A method for detecting common components in cortex moutan medicinal materials that can be transferred into liuwei dihuang oral liquid

Abstract

This invention relates to a method for detecting common components of peony bark that can be transferred to Liuwei Dihuang oral liquid. The method comprises two parts: First, using thin-layer chromatography (TLC) to detect in parallel samples 1 (test solution 1), peony bark solution 1, paeonol solution 1, and negative sample solution 1, to obtain spots of at least two peony bark components that can be transferred to Liuwei Dihuang oral liquid; second, using high-performance liquid chromatography (HPLC) to detect sample solution 2, peony bark solution 2, paeonol solution 2, paeoniflorin solution, and negative sample solution 2, to obtain fingerprint chromatograms of common characteristic peaks of at least six peony bark components that can be transferred to Liuwei Dihuang oral liquid. The TLC and HPLC fingerprinting techniques of this invention have advantages such as high efficiency, accuracy, high specificity, high separation, and simple operation, providing a reliable regulatory technology for combating illegal activities related to the manufacture and sale of counterfeit and substandard Liuwei Dihuang oral liquid.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a method for detecting common components that can be transferred from peony bark to Liuwei Dihuang oral liquid and its application. Background Technology

[0002] The classic Chinese medicine formula Liuwei Dihuang Wan (Six-Ingredient Rehmannia Pill) originated from Qian Yi's "Xiao'er Yaozheng Zhijue" (Straightforward Explanation of Pediatric Drug Syndromes) from the Song Dynasty. It is made from six Chinese herbs: Rehmannia glutinosa (processed), Cornus officinalis (processed with wine), Dioscorea opposita (processed), Paeonia suffruticosa (processed), Poria cocos (processed), and Alisma plantago-aquatica (processed). It has the effect of nourishing Yin and tonifying the kidneys. Clinically, it is used to prevent and treat kidney Yin deficiency syndrome (symptoms such as soreness and weakness of the lower back and knees, dizziness and tinnitus, night sweats and seminal emission, bone steaming fever, hot palms and soles, red tongue with little coating, and thready and rapid pulse), as well as to prevent and treat chronic nephritis, diabetes, menopausal syndrome, and other diseases caused by insufficient kidney Yin.

[0003] Liuwei Dihuang (Six-Ingredient Rehmannia Formula) organically combines three tonifying herbs (Rehmannia, Cornus officinalis, and Dioscorea opposita) and three purging herbs (Alisma plantago-aquatica, Paeonia suffruticosa, and Poria cocos), employing both tonifying and purging methods simultaneously. It tonifies without causing stagnation and nourishes without being cloying, restoring the physiological balance of kidney yin, rather than simply providing strong tonification. It embodies the principle of "strengthening the water element to control the fire element." Rehmannia glutinosa (processed): Nourishes the kidneys and replenishes essence, greatly tonifying true yin (the chief herb for tonifying kidney yin). Cornus officinalis (processed): Tonifies the liver and kidneys, astringes essence and consolidates the body (tonifies liver yin). Dioscorea opposita: Tonifies the spleen and kidneys, benefits essence and reduces urination (tonifies spleen yin). Alisma plantago-aquatica: Drains kidney turbidity, preventing the cloying effect of Rehmannia glutinosa (drains kidney turbidity). Paeonia suffruticosa: Clears liver fire, counteracts the warming effect of Cornus officinalis (drains liver fire). Poria cocos: Drains spleen dampness, assists Dioscorea opposita in its function (drains spleen dampness). Rehmannia glutinosa (processed) is the chief herb, directly entering the kidney meridian to replenish true yin; Cornus officinalis and Dioscorea opposita are the assistant herbs, tonifying the yin of the liver, spleen, and kidneys simultaneously, while also consolidating essence; Alisma plantago-aquatica, Paeonia suffruticosa, and Poria cocos are the adjuvant herbs, draining turbidity and promoting diuresis, clearing deficiency heat, and harmon

[0004] Peony bark (root bark of Paeonia suffruticosa Andrews, a plant in the Ranunculaceae family) is used in the Liuwei Dihuang formula to clear liver fire and counteract the warming effect of Cornus officinalis (to purge liver fire). It also has the effects of clearing heat and cooling blood, promoting blood circulation and removing blood stasis, anti-inflammatory, anti-thrombotic, anti-tumor, anti-arrhythmic, hypoglycemic, liver protection, and anti-allergy. Its active ingredients include paeonol, paeoniflorin, norpaeonol and gallic acid, which have anti-inflammatory effects; paeonol, benzoylpaeoniflorin, benzoyl paeoniflorin oxide, etc., which inhibit platelet aggregation; and polysaccharide components with hypoglycemic effects. Peony bark extract has significant inhibitory activity on the growth of microorganisms such as Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Salmonella typhimurium, Shigella dysenteriae, and Microsporum ferrugine.

[0005] The National Pharmacopoeia Commission's national drug standard for Liuwei Dihuang oral liquid includes a content determination section for peony bark, with each mL of peony bark containing paeonol (C9H2O) as the content. 10The dosage of O3 (total) should not be less than 0.60 mg. Some unscrupulous merchants use this as a basis to substitute cheap paeonol for peony bark in the preparation of Liuwei Dihuang oral liquid. Liuwei Dihuang is a classic formula created under the guidance of the holistic view of traditional Chinese medicine theory, based on the syndrome differentiation of kidney yin deficiency. It organically combines three tonifying herbs (Rehmannia, Cornus, and Herba) and three purging herbs (Alisma, Peony, and Poria), employing both tonifying and purging methods. Peony bark, as an "adjuvant" in Liuwei Dihuang oral liquid, plays a role in clearing deficiency heat and purging ministerial fire in the formula. For example, peony bark can be used to counteract the warming nature of Cornus officinalis. Peony bark not only provides the active ingredient paeonol to the Liuwei Dihuang formula, but also provides other active ingredients (such as paeoniflorin, gallic acid, monoterpenoid glycosides, phenols, etc.) and their synergistic effects. Unscrupulous merchants are using paeonol instead of peony bark in the preparation of Liuwei Dihuang oral liquid, which fundamentally alters the overall efficacy of peony bark in the formula. The oral liquid may lack other components from peony bark, potentially weakening its heat-clearing and blood-cooling effects, and failing to effectively balance the warming and tonifying properties of Cornus officinalis. This could affect the overall balance of hot and cold properties and therapeutic efficacy of the formula, even leading to reduced or completely altered efficacy. This practice violates the holistic view of traditional Chinese medicine, resulting in the absence of active ingredients from peony bark other than paeonol in the oral liquid. This seriously affects the quality, efficacy, and safety of the drug in clinical use. Therefore, Liuwei Dihuang oral liquid illegally prepared using paeonol instead of peony bark constitutes a counterfeit drug as defined by the Drug Administration Law and the Drug Registration Management Measures.

