A detection method of flavored sanjiaonang compound

Abstract

The application belongs to the field of traditional Chinese medicine detection, and discloses a detection method of a flavored Sanhuang compound, which comprises the following steps: determining the contents of emodin and chrysophanol in the flavored Sanhuang compound, and identifying any one or several of rhubarb, Huangqi and Huangbai in the flavored Sanhuang compound by thin layer chromatography. The application shows good stability and reliability through the thin layer chromatography identification of three main medicines in the flavored Sanhuang compound and the content determination of emodin and chrysophanol, and can be used for quality detection of the flavored Sanhuang compound.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine detection technology, and in particular relates to a detection method for a modified Sanhuang compound. Background Technology

[0002] The modified Sanhuang compound formula is an improvement on the classic Sanhuang formula, specifically composed of five herbs: rhubarb, scutellaria baicalensis, phellodendron chinense, salvia miltiorrhiza, and peppermint. It is used to treat facial inflammatory skin diseases and has shown definite clinical efficacy. Currently, the clinical treatment of facial inflammatory skin diseases mainly involves red and blue light therapy, antihistamines, and glucocorticoids. However, long-term topical use of glucocorticoids may cause adverse reactions such as skin atrophy and pigmentation. Calcineurin inhibitors, as alternatives to glucocorticoids, may also cause local skin reactions such as burning, itching, chickenpox, and vesicular eczema, and their repair of the skin barrier function is not significant, leading to a high relapse rate after discontinuation. The modified Sanhuang decoction is a pure traditional Chinese medicine preparation that overcomes some of the shortcomings of chemical drugs.

[0003] Although existing reports contain qualitative and quantitative detection methods for Sanhuang formula, there is a lack of quality testing studies specifically for modified Sanhuang compound. Furthermore, the modified Sanhuang compound contains peppermint volatile oil and other trace amounts of fat-soluble components (such as tanshinone I, tanshinone I, and tanshinone aldehyde in tanshinone), making it difficult to evaporate the sample during preparation and potentially affecting the detection results. Therefore, it is necessary to establish a suitable sample pretreatment method. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a detection method for the modified Sanhuang compound. Through thin-layer chromatography identification of the three main medicinal ingredients and determination of the contents of rhein and chrysophanol, the method has been verified in multiple batches and shows good stability and reliability. It can be used for quality detection of the modified Sanhuang compound.

[0005] The first objective of this invention is to provide a modified Sanhuang compound formula and its preparation method.

[0006] To achieve the first objective, the present invention adopts the following technical solution: The prescription of the modified Sanhuang compound formula includes, by weight parts: 1-100 parts of rhubarb, 1-100 parts of scutellaria baicalensis, 1-100 parts of phellodendron chinense, 1-100 parts of salvia miltiorrhiza, and 1-100 parts of peppermint.

[0007] Preferably, the prescription of the modified Sanhuang compound includes, by weight parts: 10-50 parts of rhubarb, 10-50 parts of scutellaria baicalensis, 10-50 parts of phellodendron chinense, 10-50 parts of salvia miltiorrhiza, and 10-50 parts of peppermint.

[0008] More preferably, the prescription of the modified Sanhuang compound includes, by weight parts: 10 parts rhubarb, 10 parts scutellaria baicalensis, 10 parts phellodendron chinense, 10 parts salvia miltiorrhiza, and 10 parts peppermint.

[0009] The preparation method of the sample solution of the modified Sanhuang compound includes: steam distillation to extract peppermint and collect volatile oil, the remaining residue is combined with other medicinal materials for water extraction and concentration, and the resulting concentrate is mixed with volatile oil to obtain the final product.

[0010] Preferably, the steam distillation extraction of peppermint includes: soaking the peppermint in 5 to 20 times its volume of water for 0.5 to 4.0 hours, and then steam distilling for 1 to 8 hours.

[0011] Preferably, the water extraction includes: extracting with 5 to 30 times the volume of water 1 to 3 times, with each extraction lasting 0.5 to 4.0 hours.

[0012] Preferably, the concentration includes: atmospheric pressure concentration or vacuum concentration, to a relative density of 1.05~1.10 (60°C).

[0013] The second objective of this invention is to provide a detection method for the modified Sanhuang compound, which uses thin-layer chromatography to identify the three main medicinal ingredients in the modified Sanhuang compound.

[0014] To achieve the second objective, the present invention adopts the following technical solution: A detection method for a modified Sanhuang compound includes (1) thin-layer chromatography identification.

[0015] (1) Thin-layer chromatography identification includes: identifying any one or more of the rhubarb, scutellaria and phellodendron in the Jiawei Sanhuang compound.

[0016] Thin-layer chromatography identification methods for rhubarb include: Preparation of test solution: Extract the sample solution of Jiawei Sanhuang compound with diethyl ether, evaporate the solvent, dissolve the residue in methanol to prepare the test solution.

[0017] Identification conditions: Silica gel G thin-layer plate, with n-hexane-ethyl acetate-formic acid in a volume ratio of (20~40):(8~12):(0.3~0.8) as the developing solvent, and the sample volume is 3~10 μL.

[0018] Thin-layer chromatography identification methods for Scutellaria baicalensis include: Preparation of test solution: Take the sample solution of Jiawei Sanhuang compound, evaporate to dryness, dissolve the residue in methanol to prepare the test solution.

[0019] Identification conditions: Polyamide film, with glacial acetic acid-water at a volume ratio of (70~80):(110~140) as the developing solvent, and a sample volume of 3~10 μL.

[0020] Thin-layer chromatography identification methods for Phellodendron bark include: Preparation of test solution: Take the sample solution of Jiawei Sanhuang compound, evaporate to dryness, dissolve the residue in methanol to prepare the test solution.

[0021] Identification conditions: Silica gel G thin-layer plate, with ethyl acetate-butanone-water-glacial acetic acid as the developing solvent in a volume ratio of (5~9):(5~9):(1~3):(1~3), and the sample volume is 3~10 μL.

