracting it with an organic solvent to prepare Notopterygium incisum reference material solution; taking the test solution and Notopterygium incisum reference material solution obtained in step S1, spotting them separately on another thin-layer plate, developing them with a second developing solvent, removing them, drying them, and examining them under ultraviolet light to identify Notopterygium incisum; the second developing solvent is chloroform and methanol in a volume ratio of 7~9:1.5~2.5; the organic solvent is methanol.

3. The thin-layer chromatography method for simultaneously identifying different medicinal flavors in a pediatric calming and brain-tonifying drug preparation according to claim 2, characterized in that, The wavelength of the ultraviolet light is 365nm.

4. The thin-layer chromatography method for simultaneously identifying different medicinal flavors in a pediatric calming and brain-tonifying drug preparation according to claim 2, characterized in that, The thin-layer chromatography method further includes: taking imperatorin reference standard, adding organic solvent to prepare imperatorin reference standard solution, and spotting it, the test solution and the Notopterygium incisum reference medicinal material solution on the same thin-layer plate before development.

5. The thin-layer chromatography method for simultaneously identifying different medicinal flavors in a pediatric calming and brain-tonifying drug preparation according to claim 4, characterized in that, Each 1 mL of the above-mentioned imperatorin reference solution contains 0.8 to 1.2 mg of reference standard.

6. The thin-layer chromatography method for simultaneously identifying different medicinal flavors in a pediatric calming and brain-tonifying drug preparation according to claim 1, characterized in that, The thin-layer chromatography method further includes: taking Cornus officinalis reference material, extracting it with an organic solvent to obtain a Cornus officinalis reference material solution; taking the test solution and the Cornus officinalis reference material solution, spotting them separately on another thin-layer plate, developing them with a third developing solvent, removing them, drying them, spraying them with a second colorimetric solvent, heating them, examining them, and identifying Cornus officinalis. The third developing solvent is ethyl acetate, ethanol, and glacial acetic acid in a volume ratio of 12~17:3~7:0.3~0.5; the second colorimetric solvent includes vanillin sulfuric acid solution, and the organic solvent is anhydrous ethanol.

7. The thin-layer chromatography method for simultaneously identifying different medicinal flavors in a pediatric calming and brain-tonifying drug preparation according to claim 1, characterized in that, The thin-layer chromatography method further includes: taking at least one of hesperidin, neohesperidin, and naringin reference standards, adding an organic solvent to prepare a first reference solution, and step S3 further includes spotting the reference solution onto the same thin-layer plate used in step S3 before development.

8. The thin-layer chromatography method for simultaneously identifying different medicinal flavors in a pediatric calming and brain-tonifying drug preparation according to claim 7, characterized in that, Each 1 mL of the first reference solution contains 0.8 to 1.2 mg of each reference standard.

9. The thin-layer chromatography method for simultaneously identifying different medicinal flavors in a pediatric calming and brain-tonifying drug preparation according to claim 1, characterized in that, In step S1 or S2, the extraction includes shaking extraction or ultrasonic extraction; And / or, step S2 includes solid-liquid separation, drying the liquid, and dissolving it with an organic solvent after extraction with an organic solvent; the organic solvent includes at least one of anhydrous ethanol, methanol, and n-hexane. And / or, in step S3, after spraying the first color developer, the heating temperature is 102~108℃.

10. The thin-layer chromatography method for simultaneously identifying different medicinal flavors in pediatric calming and brain-tonifying drug preparations according to any one of claims 1-9, characterized in that, Step S1 includes taking 0.5-15g of the sample to be tested, adding 15-60mL of methanol and sonicating for 20-40 minutes, filtering, evaporating the filtrate to dryness, and dissolving the residue in 1-3mL of methanol to obtain the test solution.

11. The thin-layer chromatography method for simultaneously identifying different medicinal flavors in pediatric calming and brain-tonifying drug preparations according to any one of claims 1-9, characterized in that, Step S2 includes taking 0.05-0.15g of tangerine peel reference material and citrus aurantium reference material, adding 15-60mL of methanol to ultrasonically treat for 20-40 minutes, filtering, evaporating the filtrate to dryness, and dissolving the residue in 1-3ml of methanol to obtain solutions of each reference material.

12. The thin-layer chromatography method for simultaneously identifying different medicinal flavors in a pediatric calming and brain-tonifying drug preparation according to claim 2 or 3, characterized in that, The preparation of the Notopterygium incisum reference herb solution includes the steps of solid-liquid separation, drying the liquid, and dissolving it with an organic solvent after extraction with an organic solvent, wherein the organic solvent is methanol.

13. The thin-layer chromatography method for simultaneously identifying different medicinal flavors in a pediatric calming and brain-tonifying drug preparation according to claim 12, characterized in that, The preparation method of the Notopterygium incisum reference herb solution includes taking 0.05~0.15g of Notopterygium incisum reference herb, adding 15~60mL of methanol or ethanol, sonicating for 20~40 minutes, filtering, evaporating the filtrate to dryness, and dissolving the residue in 1~3ml of methanol or ethanol to obtain the Notopterygium incisum reference herb solution.

14. The thin-layer chromatography method for simultaneously identifying different medicinal flavors in a pediatric calming and brain-tonifying drug preparation according to claim 6, characterized in that, The preparation of the Cornus officinalis reference herb solution includes the steps of solid-liquid separation, drying the liquid, and dissolving it with an organic solvent after extraction with an organic solvent, wherein the organic solvent is anhydrous ethanol.

15. The thin-layer chromatography method for simultaneously identifying different medicinal flavors in a pediatric calming and brain-tonifying drug preparation according to claim 14, characterized in that, The preparation method of the Cornus officinalis reference medicinal material solution includes taking 0.05~0.3g of Cornus officinalis reference medicinal material, adding 15~60mL of methanol or ethanol and sonicating for 20~40 minutes, filtering, evaporating the filtrate to dryness, and dissolving the residue in 1~3ml of methanol or ethanol to obtain the Cornus officinalis reference medicinal material solution.

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