[0006] Existing methods for detecting Liuwei Dihuang oral liquid, such as thin-layer chromatography (TLC), suffer from limitations due to improper selection of silica gel plates or TLC materials, reference standards, pretreatment, and developing solvents, resulting in the inability to form effective monitoring spots. High-performance liquid chromatography (HPLC) for detecting Liuwei Dihuang oral liquid also exhibits drawbacks due to unsuitable chromatographic conditions, reference standard selection, and pretreatment. Firstly, it detects fewer common peaks of peony bark components transferable to Liuwei Dihuang oral liquid. Secondly, it detects numerous and mixed peaks of other components in Liuwei Dihuang oral liquid, making it difficult to effectively monitor whether counterfeit drugs, such as those using paeonol instead of peony bark, are produced during the preparation of Liuwei Dihuang oral liquid. Therefore, it is necessary to establish a scientific method for detecting common components of peony bark transferable to Liuwei Dihuang oral liquid to effectively monitor its quality and efficacy, ensuring the safety and effectiveness of medication for patients. Summary of the Invention

[0007] The purpose of this invention is to provide a method for detecting common components of peony bark that can be transferred to Liuwei Dihuang oral liquid. The method comprises two parts: first, using thin-layer chromatography to detect in parallel samples 1 (test solution 1), peony bark solution 1, paeonol solution 1, and negative sample solution 1, to obtain at least two spots containing common peony bark components that can be transferred to Liuwei Dihuang oral liquid; second, using high-performance liquid chromatography to detect sample solution 2, peony bark solution 2, paeonol solution 2, paeoniflorin solution, and negative sample solution 2, to obtain fingerprint chromatograms of at least six common characteristic peaks of peony bark components that can be transferred to Liuwei Dihuang oral liquid.

[0008] In a preferred embodiment of the present invention, the preparation of the test solution 1 includes the following steps: Liuwei Dihuang oral liquid is extracted sequentially with petroleum ether and chloroform, the aqueous phase is collected, extracted with ethyl acetate, the organic phase is collected, evaporated to dryness, and dissolved in methanol to obtain the final product.

[0009] In a preferred embodiment of the present invention, the preparation of the peony bark medicinal solution 1 includes the following steps: adding water to peony bark at a mass-to-volume ratio of 1:20-50, boiling and extracting for 20-50 minutes, cooling to room temperature, filtering, extracting the filtrate with ethyl acetate, combining the organic phase extracts, drying, and dissolving in methanol to obtain the solution.

[0010] In the preferred embodiment of the present invention, in the preparation of the peony bark medicinal solution 1, the mass-to-volume ratio of peony bark to water is 1:30-40.

[0011] In a preferred embodiment of the present invention, the preparation of the paeonol solution 1 includes the following steps: preparing a paeonol solution with a concentration of 0.5-2 mg / mL using methanol, preferably 1-1.5 mg / mL.

[0012] In a preferred embodiment of the present invention, the preparation of the negative test solution 1 includes the following steps: the negative solution is extracted sequentially with petroleum ether and chloroform, the aqueous phase is collected, the solution is extracted with ethyl acetate, the organic phase is collected, the solution is evaporated to dryness, and then dissolved in methanol to obtain the solution.

[0013] In a preferred embodiment of the present invention, the thin-layer chromatography detection includes the following steps: taking sample solution 1, peony bark solution 1, paeonol solution 1, and negative sample solution 1, and developing them on a silica gel thin-layer plate, wherein the developing solvent is cyclohexane: ethyl acetate: formic acid.

[0014] In a preferred embodiment of the present invention, the silica gel thin-layer plate used in the thin-layer chromatography method is GF254.

[0015] In a preferred embodiment of the present invention, the volume ratio of cyclohexane:ethyl acetate:formic acid in the developing solvent is 2.5-3.5:1-2:0.1-0.3, preferably 3:1.5:0.2.

[0016] In the preferred technical solution of the present invention, the thin-layer chromatography detection includes the following steps: aspirate 2 - 5 μL of the test solution 1, 2 - 5 μL of the Cortex Moutan medicinal material solution 1, 1 - 2 μL of the paeonol solution 1, and 2 - 5 μL of the negative test solution 1, place them on a GF254 thin-layer plate, and develop. Among them, the developing agent is made of cyclohexane: ethyl acetate: formic acid in a volume ratio of 3:1.5:0.2.

[0017] In the preferred technical solution of the present invention, when using thin-layer chromatography to detect the common components that can be transferred from Cortex Moutan medicinal materials to Liuwei Dihuang Oral Liquid, it is qualified to obtain at least two component spots that can be transferred from Cortex Moutan medicinal materials to Liuwei Dihuang Oral Liquid. Among them, the Rf value of spot a is 0.6 - 0.9, and the Rf value of spot b is 0.5 - 0.6.

[0018] In the preferred technical solution of the present invention, in the thin-layer chromatography detection, it is qualified that the test sample shows the same component spots at the positions corresponding to the ultraviolet detection (254 nm) chromatograms of Cortex Moutan medicinal materials and paeonol.

[0019] In the preferred technical solution of the present invention, use high-performance liquid chromatography to detect the test solution 2, Cortex Moutan medicinal material solution 2, paeonol solution 2, paeoniflorin solution, and negative test solution 2. It is qualified to detect and obtain the fingerprint图谱 of at least six common characteristic peaks of Cortex Moutan components that can be transferred from Cortex Moutan medicinal materials to Liuwei Dihuang Oral Liquid.

[0020] In the preferred technical solution of the present invention, the method for obtaining the fingerprint图谱 of the common characteristic peaks of Cortex Moutan components that can be transferred from Cortex Moutan medicinal materials to Liuwei Dihuang Oral Liquid by high-performance liquid chromatography detection includes the following steps:

[0021] (1) The chromatographic column uses octadecylsilane-bonded silica gel as the filler; the mobile phase consists of acetonitrile and water, and the gradient elution conditions are:

[0022]

[0023] The detection wavelength is 230 - 254 nm; the flow rate is 0.8 - 1.2 mL / min; the column temperature is 30 - 40 °C. Detect the test solution 2, Cortex Moutan medicinal material solution 2, paeonol solution 2, paeoniflorin solution, and negative test solution 2, and record the common characteristic peaks of Cortex Moutan components that can be transferred from Cortex Moutan medicinal materials to Liuwei Dihuang Oral Liquid and their relative retention times;

[0024] (2) Collect the fingerprint图谱 of multiple batches of Liuwei Dihuang Oral Liquid, and then use the similarity evaluation system to analyze and generate the control图谱 of Liuwei Dihuang Oral Liquid;

[0025] (3) The fingerprint spectrum of the test sample is qualified if the similarity of the six characteristic peaks of the common components of peony bark with the control spectrum is ≥0.9. Among them, peak 1 is paeoniflorin, peak 6 is paeonol, and peaks 2-5 are the common characteristic peaks of peony bark components other than paeonol and paeoniflorin that can be transferred from peony bark to Liuwei Dihuang oral liquid.