[0022] Preferably, the thin-layer chromatography identification method for rhubarb includes at least one of the following conditions: Preparation of test solution: Take the sample solution of Jiawei Sanhuang compound and extract it with 1-3 times the volume of diethyl ether 1-3 times, evaporate the solvent, dissolve the residue in methanol to prepare the test solution; Preparation of emodin reference solution: Take emodin reference standard, dissolve it in methanol to prepare a reference solution with a concentration of 0.5~5.0 mg / mL; Preparation of rhubarb reference material solution: Take rhubarb reference material, add methanol at a material-to-liquid ratio of 1g:(6~20)mL, sonicate for 10~30min, filter, and take the filtrate as rhubarb reference material solution; Preparation of negative sample solution: Prepare a negative sample without rhubarb according to the ratio of the modified Sanhuang compound, and then prepare a negative sample solution according to the test sample solution preparation method; Identification conditions: Using a hexane-ethyl acetate-formic acid mixture with a volume ratio of 30:10:0.5 as the developing solvent, examine under a 365 nm UV lamp or under sunlight after fuming with ammonia for 3 min. The sample volume is 5 μL.

[0023] Preferably, the thin-layer chromatography identification method for Scutellaria baicalensis includes at least one of the following conditions: Preparation of baicalin reference solution: Dissolve baicalin reference standard in methanol to prepare a reference solution with a concentration of 0.05~0.30 mg / mL; Preparation of Scutellaria baicalensis reference material solution: Take Scutellaria baicalensis reference material, add water at a material-to-liquid ratio of 1g:(6~15)mL and decoct for 0.5~1.5h, filter, concentrate, evaporate the solvent, dissolve the residue in methanol to obtain the reference material solution; Preparation of negative sample solution: Prepare a negative sample without Scutellaria baicalensis according to the ratio of the modified Sanhuang compound formula, and then prepare a negative sample solution according to the test sample solution preparation method; Identification conditions: Develop twice with glacial acetic acid-water solution at a volume ratio of 73:127, spray with 5% ferric chloride ethanol solution, then heat at 105℃, and finally examine under sunlight. The sample volume is 4 μL.

[0024] Preferably, the thin-layer chromatography identification method for Phellodendron amurense includes at least one of the following conditions: Preparation of berberine hydrochloride reference solution: Dissolve berberine hydrochloride reference standard in methanol to prepare a reference solution with a concentration of 0.5~5.0 mg / mL; Preparation of Phellodendron bark reference material solution: Take Phellodendron bark reference material, add methanol at a material-to-liquid ratio of 1g:(6~20)mL, sonicate for 10~30min, filter, and take the filtrate as the reference material solution; Preparation of negative sample solution: Prepare a negative sample without Phellodendron bark according to the ratio of the modified Sanhuang compound, and then prepare a negative sample solution according to the test sample solution preparation method; Identification conditions: Ethyl acetate-butanone-water-glacial acetic acid (v / v) was used as the developing solvent, and the sample was examined under a 365 nm UV lamp. The sample volume was 5 μL.

[0025] The third objective of this invention is to provide another detection method for the modified Sanhuang compound formula, which determines the content of rhein and chrysophanol in the modified Sanhuang compound formula.

[0026] To achieve the third objective, the present invention adopts the following technical solution: A detection method for a modified Sanhuang compound formula includes (2) content determination.

[0027] (2) Content determination includes determining the content of rhein and chrysophanol in the modified Sanhuang compound, and the methods include: Preparation of test solution: Take the sample solution of Jiawei Sanhuang compound, evaporate to dryness, dissolve the residue in methanol to prepare the test solution.

[0028] Chromatographic conditions: Methanol was used as mobile phase A, and 0.05-0.3% phosphoric acid solution was used as mobile phase B. The detection wavelength was 250-260 nm. The gradient elution program, expressed as the volume percentage change of mobile phase A at different elution times, was as follows: 0-10 min, 12%A-25%A; 10-20 min, 25%A-40%A; 20-30 min, 40%A-77%A; 30-45 min, 77%A-90%A.

[0029] Preferably, the content determination includes at least one of the following conditions: Sample pretreatment: At 30~40℃, take the sample solution of Jiawei Sanhuang compound and filter it through filter paper, and collect the filtrate. Preparation of emodin and chrysophanol reference solutions: Emodin and chrysophanol reference standards were dissolved in methanol to prepare emodin and chrysophanol reference solutions with concentrations of 0.2-3.0 mg / mL. Preparation of negative sample solution: Prepare a negative sample without rhubarb according to the ratio of the modified Sanhuang compound (add the other four medicinal materials in the original ratio), and then prepare a negative sample solution according to the test sample solution preparation method; Chromatographic conditions: Column type C 18 The column temperature is 25~35℃, the injection volume is 8~15 μL, and the flow rate is 0.5~1.5 mL / min.

[0030] Preferably, the filter paper is cellulose filter paper, meeting the requirements for qualitative analysis filter paper in GB / T 1914-2017 "Chemical Analysis Filter Paper", with specifications including: basis weight 76~85 g / m³. 2 70~150mm in diameter.

[0031] Preferably, in the sample pretreatment step, the filter paper is used after being treated with a fat-soluble compound. The specific method includes: mixing the fat-soluble compound with an organic solvent evenly, then immersing the filter paper in the mixture for 20-40 minutes, and then evaporating the solvent to obtain the treated filter paper.

[0032] Preferably, the fat-soluble compound is any one or more of cholesterol nonanoate, stearic acid, palmitic acid, and palmitic acid monoglyceride. More preferably, the fat-soluble compound is cholesterol nonanoate.

[0033] Preferably, the amount of the fat-soluble compound added is 0.1 to 0.5% of the mass of the filter paper.

[0034] Preferably, the organic solvent is any one or more of diethyl ether, petroleum ether, ethanol, tetrahydrofuran, acetone, and carbon tetrachloride.

[0035] More preferably, the chromatographic conditions include: using methanol as mobile phase A, using 0.1% phosphoric acid solution as mobile phase B, a detection wavelength of 254 nm, and a chromatographic column of type C. 18 The column (250 mm × 4.6 mm, 5 µm) was used, with a column temperature of 30 °C, an injection volume of 10 μL, and a flow rate of 1.0 mL / min.

[0036] Preferably, when performing either (1) thin-layer chromatography identification or (2) content determination on the Jiawei Sanhuang compound according to the above detection methods, at least one of the following conditions must be met: A. The thin-layer chromatography of the sample to be tested shows spots of the same color at the corresponding positions as those of the reference standards of emodin, baicalin, or berberine hydrochloride. B. The thin-layer chromatogram of the sample to be tested shows spots of the same color at the corresponding positions as those of the reference herbs rhubarb, scutellaria baicalensis, or phellodendron amurense. C. The total content of emodin and chrysophanol in the sample to be tested shall not be less than 0.11 mg / mL.

[0037] Technical effects of the present invention: 1. The modified Sanhuang compound of this invention consists of five medicinal herbs: rhubarb, scutellaria baicalensis, phellodendron chinense, salvia miltiorrhiza, and peppermint. The volatile oil contained in peppermint is an important component for exerting the efficacy of this formula. To overcome the problem of easy loss of volatile components in traditional decoction processes, steam distillation is used to extract the aromatic water separately, ensuring that the effective substances are preserved to the greatest extent. Therefore, the proposed process is to first extract the peppermint aromatic water separately, then decoct and concentrate the peppermint residue with the remaining medicinal herbs, and finally mix the concentrated liquid with the aromatic water to retain the characteristics of the original formula.

[0038] 2. In the modified Sanhuang compound formula of this invention, rhubarb is the principal ingredient, which has the functions of promoting blood circulation, removing blood stasis, and regulating menstruation. Scutellaria baicalensis is the assistant ingredient, which has the effects of clearing heat and drying dampness, purging fire and detoxifying. Phellodendron amurense is added to purge fire and remove dampness, detoxify and heal sores. Therefore, rhubarb, scutellaria baicalensis and phellodendron amurense are selected for qualitative identification by thin-layer chromatography.

[0039] This invention, in identifying the above-mentioned medicinal materials, focuses on the preparation of the test solution, the preparation of the reference medicinal materials, the type of developing solvent, and the inspection conditions. Using the optimal conditions determined above, a spectrum with clear and round spots was obtained. This method avoids the use of toxic reagents such as chloroform and toluene, and is suitable for the identification of rhubarb, scutellaria, and phellodendron in the modified Sanhuang compound formula.

[0040] 3. This invention uses HPLC to determine the content of the modified Sanhuang compound, establishing a standard for the content of components in the modified Sanhuang compound with the total content of rhein and chrysophanol as the evaluation index. The results show that the target peak is well separated from other components without interference, and methodological validation demonstrates that the content determination method is stable and reliable.

[0041] 4. The present invention optimizes the sample pretreatment steps for HPLC content determination. The sample (Jiawei Sanhuang compound sample solution) is filtered using standard laboratory filter paper. This filter paper, treated with lipid-soluble compounds, not only effectively removes impurities but also effectively separates the oily components (volatile oils and lipid-soluble components) in Jiawei Sanhuang compound, reducing the impact of oily components on the sample evaporation process and detection results. Furthermore, these lipid-soluble compounds are difficult to dissolve in aqueous media and will not leave residues in the Jiawei Sanhuang compound (aqueous medium), thus having no interference with the detection process.

[0042] In the process of treating the filter paper, the preferred fat-soluble compound used in this invention is cholesterol nonanoate. This results in a shorter evaporation time for the treated Jiawei Sanhuang compound sample solution (indicating more thorough removal of oily components), which not only saves detection time but also reduces the loss of active ingredients, leading to a higher measured content of active ingredients.

[0043] 5. This invention establishes a quality detection method based on thin-layer chromatography (TLC) for qualitative identification and high-performance liquid chromatography (HPLC) for the modified Sanhuang compound. The method has good reproducibility and can provide a quality basis for the subsequent research and development of this compound. Attached Figure Description

[0044] Figure 1 The thin-layer chromatographic identification diagram of rhubarb in the modified Sanhuang compound of Example 2 of the present invention is shown in the following figure (A is the result of examination under 365nm ultraviolet light, and B is the result of examination under sunlight after ammonia fuming; wherein: in A and B, 1 represents the reference standard, 2 represents the reference medicinal material, 3-5 represent the test sample, and 6 represents the negative sample). Figure 2 The results of the test solution preparation method in Example 2 of this invention are as follows (A is the chromatogram of test solution 1, where 1-3 represent the test sample and 4-5 represent the reference sample; B is the chromatogram of test solution 2, where 1 represents the reference sample, 2 represents the reference medicinal material, 3-5 represent the test sample, and 6 represents the negative sample). Figure 3 This is a thin-layer chromatographic identification diagram of Scutellaria baicalensis in the modified Sanhuang compound of Example 3 of the present invention (1 represents the reference standard, 2 represents the reference medicinal material, 3-5 represent the test sample, and 6 represents the negative sample). Figure 4 The results of the investigation on the preparation method of Scutellaria baicalensis reference medicinal material solution in Example 3 of the present invention are shown (1 represents reference standard, 2 represents reference medicinal material, 3-5 represent test sample, 6 represents negative sample). Figure 5 The results of the development method investigation in Example 3 of the present invention are shown (1 represents negative sample, 2 and 5 represent control samples, and 3 and 4 represent test samples). Figure 6 The thin-layer chromatographic identification diagram of Phellodendron bark in the modified Sanhuang compound of Example 4 of this invention is shown in the following figure (A is the result of examination under 365nm ultraviolet light, and B is the result of examination under sunlight; wherein: in A and B, 1 represents the reference standard, 2 represents the reference medicinal material, 3-5 represent the test sample, and 6 represents the negative sample). Figure 7 The HPLC chromatogram for the specificity investigation of the content determination of the modified Sanhuang compound in Example 5 of the present invention (1 represents rhein, 2 represents chrysophanol). Figure 8 The water bath evaporation conditions were investigated in the test solution preparation method of Example 5 of the present invention (A is the water bath evaporation condition of test solution 3, and B is the water bath evaporation condition of test solution 1). Figure 9 The HPLC chromatogram of the mobile phase in Example 5 of the present invention is shown (1 represents emodin, 2 represents chrysophanol). Detailed Implementation

[0045] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. 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.