[0026] In a preferred embodiment of the present invention, the length of the chromatographic column is 100-300 mm, the diameter of the chromatographic column is 3-5 mm, and the particle size of the packing material in the chromatographic column is 3-5 μm, preferably 250 mm × 4.6 mm, 5 μm.

[0027] In a preferred embodiment of the present invention, the chromatographic column is an Agile ZORBAXSB-C18.

[0028] In a preferred embodiment of the present invention, the preparation of the test solution 2 includes the following steps: adding methanol according to the volume ratio of Liuwei Dihuang oral liquid to methanol of 1:3-5, weighing, ultrasonically extracting for 10-30 min, cooling to room temperature, replenishing the lost amount with methanol, shaking well, filtering through a 0.45 μm microporous membrane, and taking the filtrate.

[0029] In a preferred embodiment of the present invention, the preparation of the paeonol solution 2 includes the following steps: taking paeonol, dissolving it in methanol to prepare paeonol solution 2.

[0030] In a preferred embodiment of the present invention, the preparation of the paeoniflorin solution includes the following steps: taking paeoniflorin and dissolving it in methanol to obtain the solution.

[0031] In a preferred embodiment of the present invention, the preparation of the peony bark medicinal solution 2 includes the following steps: adding methanol at a mass-to-volume ratio of peony bark to methanol of 1:30-50 (m / v), ultrasonically extracting for 10-30 min, filtering, and taking the filtrate to obtain the solution.

[0032] In the preferred embodiment of the present invention, the negative test solution 2 is prepared as follows: methanol is added at a volume ratio of negative solution to methanol of 1:3-5 (v / v), weighed, ultrasonically extracted for 10-30 min, cooled to room temperature, the amount lost is replenished with methanol, shaken well, filtered through a 0.45 μm microporous membrane, and the filtrate is obtained.

[0033] In the preferred embodiment of the present invention, the relative retention times of the six common peaks in the fingerprint spectrum of the common characteristic peaks of the peony bark components that can be transferred to Liuwei Dihuang oral liquid are 0.50, 1.00, 1.25, 1.33, 1.47, and 1.50, respectively.

[0034] In a preferred embodiment of the present invention, the resolution of the six common peaks in the fingerprint spectrum of the common characteristic peaks of the peony bark components that can be transferred to Liuwei Dihuang oral liquid is ≥1.2.

[0035] In a preferred embodiment of the present invention, the similarity between the fingerprint spectrum and the reference spectrum is 0.942-0.998.

[0036] The purpose of this invention is to provide a method for detecting common components of peony bark that can be transferred to Liuwei Dihuang oral liquid using thin-layer chromatography. The method involves parallel detection of test solution 1, peony bark solution 1, paeonol solution 1, and negative test solution 1 using thin-layer chromatography to detect at least two spots of peony bark components that can be transferred to Liuwei Dihuang oral liquid.

[0037] In a preferred embodiment of the present invention, the preparation of the test solution 1 includes the following steps: Liuwei Dihuang oral liquid is extracted sequentially with petroleum ether and chloroform, the aqueous phase is collected, extracted with ethyl acetate, the organic phase is collected, evaporated to dryness, and dissolved in methanol to obtain the final product.

[0038] In a preferred embodiment of the present invention, the preparation of the peony bark medicinal solution 1 includes the following steps: adding water to peony bark at a mass-to-volume ratio of 1:20-50, boiling and extracting for 20-50 minutes, cooling to room temperature, filtering, extracting the filtrate with ethyl acetate, combining the organic phase extracts, drying, and dissolving in methanol to obtain the solution.

[0039] In the preferred embodiment of the present invention, in the preparation of the peony bark medicinal solution 1, the mass-to-volume ratio of peony bark to water is 1:30-40.

[0040] In a preferred embodiment of the present invention, the preparation of the paeonol solution 1 includes the following steps: preparing a paeonol solution with a concentration of 0.5-2 mg / mL using methanol, preferably 1-1.5 mg / mL.

[0041] In a preferred embodiment of the present invention, the preparation of the negative test solution 1 includes the following steps: the negative solution is extracted sequentially with petroleum ether and chloroform, the aqueous phase is collected, the solution is extracted with ethyl acetate, the organic phase is collected, the solution is evaporated to dryness, and then dissolved in methanol to obtain the solution.

[0042] In a preferred embodiment of the present invention, the thin-layer chromatography detection includes the following steps: taking sample solution 1, peony bark solution 1, paeonol solution 1, and negative sample solution 1, and developing them on a silica gel thin-layer plate, wherein the developing solvent is cyclohexane: ethyl acetate: formic acid.

[0043] In a preferred embodiment of the present invention, the silica gel thin-layer plate used in the thin-layer chromatography method is GF254.

[0044] In the preferred technical solution of the present invention, the volume ratio of cyclohexane: ethyl acetate: formic acid in the developing agent is 2.5 - 3.5: 1 - 2: 0.1 - 0.3, preferably 3: 1.5: 0.2.

[0045] In the preferred technical solution of the present invention, the thin layer chromatography detection includes the following steps: Pipette 2 - 5 μL of the test solution 1, 2 - 5 μL of the Cortex Moutan medicinal material solution 1, 1 - 2 μL of the paeonol solution 1, and 2 - 5 μL of the negative test solution 1, place them on a GF254 thin layer plate, and develop. Among them, the developing agent is made of cyclohexane: ethyl acetate: formic acid according to a volume ratio of 3: 1.5: 0.2.

[0046] In the preferred technical solution of the present invention, when using thin layer chromatography to detect the common components that can be transferred from Cortex Moutan medicinal materials to Liuwei Dihuang Oral Liquid, it is qualified to obtain at least two component spots that can be transferred from Cortex Moutan medicinal materials to Liuwei Dihuang Oral Liquid. Among them, the Rf value of spot a is 0.6 - 0.9, and the Rf value of spot b is 0.5 - 0.6.

[0047] In the preferred technical solution of the present invention, in the thin layer chromatography detection, it is qualified that the test sample shows the same component spots at the positions corresponding to the ultraviolet detection (254 nm) chromatograms of Cortex Moutan medicinal materials and paeonol.