[0046] Unless otherwise specified in the following examples, the implementation conditions are generally those of routine experiments, and the raw materials are all commercially available or prepared by conventional methods in the art.

[0047] Example 1: Preparation of a sample solution of the modified Sanhuang compound formula The ingredient ratio of the modified Sanhuang compound is as follows: rhubarb 10g, scutellaria baicalensis 10g, phellodendron chinense 10g, salvia miltiorrhiza 10g, and mint 10g.

[0048] Preparation method of sample solution of the modified Sanhuang compound: S1, Peppermint volatile oil extract Weigh the prescribed amount of peppermint herbs, add 10 times their volume of water and soak for 1 hour. Extract by steam distillation for 2 hours and collect the volatile oil. Three parallel groups were set up for peppermint volatile oil extraction. The yield of volatile oil was determined to be 1.11 mL / 100g (RSD=1.80%), indicating that the process is stable and reliable.

[0049] S2, Water extraction of remaining dregs and other medicinal materials After steam distillation extraction of peppermint, the remaining residue was combined with other medicinal materials and extracted twice, each time for 1 hour. For the first extraction, 10 times the volume of water was added, and for the second extraction, 8 times the volume of water was added. The extracts were collected after extraction. Three parallel groups were set up for water extraction. The average dry extract yield was 26.24% (RSD=1.62%), and the average total content of rhein and chrysophanol was 0.5826 mg / g (RSD=2.09%), indicating that the process is stable and reliable.

[0050] S3, Concentration Process Take the extract collected by the above water extraction and concentrate it under reduced pressure to a relative density of 1.05~1.10 (60 ℃) to obtain a concentrated solution.

[0051] S4, Mix Take the concentrated solution obtained above and mix it with the volatile oil to obtain the modified Sanhuang compound sample solution. In this step, an appropriate amount of pharmaceutically acceptable excipients may also be added as needed.

[0052] Example 2: Thin-layer chromatographic identification of modified Sanhuang compound formula - identification of rhubarb 1. Solution preparation Preparation of test solution: Take 20 mL of the Jiawei Sanhuang compound sample solution, extract twice with ether (20 mL each time), combine the two ether extracts, evaporate the solvent in a water bath, dissolve the residue in 1 mL of methanol to prepare the test solution.

[0053] Preparation of emodin reference solution: Take an appropriate amount of emodin reference standard, dissolve it in methanol and dilute it to the mark to prepare a 1 mg / mL emodin reference solution.

[0054] Preparation of rhubarb reference material solution: Take 1 g of rhubarb reference material, place it in a stoppered conical flask, add 10 mL of methanol, sonicate for 20 min, filter, and take the filtrate as rhubarb reference material solution.

[0055] Preparation of negative sample solution: Prepare a negative sample without rhubarb according to the ratio of the modified Sanhuang compound, and then prepare a negative sample solution according to the test sample solution preparation method.

[0056] 2. Inspection Method Following the thin-layer chromatography procedure, 5 μL of each of the above solutions were spotted onto the same silica gel G thin-layer plate. The plate was developed using n-hexane-ethyl acetate-formic acid (v / v 30:10:0.5) as the developing solvent. After drying, the plates were examined under a 365 nm UV lamp. Results are shown below. Figure 1 The chromatogram of the test sample showed the same orange-yellow fluorescent spots at the corresponding positions as the chromatograms of the reference sample and the reference medicinal material. No interference was observed with the negative control (see...). Figure 1 (A in the middle).

[0057] In addition, after spotting and fumigation with ammonia for 3 minutes, identical red spots appeared under sunlight, and the negative control showed no interference (see...). Figure 1 (B in the middle).

[0058] 3. Investigation of the preparation method of the test solution Preparation of test solution 1: Take 20 mL of the sample solution of Jiawei Sanhuang compound, evaporate the solvent in a water bath, dissolve the residue in 1 mL of methanol, and prepare test solution 1.

[0059] Preparation of test solution 2: Same as the test solution preparation method in Part 1 of this embodiment, take 20 mL of the Jiawei Sanhuang compound sample solution, extract twice with ether (20 mL each time), combine the two ether extracts, evaporate the solvent in a water bath, dissolve the residue in 1 mL of methanol to prepare test solution 2.

[0060] Take test solution 1 and test solution 2 respectively, and examine them under a 365nm ultraviolet lamp according to the other solution preparation methods in Part 1 and the inspection methods in Part 2 of this embodiment for thin-layer chromatography identification. The results are shown in [Figure Number]. Figure 2During the initial condition exploration, when identifying test solution 1, only the test sample and the reference sample were tested. It was found that test solution 1 contained more impurities, the chromatographic separation effect was poor, and the spots were prone to tailing (see...). Figure 2 (A in the text). Ether extraction was then performed (but not due to the high toxicity of chloroform, etc.), resulting in test solution 2 with less interference from impurities, clearer chromatographic spots, and better separation (see A in the text). Figure 2 (B in the middle).

[0061] Based on the above findings, the chromatogram obtained from test solution 2 showed better results.

[0062] Example 3: Thin-layer chromatographic identification of modified Sanhuang compound formula - identification of Scutellaria baicalensis. 1. Solution preparation Preparation of test solution: Take 20 mL of the sample solution of Jiawei Sanhuang compound, evaporate to dryness in a water bath, dissolve the residue in 20 mL of methanol to prepare the test solution.

[0063] Preparation of baicalin reference solution: Take an appropriate amount of baicalin reference standard, dissolve it in methanol and dilute it to the mark to prepare a 0.1 mg / mL baicalin reference solution.

[0064] Preparation of Scutellaria baicalensis reference material solution: Take 10g of Scutellaria baicalensis reference material, add 80ml of water and decoct for 1.0h, filter, concentrate to 20mL, evaporate the solvent in a water bath, dissolve the residue in 1mL of methanol to obtain Scutellaria baicalensis reference material solution.

[0065] Preparation of negative sample solution: Prepare a negative sample without Scutellaria baicalensis according to the ratio of the modified Sanhuang compound, and then prepare a negative sample solution according to the test sample solution preparation method.

[0066] 2. Inspection Method Following the thin-layer chromatography procedure, 4 μL of each of the above solutions were spotted onto the same polyamide film. Developing was performed using glacial acetic acid-water (73:127 v / v) as the developing solvent. After drying, the films were developed again using the same solvent and dried. A 5% ferric chloride ethanol solution was then sprayed evenly, followed by heating at 105°C and examination under sunlight until the spots were clearly visible. Results are shown below. Figure 3 The chromatogram of the test sample showed the same blue spots at the corresponding positions as the chromatograms of baicalin reference standard and Scutellaria baicalensis reference material, with no interference from the negative control.

[0067] 3. Investigation on the preparation method of Scutellaria baicalensis reference herb solution Preparation of Scutellaria baicalensis reference solution 1: Take 10g of Scutellaria baicalensis reference material, add 10mL of methanol, sonicate for 20min, filter, and obtain Scutellaria baicalensis reference solution 1. Thin-layer chromatography was performed using this solution, with other conditions the same as in Part 1 of this embodiment for solution preparation and Part 2 for inspection. The results are shown in [Figure Number]. Figure 4It is obvious that the spots of the control medicinal material have not unfolded, and there are phenomena such as overlap, poor separation, and unclear display.

[0068] Preparation of Scutellaria baicalensis reference solution 2: Following the same method as in Part 1 of this embodiment, 10g of Scutellaria baicalensis reference material was decocted in 80ml of water for 1.0h, filtered, concentrated to 20mL, and the solvent was evaporated in a water bath. The residue was dissolved in 1mL of methanol to obtain Scutellaria baicalensis reference solution 2. Thin-layer chromatography was performed using this solution, with the remaining conditions the same as in Part 1 of this embodiment for solution preparation and Part 2 for inspection. The results showed clear spots and good separation (see...). Figure 3 ).

[0069] 4. Examination of the deployment method Development once: Prepare each solution according to Part 1 of this embodiment. Take 4 μL of each solution and spot them onto the same polyamide film. Develop using glacial acetic acid-water (volume ratio 73:127) as the developing solvent. After drying, uniformly spray with 5% ferric chloride ethanol solution, then heat at 105°C and examine under sunlight until the spots are clearly visible. Results are shown below. Figure 5 It is evident that the separation effect of each spot is relatively poor.

[0070] Expand twice: The solutions were prepared according to Part 1 of this embodiment, and thin-layer chromatography was performed using the inspection method described in Part 2 of this embodiment. The results showed improved separation of the spots (see...). Figure 3 ).

[0071] Based on the above findings, the chromatogram obtained by using Scutellaria baicalensis reference solution 2 and developing twice with the developing solvent, i.e., following the methods in Parts 1 and 2 of this embodiment, is of good quality.

[0072] Example 4: Thin-layer chromatographic identification of modified Sanhuang compound formula - identification of Phellodendron bark. 1. Solution preparation Preparation of test solution: Take 20 mL of the sample solution of Jiawei Sanhuang compound, evaporate to dryness in a water bath, dissolve the residue in 20 mL of methanol to prepare the test solution.

[0073] Preparation of berberine hydrochloride reference solution: Take an appropriate amount of berberine hydrochloride reference standard, dissolve it in methanol and dilute it to the mark to prepare a 1 mg / mL berberine hydrochloride reference solution.

[0074] Preparation of Phellodendron bark reference material solution: Take 1g of Phellodendron bark reference material, place it in a stoppered conical flask, add 10mL of methanol, sonicate for 20min, filter, and take the filtrate as Phellodendron bark reference material solution.

[0075] Preparation of negative sample solution: Prepare a negative sample without Phellodendron bark according to the ratio of the modified Sanhuang compound, and then prepare a negative sample solution according to the test sample solution preparation method.

[0076] 2. Inspection Method Following the thin-layer chromatography procedure, 5 μL of each of the above solutions were spotted onto the same silica gel G thin-layer plate. The plate was developed using ethyl acetate-butanone-water-glacial acetic acid (volume ratio 7:7:2:2) as the developing solvent. After drying, the plates were examined under a 365 nm UV lamp. Results are shown below. Figure 6 In the A section, the chromatogram of the test sample showed the same green fluorescent spots at the corresponding positions as the chromatograms of berberine hydrochloride reference standard and Phellodendron amurense reference medicinal material, with no interference from the negative control.

[0077] 3. Examination of conditions Inspection Condition 1: Prepare each solution according to Part 1 of this embodiment, spot 4 μL of each solution, and inspect directly under sunlight. Other conditions are the same as the inspection method in Part 2 of this embodiment. Results are shown in [the original text]. Figure 6 In B, the spots are not clearly visible.

[0078] Inspection Condition 2: Prepare each solution according to Part 1 of this embodiment, spot 4 μL of each solution, and inspect under a 365 nm UV lamp. Other conditions are the same as the inspection method in Part 2 of this embodiment. The results show good chromatographic separation and clear spot display (see...). Figure 6 (A in the middle).

[0079] Based on the above findings, chromatograms obtained by examining the images under a 365nm ultraviolet lamp, i.e., following the methods described in Parts 1 and 2 of this embodiment, showed the best results.

[0080] Example 5: Content Determination of Modified Sanhuang Compound 1. Chromatographic conditions The chromatographic column is C10. 18 The column was 250 mm × 4.6 mm, 5 µm; the mobile phase was methanol (A) and 0.1% phosphoric acid solution (B) as the mass percentage, and the gradient elution program was set according to Table 1 below; the flow rate was set to 1.0 mL / min; the detection wavelength was 254 nm; the column temperature was 30 °C; and the injection volume was 10 µL.