[0048] The purpose of the present invention is to provide a method for detecting the common components that can be transferred from Cortex Moutan medicinal materials to Liuwei Dihuang Oral Liquid by high performance liquid chromatography. Use high performance liquid chromatography to detect the test solution 2, Cortex Moutan medicinal material solution 2, paeonol solution 2, paeoniflorin solution, and negative test solution 2, in order to detect and obtain a fingerprint图谱 of at least six common characteristic peaks of Cortex Moutan components that can be transferred from Cortex Moutan medicinal materials to Liuwei Dihuang Oral Liquid. <00001--1>

[0049] In the preferred technical solution of the present invention, the method for obtaining the fingerprint图谱 of the common characteristic peaks of Cortex Moutan components that can be transferred from Cortex Moutan medicinal materials to Liuwei Dihuang Oral Liquid by high performance liquid chromatography detection includes the following steps:

[0050] (1) The chromatographic column uses octadecylsilyl bonded silica gel as the filler; the mobile phase is composed of acetonitrile and water, and the gradient elution conditions are:

[0051] <000--1>

[0052] The detection wavelength is from 230 - 254 nm; the flow rate is 0.8 - 1.2 mL / min; the column temperature is 30 - 40 °C. Detect the test solution 2, Cortex Moutan medicinal material solution 2, paeonol solution 2, paeoniflorin solution, and negative test solution 2, and record the common characteristic peaks of Cortex Moutan components that can be transferred from Cortex Moutan medicinal materials to Liuwei Dihuang Oral Liquid and their relative retention times;

[0053] (2) Collect fingerprint chromatograms of multiple batches of Liuwei Dihuang oral liquid, and then use a similarity evaluation system to analyze and generate a comparison chromatogram of Liuwei Dihuang oral liquid;

[0054] (3) The fingerprint spectrum of the test sample is qualified if the similarity of the six characteristic peaks of the common components of peony bark with the control spectrum is ≥0.9. Among them, peak 1 is paeoniflorin, peak 6 is paeonol, and peaks 2-5 are the common characteristic peaks of peony bark components other than paeonol and paeoniflorin that can be transferred from peony bark to Liuwei Dihuang oral liquid.

[0055] In a preferred embodiment of the present invention, the length of the chromatographic column is 100-300 mm, the diameter of the chromatographic column is 3-5 mm, and the particle size of the packing material in the chromatographic column is 3-5 μm, preferably 250 mm × 4.6 mm, 5 μm.

[0056] In a preferred embodiment of the present invention, the chromatographic column is an Agile ZORBAXSB-C18.

[0057] In a preferred embodiment of the present invention, the preparation of the test solution 2 includes the following steps: adding methanol according to the volume ratio of Liuwei Dihuang oral liquid to methanol of 1:3-5, weighing, ultrasonically extracting for 10-30 min, cooling to room temperature, replenishing the lost amount with methanol, shaking well, filtering through a 0.45 μm microporous membrane, and taking the filtrate.

[0058] In a preferred embodiment of the present invention, the preparation of the paeonol solution 2 includes the following steps: taking paeonol, dissolving it in methanol to prepare paeonol solution 2.

[0059] In a preferred embodiment of the present invention, the preparation of the paeoniflorin solution includes the following steps: taking paeoniflorin and dissolving it in methanol to obtain the solution.

[0060] In a preferred embodiment of the present invention, the preparation of the peony bark medicinal solution 2 includes the following steps: adding methanol at a mass-to-volume ratio of peony bark to methanol of 1:30-50 (m / v), ultrasonically extracting for 10-30 min, filtering, and taking the filtrate to obtain the solution.

[0061] In the preferred embodiment of the present invention, the negative test solution 2 is prepared as follows: methanol is added at a volume ratio of negative solution to methanol of 1:3-5 (v / v), weighed, ultrasonically extracted for 10-30 min, cooled to room temperature, the amount lost is replenished with methanol, shaken well, filtered through a 0.45 μm microporous membrane, and the filtrate is obtained.

[0062] In the preferred embodiment of the present invention, the relative retention times of the six common peaks in the fingerprint spectrum of the common characteristic peaks of the peony bark components that can be transferred to Liuwei Dihuang oral liquid are 0.50, 1.00, 1.25, 1.33, 1.47, and 1.50, respectively.

[0063] In a preferred embodiment of the present invention, the resolution of the six common peaks in the fingerprint spectrum of the common characteristic peaks of the peony bark components that can be transferred to Liuwei Dihuang oral liquid is ≥1.2.

[0064] In a preferred embodiment of the present invention, the similarity between the fingerprint spectrum and the reference spectrum is 0.942-0.998.

[0065] Another object of the present invention is to provide a fingerprint spectrum of the common characteristic peaks of peony bark components that can be transferred to Liuwei Dihuang oral liquid for monitoring the use of paeonol or any one or a combination of peony bark in the preparation of Liuwei Dihuang oral liquid.

[0066] In a preferred embodiment of the present invention, high performance liquid chromatography is used to detect the test solution 2, peony bark medicinal material solution 2, paeonol solution 2, paeoniflorin solution and negative test solution 2, so as to obtain the fingerprint spectrum of at least six peony bark components that can be transferred to Liuwei Dihuang oral liquid.

[0067] In a preferred embodiment of the present invention, the method for detecting the fingerprint spectrum of common characteristic peaks of peony bark components that can be transferred to Liuwei Dihuang oral liquid by high performance liquid chromatography includes the following steps:

[0068] (1) The chromatographic column was packed with octadecylsilane-bonded silica gel; the mobile phase consisted of acetonitrile and water, and the gradient elution conditions were as follows:

[0069]

[0070] The detection wavelength is 230-254 nm; the flow rate is 0.8-1.2 mL / min; the column temperature is 30-40℃. The test solution 2, peony bark solution 2, paeonol solution 2, paeoniflorin solution, and negative test solution 2 are detected. The common characteristic peaks of peony bark components that can be transferred from peony bark to Liuwei Dihuang oral liquid and their relative retention times are recorded.

[0071] (2) Collect fingerprints of common characteristic peaks of peony bark components that can be transferred to Liuwei Dihuang oral liquid from multiple batches of peony bark medicinal materials, and then use a similarity evaluation system to analyze and generate a comparison spectrum of Liuwei Dihuang oral liquid.

[0072] (3) The fingerprint spectrum of the test sample is qualified if the similarity of the six characteristic peaks of the common components of peony bark with the control spectrum is ≥0.9. Among them, peak 1 is paeoniflorin, peak 6 is paeonol, and peaks 2-5 are the common characteristic peaks of peony bark components other than paeonol and paeoniflorin that can be transferred from peony bark to Liuwei Dihuang oral liquid.

[0073] In a preferred embodiment of the present invention, the length of the chromatographic column is 100-300 mm, the diameter of the chromatographic column is 3-5 mm, and the particle size of the packing material in the chromatographic column is 3-5 μm, preferably 250 mm × 4.6 mm, 5 μm.

[0074] In a preferred embodiment of the present invention, the chromatographic column is an Agile ZORBAXSB-C18.