[0081] Table 1 Gradient elution program

[0082] 2. Solution preparation (1) Preparation of test solution Mix cholesterol nonanoate with ether until homogeneous, then place the mixture onto cellulose filter paper (quantitative basis 80 g / m³). 2The filter paper was impregnated with a 90mm diameter sample for 30 minutes, and the solvent was evaporated to obtain the treated filter paper. The amount of cholesterol nonanoate added was 0.3% of the filter paper mass. At 35℃, 10mL of the Jiawei Sanhuang compound sample solution was filtered through the treated filter paper. The filtrate was collected, evaporated to dryness in a water bath, and then diluted to 50mL with methanol. After centrifugation, the solution was filtered through a 0.22μm microporous membrane to obtain the final product.

[0083] (2) Preparation of reference solution Take the reference standards of emodin and chrysophanol, place them in a 25 mL brown volumetric flask, add methanol to dissolve them by sonication, and then dilute to the mark to prepare a mixed reference solution with a concentration of 0.4096 mg / mL of emodin and 0.4208 mg / mL of chrysophanol.

[0084] (3) Preparation of negative sample solution According to the formula of the modified Sanhuang compound, a negative sample without rhubarb was prepared (the other four medicinal materials were added in the original proportions). Then, a negative sample solution was prepared according to the test sample solution preparation method.

[0085] 3. Methodological Examination (1) Linear examination Take an appropriate amount of the mixed reference solution, dilute it serially with methanol, and inject it for determination according to the chromatographic conditions in Part 1 of this embodiment. Perform linear regression analysis with the reference concentration (X, mg / mL) as the abscissa and the peak area (Y) as the ordinate to establish a regression equation.

[0086] The regression equation for emodin is Y = 82694X - 10.154 (R²). 2 =0.9998), indicating a good linear relationship within the range of 0.0083~0.0416 mg / mL. Rhein Y = 123745X + 4.7043 (R² = 0.9998) 2 = 0.9998), indicating that it has a good linear relationship in the range of 0.0043~0.0215 mg / mL.

[0087] (2) Specificity examination Take appropriate amounts of the mixed reference solution, test solution, and negative sample solution, respectively, and inject them for analysis according to the chromatographic conditions described in Part 1 of this embodiment. Record the chromatograms as shown below. Figure 7 The results showed that the retention times of the chromatographic peaks of emodin and chrysophanol in the test sample were consistent with the corresponding peaks of the reference sample, the resolution was greater than 1.5, the theoretical plate number met the requirements, and no interference was observed in the negative control, indicating good specificity.

[0088] (3) Precision test Take an appropriate amount of the mixed reference solution and inject it six times consecutively under the chromatographic conditions described in Part 1 of this example, recording the peak area for each injection. The calculation results show that the peak area RSDs of emodin and chrysophanol are 0.51% and 0.72%, respectively, indicating good instrument precision.

[0089] (4) Repeatability test Six test solutions were prepared in parallel using the same batch of Jiawei Sanhuang compound sample solution, following the test solution preparation method in Part 2 of this embodiment. The solutions were then determined under the chromatographic conditions in Part 1 of this embodiment. The total content of rhein and chrysophanol had an RSD of 1.68%, indicating good repeatability.

[0090] (5) Stability test Six portions of the test solution were accurately measured and injected for analysis at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h, and the peak areas were recorded. The peak area RSDs of emodin and chrysophanol were 1.29% and 1.61%, respectively, indicating good stability within 24 h.

[0091] (6) Recovery test Rhein and emodin reference standards were prepared by adding methanol to prepare rhein reference standard solutions with a concentration of 0.0959 mg / mL and emodin reference standard solutions with a concentration of 0.1743 mg / mL, respectively.

[0092] Six aliquots of the test solution with known content (1 mL each) were accurately measured, and 1 mL each of rhein and chrysophanol reference solutions were accurately added. The solutions were injected and analyzed according to the chromatographic conditions described in Part 1 of this embodiment, and the recoveries of rhein and chrysophanol were calculated. The results showed that the average recoveries of rhein and chrysophanol were 99.86% and 100.01%, respectively, with RSDs of 1.82% and 1.56%, respectively.

[0093] (7) Content determination Three batches of pilot-scale sample solutions of the modified Sanhuang compound (batch numbers 20241101, 20241102, and 20241103) were taken and subjected to high-performance liquid chromatography (HPLC) according to the content determination method determined above. The total content of rhein and chrysophanol was (0.1348±0.0046) mg / mL.

[0094] 4. Investigation of the preparation method of the test solution (1) Preparation of different test solutions Preparation of test solution 1: Take 10 mL of the Jiawei Sanhuang compound sample solution, evaporate to dryness in a water bath, add methanol to make up to 50 mL, centrifuge, and filter through a 0.22 μm microporous membrane to obtain the test solution.

[0095] Preparation of test solution 2: At 35℃, take the sample solution of Jiawei Sanhuang compound and pass it through cellulose filter paper (quantitative 80g / m). 2 Filter the solution (90 mm in diameter), take 10 mL of the filtrate, evaporate it to dryness in a water bath, add methanol to make up to 50 mL, centrifuge, and then filter it through a 0.22 μm microporous membrane to obtain the final product.

[0096] Preparation of test solution 3: Mix cholesterol nonanoate with diethyl ether until homogeneous, then place the mixture onto cellulose filter paper (quantitative 80 g / m²). 2 The filter paper was impregnated with a 90mm diameter sample for 30 minutes, and the solvent was evaporated to obtain the treated filter paper. The amount of cholesterol nonanoate added was 0.3% of the filter paper mass. At 35℃, the sample solution of the Jiawei Sanhuang compound was filtered through the treated filter paper. 10 mL of the filtrate was taken, evaporated to dryness in a water bath, and then methanol was added to make up to 50 mL. After centrifugation, it was filtered through a 0.22μm microporous membrane to obtain the final product.