[0075] In a preferred embodiment of the present invention, the preparation of the test solution 2 includes the following steps: adding methanol according to the volume ratio of Liuwei Dihuang oral liquid to methanol of 1:3-5, weighing, ultrasonically extracting for 10-30 min, cooling to room temperature, replenishing the lost amount with methanol, shaking well, filtering through a 0.45 μm microporous membrane, and taking the filtrate.

[0076] In a preferred embodiment of the present invention, the preparation of the paeonol solution 2 includes the following steps: taking paeonol, dissolving it in methanol to prepare paeonol solution 2.

[0077] In a preferred embodiment of the present invention, the preparation of the paeoniflorin solution includes the following steps: taking paeoniflorin and dissolving it in methanol to obtain the solution.

[0078] In a preferred embodiment of the present invention, the preparation of the peony bark medicinal solution 2 includes the following steps: adding methanol at a mass-to-volume ratio of peony bark to methanol of 1:30-50 (m / v), ultrasonically extracting for 10-30 min, filtering, and taking the filtrate to obtain the solution.

[0079] In the preferred embodiment of the present invention, the negative test solution 2 is prepared as follows: methanol is added at a volume ratio of negative solution to methanol of 1:3-5 (v / v), weighed, ultrasonically extracted for 10-30 min, cooled to room temperature, the amount lost is replenished with methanol, shaken well, filtered through a 0.45 μm microporous membrane, and the filtrate is obtained.

[0080] In the preferred embodiment of the present invention, the relative retention times of the six common peaks in the fingerprint spectrum of the common components of peony bark that can be transferred to Liuwei Dihuang oral liquid are 0.50, 1.00, 1.25, 1.33, 1.47 and 1.50, respectively.

[0081] In a preferred embodiment of the present invention, the resolution of the six common peaks in the fingerprint spectrum of the common characteristic peaks of the peony bark components that can be transferred to Liuwei Dihuang oral liquid is ≥1.2.

[0082] In a preferred embodiment of the present invention, the similarity between the fingerprint spectrum and the reference spectrum is 0.942-0.998.

[0083] To clearly define the scope of protection of this invention, the following terms are defined as follows:

[0084] 1. The common components of peony bark that can be transferred to Liuwei Dihuang oral liquid in this invention refer to the detectable common components of peony bark contained in Liuwei Dihuang oral liquid prepared according to the composition and preparation method of Liuwei Dihuang oral liquid.

[0085] 2. In this invention, the test solution, peony bark medicinal material solution, paeonol solution, and negative test solution used for thin-layer chromatography detection are named Test Solution 1, Peony Bark Medicinal Material Solution 1, Paeonol Solution 1, and Negative Test Solution 1, respectively, in order to detect at least two peony bark component spots that can be transferred to Liuwei Dihuang Oral Liquid.

[0086] 3. In this invention, the test solution, peony bark medicinal material solution, paeonol solution, and negative test solution used for high performance liquid chromatography detection are named test solution 2, peony bark medicinal material solution 2, paeonol solution 2, and negative test solution 2, in order to detect and obtain fingerprint chromatograms of at least six peony bark components that can be transferred to Liuwei Dihuang oral liquid.

[0087] 4. The negative solution of the present invention refers to the oral liquid prepared according to the formula and preparation method of Liuwei Dihuang oral liquid after removing the peony bark from the formula of Liuwei Dihuang oral liquid.

[0088] 5. The peony bark medicinal solution described in this invention is prepared using commercially available peony bark medicinal material.

[0089] 6. The preparation of the peony bark medicinal solution 1 of the present invention includes the following steps: Peony bark: water at a mass-volume ratio of 1:30, add water to the required amount of peony bark, boil and extract for 0.5h-1.0h, cool to room temperature, filter, add ethyl acetate to the filtrate at a mass-volume ratio of peony bark: ethyl acetate at 1:10, shake and extract twice, combine the two ethyl acetate extracts, evaporate the solvent, add 1mL of methanol to dissolve, and obtain the solution.

[0090] 7. The preparation of the peony bark medicinal solution 2 of the present invention includes the following steps: Peony bark and methanol are added at a mass-volume ratio of 1:40, ultrasonic treatment (power 250w, frequency 50kHz) for 0.5h-1.0h, then filtered, and the filtrate is obtained.

[0091] Unless otherwise stated, when this invention relates to percentages between liquids, the percentage is volume / volume percentage; when this invention relates to percentages between liquids and solids, the percentage is volume / weight percentage; when this invention relates to percentages between solids and liquids, the percentage is weight / volume percentage; the remainder is weight / weight percentage.

[0092] Compared with the prior art, the present invention has the following beneficial technical effects:

[0093] 1. This invention is the first to employ a systematic solvent extraction method to purify and test samples, and scientifically optimizes the test samples, their preparation, and testing conditions (such as silica gel plates and developing solvents). It also establishes for the first time a thin-layer chromatography (TLC) and HPLC fingerprinting method for detecting common characteristic components of peony bark that can be transferred to Liuwei Dihuang oral liquid. Firstly, paeonol and peony bark are used as dual controls for TLC detection, obtaining spots of at least two components (such as spot a and spot b) that can be transferred from peony bark to Liuwei Dihuang oral liquid. Secondly, a fingerprint spectrum of at least six common characteristic peaks of peony bark components that can be transferred to Liuwei Dihuang oral liquid is created using HPLC, enabling rapid and accurate monitoring of whether peony bark or paeonol is used in the preparation of Liuwei Dihuang oral liquid.

[0094] 2. The thin-layer chromatography and HPLC fingerprinting method for detecting common components of peony bark that can be transferred to Liuwei Dihuang oral liquid have the advantages of accurate monitoring, good specificity, high separation, and simple operation. It can simplify the identification process, save monitoring costs, and can be used for quality testing in Liuwei Dihuang oral liquid to ensure the effectiveness of the treatment. It provides an efficient, accurate, reliable and powerful regulatory technology for severely cracking down on illegal activities of manufacturing and selling counterfeit and substandard Liuwei Dihuang oral liquid. Attached Figure Description

[0095] Figure 1 Using paeonol and peony bark as reference standards, and by examining specificity, common component spots that can be transferred from peony bark to Liuwei Dihuang oral liquid were identified. The thin-layer chromatography method of this invention was used to detect the thin-layer chromatographic results of at least two thin-layer spots that can be transferred from peony bark to Liuwei Dihuang oral liquid.

[0096] Figure 2 Using paeonol and paeoniflorin as reference standards, the HPLC fingerprint of the effective components in peony bark that can be transferred to Liuwei Dihuang oral liquid was detected by the high performance liquid chromatography method of the present invention. Peak 1 is paeoniflorin and peak 6 is paeonol.

[0097] Figure 3 Using peony bark as a reference standard, and through specificity analysis, the common components that can be transferred from peony bark to Liuwei Dihuang oral liquid were identified. The fingerprint spectrum of the six common characteristic peaks of peony bark components that can be transferred to Liuwei Dihuang oral liquid was obtained by high performance liquid chromatography of the present invention. Among them, peak 1 is paeoniflorin, peak 6 is paeonol, and peaks 2-5 are other common components that can be transferred besides paeoniflorin and paeonol.

[0098] Figure 4 shows the fingerprint matching diagram of the common characteristic peaks of the six peony bark components that can be transferred from peony bark to Liuwei Dihuang oral liquid obtained by the high performance liquid chromatography method of the present invention in 10 batches of Liuwei Dihuang oral liquid (see figure). Figure 4a ) and reference atlas (see Figure 4b Among them, peak 1 is paeoniflorin, peak 6 is paeonol, and peaks 2-5 are other common components that can be transferred besides paeoniflorin and paeonol.

[0099] Figure 5 Using the reference chromatograms generated from 10 batches of Liuwei Dihuang oral liquid as a reference chromatogram, the high performance liquid chromatography method of this invention was used to verify the fingerprint chromatograms and batch verification results of the six common characteristic peaks of peony bark components that can be transferred from 5 batches of Liuwei Dihuang oral liquid. Among them, peak 1 is paeoniflorin, peak 6 is paeonol, and peaks 2-5 are other common components that can be transferred besides paeoniflorin and paeonol. Detailed Implementation

[0100] The technical solution of the present invention will be further described in detail below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0101] 1. The 15 batches of Liuwei Dihuang oral liquid used for testing in this invention were numbered S1-S15 in sequence.

[0102] 2. Preparation of the negative solution of this invention: Take 144g of Rehmannia glutinosa (processed), 72g of Cornus officinalis (processed), 72g of Dioscorea opposita, 54g of Poria cocos, and 54g of Alisma plantago-aquatica:

[0103] (1) Mix the required amount of processed Cornus officinalis with 95% ethanol at a mass-volume ratio of 1:4, reflux and extract 3 times, each time for 1 hour. Concentrate the collected and combined ethanol extracts until there is no alcohol taste, filter, and obtain Cornus officinalis filtrate, Cornus officinalis filter matter and medicinal residue.

[0104] (2) Add the Cornus officinalis residue collected in step (1) and the required amount of the other four herbs (Rehmannia glutinosa, Dioscorea opposita, Poria cocos, Alisma plantago-aquatica) and water at a mass-volume ratio of 1:8 (m / v), decoct three times. The first decoction is for 2 hours, and the second and third decoctions are for 1 hour each. Filter, collect and combine the three decoction filtrates, concentrate them to a raw herb content of 1:1 (W / V), add ethanol to a content of 65% alcohol, mix evenly with the Cornus officinalis filtrate obtained in step (1), refrigerate, filter, concentrate the filtrate until there is no alcohol taste, add water to adjust to a volume ratio of 1:1, refrigerate, filter, and collect the filtrate;

[0105] (3) Dissolve 0.2g of sucralose in 10mL of water and pour it into the filtrate in step (2); dissolve 3.5g of sodium alginate in 100mL of hot water to obtain sodium alginate solution; dissolve the cornus officinalis filter from step (1) and 3g of sodium benzoate in 5mL of 95% ethanol, add the above sodium alginate solution and the above filtrate with added sucralose, stir well, boil for 0.5h, adjust the pH to 4-6, filter, add distilled water to the filtrate to make up to 1000mL, stir well, and the product is obtained.

[0106] 3. Preparation of thin-layer chromatography detection solution

[0107] Preparation of test solution 1: Take 10 mL of Liuwei Dihuang oral liquid and extract it twice with petroleum ether and chloroform, 10 mL each time. Discard the petroleum ether layer and chloroform layer. Then add ethyl acetate and shake to extract twice, 10 mL each time. Combine the two ethyl acetate extraction layers, evaporate the solvent, and dissolve in 1 mL of methanol to obtain the test solution.

[0108] Preparation of negative test solution 1: Take 10 mL of negative solution and extract it twice with petroleum ether and chloroform, 10 mL each time. Discard the petroleum ether layer and chloroform layer. Then add ethyl acetate and shake to extract twice, 10 mL each time. Combine the two ethyl acetate extraction layers, evaporate the solvent, and dissolve in 1 mL of methanol to obtain the solution.

[0109] Preparation of peony bark medicinal solution 1: 1g of peony bark was boiled in 30mL of water for 30min, cooled to room temperature, filtered, and the filtrate was extracted twice with 10mL of ethyl acetate each time. The two ethyl acetate extracts were combined, the solvent was evaporated, and the solution was dissolved in 1mL of methanol to obtain the solution.

[0110] Preparation of Paeonol Solution 1: Paeonol was prepared into a 1 mg / mL solution using methanol.

[0111] 4. Preparation of solutions for high performance liquid chromatography (HPLC) detection

[0112] Preparation of test solution 2: Accurately transfer 5 mL of Liuwei Dihuang oral liquid, add 20 mL of methanol, weigh, sonicate for 20 min, cool to room temperature, replenish the lost amount with methanol, shake well, filter through a 0.45 μm microporous membrane, and take the filtrate.

[0113] Preparation of negative test solution 2: Take 5 mL of negative solution, add 20 mL of methanol, weigh, extract by sonication for 20 min, cool to room temperature, replenish the lost amount with methanol, shake well, filter through a 0.45 μm microporous membrane, and take the filtrate.

[0114] Preparation of peony bark medicinal solution 2: 0.5g of peony bark was treated with 20mL of methanol by ultrasonication (power 250w, frequency 50kHz) for 20min, filtered, and the filtrate was collected.

[0115] Preparation of Paeonol Solution 2: Paeonol was dissolved in methanol to prepare Paeonol solution.

[0116] Preparation of paeoniflorin solution: Paeoniflorin was dissolved in methanol to prepare paeoniflorin solution.

[0117] Example 1 This invention relates to a method for detecting common components of peony bark that can be transferred to Liuwei Dihuang oral liquid using thin-layer chromatography.

[0118] A method for detecting common components of peony bark that can be transferred to Liuwei Dihuang oral liquid using thin-layer chromatography includes the following steps:

[0119] 1. In accordance with the relevant provisions of thin-layer chromatography (General Rule 0502 of the Chinese Pharmacopoeia (2025 Edition), sample test samples S1-S3 and prepare test solution 1 for each sample.

[0120] 2. Take 2 μL of each of the following test solutions (S1-S3), 2 μL of peony bark solution, 1 μL of paeonol solution, and 2 μL of negative test solution, and spot them separately onto a silica gel GF254 thin-layer plate. The developing solvent is prepared by cyclohexane:ethyl acetate:formic acid in a volume ratio of 3:1.5:0.2. Develop, remove, air dry, and examine under ultraviolet light (254 nm). See below for results. Figure 1 . Figure 1 From left to right, samples 1-6 are, respectively, paeonol solution 1, peony bark medicinal material solution 1, test solution 1 of S1, test solution 1 of S2, test solution 1 of S3, and negative test solution 1.