[0097] Preparation of test solution 4: The method is the same as that for test solution 3, except that cholesterol nonanoate is replaced with stearic acid and ether is replaced with acetone.

[0098] Preparation of test solution 5: The method is the same as that for test solution 3, except that cholesterol nonanoate is replaced with palmitic acid.

[0099] Preparation of test solution 6: The method is the same as that for test solution 3, except that cholesterol nonanoate is replaced with palmitate monoglyceride and ether is replaced with ethanol.

[0100] (2) Total content of emodin and chrysophanol Six test solutions prepared above were taken and subjected to high-performance liquid chromatography (HPLC) for content determination according to the method determined in this embodiment. The results of the total content of emodin and chrysophanol are shown in Table 2. It can be seen that the total content of emodin and chrysophanol in test solutions 3 to 6 is relatively high, with the content determination result of test solution 3 being even higher.

[0101] Table 2. Results of content determination for different test solutions

[0102] (3) Water bath evaporation during the preparation of the test solution Six test solutions were prepared according to the above method. Under the same water bath conditions, the evaporation of the Jiawei Sanhuang compound sample solution was observed. The evaporation time (starting when the water temperature reached 100℃ and continuing until a dry paste was formed) and the properties of the residue after evaporation were recorded. The results are shown in Table 3 and... Figure 8It can be seen that the water bath evaporation of test solutions 2 to 6 was relatively good, with evaporation time ranging from 45 to 66 minutes. The color and surface properties of the residue after evaporation were normal (taking test solution 3 as an example). Figure 8 Solution A in the sample requires a longer drying time (76 min) to evaporate, and the residue after evaporation is darker in color, has a rough surface, and is unevenly dried (see A in the sample A). Figure 8 (B in the sample). Among them, test solution 3 showed better evaporation in a water bath, requiring only 45 minutes to dry.

[0103] Table 3. Water bath evaporation during the preparation of different test solutions

[0104] 5. Investigation of the mobile phase Mobile phase 1: Acetonitrile-water (80:20 v / v) was used as the mobile phase for isocratic elution for 45 min. Other conditions were the same as those described in Part 1 of this embodiment. The mixed reference solution and test solution 3 were subjected to high-performance liquid chromatography (HPLC) for detection. The results are shown in [Figure 1]. Figure 9 The chromatographic peaks of emodin and chrysophanol in the test sample interfered with each other.

[0105] Mobile phase 2: The chromatographic conditions were the same as in Part 1 of this embodiment, using methanol (A) and 0.1% phosphoric acid solution (B) as the mobile phase, and the gradient elution procedure was followed according to Table 1. The mixed reference solution, test solution 3, and negative sample solution were subjected to high-performance liquid chromatography (HPLC) detection. The results are shown in the table below. Figure 7 The chromatographic peaks of emodin and chrysophanol in the test sample were well separated, and no interference was observed.

[0106] Based on the above findings, the test solution 3 and mobile phase 2 were selected, i.e., the method described in Parts 1 and 2 of this embodiment was followed, and the liquid chromatography determination yielded better results.

[0107] Example 6 Evaluation of the test results of the modified Sanhuang compound formula 1. Qualitative identification of rhubarb, scutellaria, and phellodendron The sample to be tested shall be identified according to the methods of Examples 2 to 4, and at least one of the following conditions must be met: A. The thin-layer chromatography of the sample to be tested shows spots of the same color at the corresponding positions as those of the reference standards of emodin, baicalin, or berberine hydrochloride.

[0108] B. The thin-layer chromatogram of the sample to be tested shows spots of the same color at the corresponding positions as those of the reference herbs rhubarb, scutellaria baicalensis, or phellodendron amurense.

[0109] 2. Control of emodin and chrysophanol content In Example 5 above, the total content of rhein and chrysophanol in three batches (different batches of medicinal materials) of the modified Sanhuang compound sample solution was (0.1348±0.0046) mg / mL. Considering the differences in the source of medicinal materials, production and storage, the limit for the total amount of rhein and chrysophanol per milliliter of this product is set at not less than 80% of the average value, that is, not less than 0.11 mg.

[0110] Therefore, the content of the sample to be tested was determined according to the method in Example 5, and the total content of rhein and chrysophanol in the sample to be tested was not less than 0.11 mg / mL.

[0111] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting a modified Sanhuang compound, characterized in that: The modified Sanhuang compound formula comprises, by weight, 10 parts rhubarb, 10 parts scutellaria baicalensis, 10 parts phellodendron chinense, 10 parts salvia miltiorrhiza, and 10 parts peppermint. The detection method includes determining the content of rhein and chrysophanol in the modified Sanhuang compound, and the determination method includes: Preparation of test solution: Take the sample solution of Jiawei Sanhuang compound, evaporate it to dryness after pretreatment, dissolve the residue in methanol to prepare the test solution; The pretreatment method for the sample solution of Jiawei Sanhuang compound includes: taking the sample solution of Jiawei Sanhuang compound and filtering it through filter paper, and taking the filtrate; the filter paper is used after being treated with a fat-soluble compound, and the specific method includes: taking the fat-soluble compound and organic solvent, mixing them evenly, then immersing the filter paper in the mixture, evaporating it, and obtaining the treated filter paper. The amount of the fat-soluble compound added is 0.1-0.5% of the filter paper mass; the time for impregnating the filter paper after the fat-soluble compound and organic solvent are mixed evenly is 20-40 minutes. When the fat-soluble compound is cholesterol nonanoate, the organic solvent is diethyl ether; or when the fat-soluble compound is stearic acid, the organic solvent is acetone; or when the fat-soluble compound is palmitic acid, the organic solvent is diethyl ether; or when the fat-soluble compound is palmitic acid monoglyceride, the organic solvent is ethanol. Chromatographic conditions: Column type C 18 The column was set at a temperature of 25–35 °C, an injection volume of 8–15 μL, and a flow rate of 0.5–1.5 mL / min. Methanol was used as mobile phase A, and 0.05–0.3% phosphoric acid solution was used as mobile phase B. The detection wavelength was 250–260 nm. The gradient elution program, expressed as the volume percentage change of mobile phase A at different elution times, was as follows: 0–10 min, 12% A–25% A; 10–20 min, 25% A–40% A; 20–30 min, 40% A–77% A; 30–45 min, 77% A–90% A. The preparation method of the sample solution of the modified Sanhuang compound includes: steam distillation to extract peppermint and collect volatile oil, the remaining residue is combined with other medicinal materials for water extraction and concentration, and the resulting concentrate is mixed with volatile oil to obtain the final product.