[0121] This invention uses paeonol and peony bark as reference standards, and determines the transmissible thin-layer spots by examining their specificity. Thin-layer chromatography was used to detect at least two types of thin-layer spots in the obtained peony bark solution 1 that can be transmissible to Liuwei Dihuang oral liquid. Figure 1 The results showed that the thin-layer chromatography of the S1-S3 test solutions showed the same fluorescent spots at the corresponding positions as those of the peony bark solution 1 and paeonol solution 1 in the thin-layer chromatography, and the spots were clear and well separated. Spot a was paeonol, and spot b was other components of peony bark that could be transferred to Liuwei Dihuang oral liquid besides paeonol.

[0122] Example 2 This invention employs HPLC detection to construct an HPLC fingerprint of the effective components of peony bark that can be transferred to Liuwei Dihuang oral liquid.

[0123] Accurately pipette 10 μL each of paeonol solution 2, paeoniflorin solution, and test solution 2, and inject them into a high-performance liquid chromatograph (Agilent Technologies, Inc., Model 1260) for high-performance separation and detection. The chromatographic column was an Agilent ZORBAX SB-C18 (250 mm × 4.6 mm, 5 μm) with octadecylsilane-bonded silica gel as the packing material; acetonitrile was used as mobile phase A, and water was used as mobile phase B. The gradient elution conditions are shown in Table 1.

[0124] Table 1

[0125]

[0126] The detection wavelength was 254 nm, the flow rate was 1 mL / min, and the column temperature was 40 °C. The detection results are shown below. Figure 2 .

[0127] Figure 2 The results showed that, using paeonol and paeoniflorin as reference standards, the HPLC fingerprint of the effective components of peony bark that can be transferred to Liuwei Dihuang oral liquid was obtained by high performance liquid chromatography. Among them, peak 1 was paeoniflorin and peak 6 was paeonol.

[0128] Example 3 This invention employs HPLC to investigate the specificity of HPLC fingerprints of common characteristic peaks of peony bark components that can be transferred to Liuwei Dihuang oral liquid.

[0129] Referring to the high-performance liquid chromatography (HPLC) method in Example 2, the negative test solution 2, the peony bark medicinal material solution 2, and the test solution 2 were precisely pipetted and injected into the HPLC instrument (Agilent Technologies, Inc., Model 1260) for high-performance separation and detection. The chromatographic column was an Agilent ZORBAX SB-C18 (250 mm × 4.6 mm, 5 μm) with octadecylsilane-bonded silica gel as the packing material; acetonitrile was used as mobile phase A, and water was used as mobile phase B. The gradient elution conditions are shown in Table 1. The detection wavelength was 254 nm, the flow rate was 1 mL / min, and the column temperature was 40 °C.

[0130] Table 1

[0131]

[0132] The common characteristic peaks and their relative retention times of peony bark components that can be transferred from peony bark to Liuwei Dihuang oral liquid were recorded. The results are shown in […]. Figure 3 .

[0133] Figure 3The results showed that, using peony bark solution 2 as a control, the specificity of the transmissible components was examined through negative sample solution 2, thus identifying the transmissible components. The fingerprint spectrum of the six peony bark components transmissible to Liuwei Dihuang oral liquid, obtained by high-performance liquid chromatography of this invention, was obtained. Peak 1 is paeoniflorin, peak 6 is paeonol, and peaks 2-5 are other transmissible common components besides paeoniflorin and paeonol.

[0134] Example 4 This invention employs an HPLC fingerprint analysis of common characteristic peaks of peony bark components that can be transferred to Liuwei Dihuang oral liquid.

[0135] 1. Precision test: Take 2g of the test solution and inject it 6 times consecutively, recording the retention time and peak area. The relative peak area and relative retention time of the six common peaks are all less than 5%, indicating that the instrument has good precision;

[0136] 2. Repeatability test: Six parallel test solutions were prepared using the same batch of test solution 2, and each solution was injected. The retention time and peak area were recorded. The results showed that the relative peak area and relative retention time of each common peak were less than 5%, indicating good repeatability of the method.

[0137] 3. Stability test: Take 2 samples of the test solution and inject them at 0h, 2h, 4h, 12h, 24h, and 48h, respectively, and record the retention time and peak area. The relative peak area and relative retention time of the six common peaks are all less than 5%, indicating that the test solution has good stability after 48h.

[0138] Example 5 This invention uses HPLC to obtain the HPLC fingerprint of common characteristic peaks of peony bark components that can be transferred to Liuwei Dihuang oral liquid.

[0139] This invention uses HPLC detection to obtain the HPLC fingerprint of common characteristic peaks of peony bark components that can be transferred to Liuwei Dihuang oral liquid, including the following steps:

[0140] 1. The 15 batches of Liuwei Dihuang oral liquid were numbered S1-S15 respectively. After preparing their test solutions 2, they were injected into the samples respectively.

[0141] 2. Accurately pipette 10 μL each of paeonol solution 2, paeoniflorin solution, peony bark medicinal material solution 2, and test sample solution 2, and obtain the HPLC fingerprint of the common characteristic peaks of peony bark components that can be transferred from peony bark medicinal material to peony bark components in 15 batches of Liuwei Dihuang oral liquid.

[0142] 3. The fingerprint spectra of S1-S10 were analyzed using the "Similarity Evaluation System for Fingerprint Spectra of Traditional Chinese Medicine (Version 2012A)". Using the S1 sample as the reference spectra, with a time window width of 0.5 min, multi-point correction and Mark peak matching were performed on the chromatographic peaks to generate fingerprint spectra matching diagrams for Liuwei Dihuang oral liquid (S1-S10) (see Figure 4(a), where R is the control chromatogram) and control chromatogram (see Figure 4(b)). The similarity was 0.942-0.998. The similarity of the fingerprint spectra of Liuwei Dihuang oral liquid (S11-S15) with the control chromatogram was ≥0.9. The results are shown in Table 2 and... Figure 5 (where R is the reference spectrum).

[0143] Table 2. Similarity Evaluation Results of 15 Batches of Liuwei Dihuang Oral Liquid

[0144]

[0145]

[0146] Figure 4 shows the fingerprint matching diagrams of at least six common characteristic peaks of peony bark components that can be transferred from peony bark to Liuwei Dihuang oral liquid obtained by the high performance liquid chromatography method of the present invention in 10 batches of Liuwei Dihuang oral liquid (see Figure 4). Figure 4a ) and comparison atlas (see Figure 4b Among them, peak 1 is paeoniflorin, peak 6 is paeonol, and peaks 2-5 are other common components that can be transferred besides paeoniflorin and paeonol.

[0147] Figure 5 The results showed that the high performance liquid chromatography method of the present invention was used to detect the fingerprint spectrum of at least six common characteristic peaks of peony bark components that can be transferred from peony bark to Liuwei Dihuang oral liquid in 10 batches of Liuwei Dihuang oral liquid, and the verification results of 5 batches. Among them, peak 1 is paeoniflorin, peak 6 is paeonol, and peaks 2-5 are other common components that can be transferred besides paeoniflorin and paeonol.

[0148] The HPLC fingerprint spectrum of the peony bark components that can be transferred to Liuwei Dihuang oral liquid, obtained by HPLC detection in this invention, should show at least six common peaks. The relative retention times of the six common peaks are 0.50, 1.00, 1.25, 1.33, 1.47, and 1.50, respectively. Among them, peak 1 is paeoniflorin and peak 6 is paeonol. If the similarity between the fingerprint spectrum of the test sample and the control fingerprint spectrum is ≥0.9, it can be judged as qualified.

[0149] The above description of specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention, and as long as they do not depart from the spirit of the present invention, they should all fall within the scope of protection of the claims of the present invention.

Claims

1. A method for detecting common components of peony bark that can be transferred to Liuwei Dihuang oral liquid, comprising: firstly, using thin-layer chromatography to detect in parallel test solution 1, peony bark solution 1, paeonol solution 1, and negative test solution 1 to obtain at least two spots of peony bark components that can be transferred to Liuwei Dihuang oral liquid; secondly, using high-performance liquid chromatography to detect test solution 2, peony bark solution 2, paeonol solution 2, paeoniflorin solution, and negative test solution 2 to obtain fingerprint chromatograms of common characteristic peaks of at least six peony bark components that can be transferred to Liuwei Dihuang oral liquid.

2. The method according to claim 1, wherein the thin-layer chromatography detection comprises the following steps: taking test solution 1, peony bark solution 1, paeonol solution 1 and negative test solution 1, and developing them on a silica gel thin-layer plate, wherein the developing solvent is cyclohexane: ethyl acetate: formic acid.

3. The method according to any one of claims 1-2, wherein the volume ratio of cyclohexane:ethyl acetate:formic acid in the developing solvent is 2.5-3.5:1-2:0.1-0.3, preferably 3:1.5:0.

2.

4. For the method according to any one of claims 1-3, thin layer chromatography is used to detect the common components that the Cortex Moutan medicinal material can transfer into the Liuwei Dihuang Oral Liquid. It is qualified if at least two component spots that the Cortex Moutan medicinal material can transfer into the Liuwei Dihuang Oral Liquid are obtained through the detection. Among them, The Rf value of spot a is 0.6-0.9, and the Rf value of spot b is 0.5-0.

6.

5. The method according to any one of claims 1-4, wherein the method for detecting the fingerprint spectrum of common characteristic peaks of peony bark components that can be transferred to Liuwei Dihuang oral liquid by high performance liquid chromatography comprises the following steps: (1) The chromatographic column was packed with octadecylsilane-bonded silica gel; the mobile phase consisted of acetonitrile and water, and the gradient elution conditions were as follows: The detection wavelength is 230-254 nm, the flow rate is 0.8-1.2 mL / min, the column temperature is 30-40 ℃, and the test solution 2, peony bark solution 2, paeonol solution 2, paeoniflorin solution, and negative test solution 2 are detected. The common characteristic peaks of peony bark components that can be transferred from peony bark to Liuwei Dihuang oral liquid and their relative retention times are recorded. (2) Collect fingerprints of common characteristic peaks of peony bark components that can be transferred to Liuwei Dihuang oral liquid from multiple batches of peony bark medicinal materials, and then use a similarity evaluation system to analyze and generate a comparison spectrum of Liuwei Dihuang oral liquid. (3) The similarity between the six characteristic peaks of the common components of the test sample fingerprint and the control fingerprint should be ≥ 0.9 for passing the test. Among them, Peak 1 is paeoniflorin, peak 6 is paeonol, and peaks 2-5 are characteristic peaks of the paeonol components (excluding paeonol and paeoniflorin) that can be transferred from paeonol to Liuwei Dihuang oral liquid.

6. A method for detecting common components of peony bark that can be transferred to Liuwei Dihuang oral liquid using thin-layer chromatography, wherein the method employs thin-layer chromatography to detect in parallel samples 1, peony bark solution 1, paeonol solution 1, and negative sample solution 1, so as to obtain at least two spots of peony bark components that can be transferred to Liuwei Dihuang oral liquid.

7. The method according to claim 6, wherein the thin-layer chromatography detection comprises the following steps: taking test solution 1, peony bark solution 1, paeonol solution 1 and negative test solution 1, and developing them on a silica gel thin-layer plate, wherein the developing solvent is cyclohexane: ethyl acetate: formic acid.

8. A method for detecting the fingerprint chromatogram of common characteristic peaks of peony bark components that can be transferred to Liuwei Dihuang oral liquid using high performance liquid chromatography, wherein the method employs high performance liquid chromatography to detect test solution 2, peony bark solution 2, paeonol solution 2, paeoniflorin solution and negative test solution 2, so as to obtain the fingerprint chromatogram of common characteristic peaks of at least six peony bark components that can be transferred to Liuwei Dihuang oral liquid.

9. The method of claim 8, wherein the method for detecting the fingerprint spectrum of common characteristic peaks of peony bark components that can be transferred to Liuwei Dihuang oral liquid by high performance liquid chromatography comprises the following steps: (1) The chromatographic column was packed with octadecylsilane-bonded silica gel; the mobile phase consisted of acetonitrile and water, and the gradient elution conditions were as follows: The detection wavelength is 230-254 nm; the flow rate is 0.8-1.2 mL / min; the column temperature is 30-40℃. The test solution 2, peony bark solution 2, paeonol solution 2, paeoniflorin solution, and negative test solution 2 are detected. The common characteristic peaks of peony bark components that can be transferred from peony bark to Liuwei Dihuang oral liquid and their relative retention times are collected. (2) Collect fingerprint spectrum of common characteristic peaks of peony bark component in multiple batches of Liuwei Dihuang oral liquid, and then use similarity evaluation system to analyze and generate Liuwei Dihuang oral liquid control spectrum; (3) The similarity of the 6 characteristic peaks of the common components of the test sample fingerprint to the control fingerprint is ≥ 0.9 as qualified. Among them, Peak 1 is paeoniflorin, peak 6 is paeonol, and peaks 2-5 are characteristic peaks of the paeonol components (excluding paeonol and paeoniflorin) that can be transferred from paeonol to Liuwei Dihuang oral liquid.

10. The fingerprint spectrum of the common characteristic peaks of the peony bark medicinal material as described in any one of claims 8-9 can be used to monitor the use of paeonol or any one or a combination of peony bark in the preparation of Liuwei Dihuang oral liquid.

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