2. The detection method according to claim 1, characterized in that: The method for determining the content of emodin and chrysophanol in the modified Sanhuang compound is as follows: The temperature at which the sample solution of the modified Sanhuang compound was filtered through filter paper was 30~40℃. The filter paper specifications include: basis weight 76~85 g / m³ 2 Diameter 70~150mm; Preparation of emodin and chrysophanol reference solutions: Emodin and chrysophanol reference standards were dissolved in methanol to prepare emodin and chrysophanol reference solutions with concentrations of 0.2-3.0 mg / mL. Preparation of negative sample solution: Prepare a negative sample without rhubarb according to the ratio of the modified Sanhuang compound, and then prepare a negative sample solution according to the test sample solution preparation method.

3. The detection method according to claim 1, characterized in that: The fat-soluble compound is cholesterol nonanoate.

4. The detection method according to claim 1, characterized in that: It also includes thin-layer chromatography identification of any one or more of the rhubarb, scutellaria, and phellodendron in the Jiawei Sanhuang compound formula; Thin-layer chromatography methods for identifying rhubarb include: Preparation of test solution: Extract the sample solution of Jiawei Sanhuang compound with diethyl ether, evaporate the solvent, dissolve the residue in methanol to prepare the test solution; Identification conditions: Silica gel G thin-layer plate, with hexane-ethyl acetate-formic acid in a volume ratio of (20~40):(8~12):(0.3~0.8) as the developing solvent, and the sample volume is 3~10 μL; Thin-layer chromatography identification methods for Scutellaria baicalensis include: Preparation of test solution: Take the sample solution of Jiawei Sanhuang compound, evaporate to dryness, dissolve the residue in methanol to prepare the test solution; Identification conditions: Polyamide film, with glacial acetic acid-water at a volume ratio of (70~80):(110~140) as the developing solvent, and a sample volume of 3~10 μL; Thin-layer chromatography identification methods for Phellodendron bark include: Preparation of test solution: Take the sample solution of Jiawei Sanhuang compound, evaporate to dryness, dissolve the residue in methanol to prepare the test solution; Identification conditions: Silica gel G thin-layer plate, with ethyl acetate-butanone-water-glacial acetic acid in a volume ratio of (5~9):(5~9):(1~3):(1~3) as the developing solvent, and the sample volume is 3~10 μL.

5. The detection method according to claim 1, characterized in that: The steam distillation extraction of peppermint includes: soaking the peppermint in 5 to 20 times its volume of water for 0.5 to 4.0 hours, and then steam distilling for 1 to 8 hours; The water extraction includes: extracting with 5 to 30 times the volume of water 1 to 3 times, with each extraction lasting 0.5 to 4.0 hours; The concentration includes: atmospheric pressure concentration or vacuum concentration, to a relative density of 1.05~1.

10.

6. The detection method according to claim 4, characterized in that: The thin-layer chromatography identification method for rhubarb is as follows: Preparation of test solution: Take the sample solution of Jiawei Sanhuang compound and extract it with 1-3 times the volume of diethyl ether 1-3 times, evaporate the solvent, dissolve the residue in methanol to prepare the test solution; Preparation of emodin reference solution: Take emodin reference standard and dissolve it in methanol to prepare a reference solution with a concentration of 0.5~5.0 mg / mL; Preparation of rhubarb reference material solution: Take rhubarb reference material, add methanol at a material-to-liquid ratio of 1g:(6~20)mL, sonicate for 10~30min, filter, and take the filtrate as rhubarb reference material solution; Preparation of negative sample solution: Prepare a negative sample without rhubarb according to the ratio of the modified Sanhuang compound, and then prepare a negative sample solution according to the test sample solution preparation method; Identification conditions: Using a hexane-ethyl acetate-formic acid mixture with a volume ratio of 30:10:0.5 as the developing solvent, examine under a 365 nm UV lamp or under sunlight after fuming with ammonia for 3 min. The sample volume is 5 μL.

7. The detection method according to claim 4, characterized in that: The thin-layer chromatography identification method for Scutellaria baicalensis is as follows: Preparation of baicalin reference solution: Dissolve baicalin reference standard in methanol to prepare a reference solution with a concentration of 0.05~0.30 mg / mL; Preparation of Scutellaria baicalensis reference material solution: Take Scutellaria baicalensis reference material, add water at a material-to-liquid ratio of 1g:(6~15)mL and decoct for 0.5~1.5h, filter, concentrate, evaporate the solvent, dissolve the residue in methanol to obtain the reference material solution; Preparation of negative sample solution: Prepare a negative sample without Scutellaria baicalensis according to the ratio of the modified Sanhuang compound, and then prepare a negative sample solution according to the test sample solution preparation method; Identification conditions: Develop twice with glacial acetic acid-water solution at a volume ratio of 73:127, spray with 5% ferric chloride ethanol solution, then heat at 105℃, and finally examine under sunlight. The sample volume is 4 μL.

8. The detection method according to claim 4, characterized in that: The thin-layer chromatography identification method for Phellodendron bark is as follows: Preparation of berberine hydrochloride reference solution: Dissolve berberine hydrochloride reference standard in methanol to prepare a reference solution with a concentration of 0.5–5.0 mg / mL; Preparation of Phellodendron bark reference material solution: Take Phellodendron bark reference material, add methanol at a material-to-liquid ratio of 1g:(6~20)mL, sonicate for 10~30min, filter, and take the filtrate as the reference material solution; Preparation of negative sample solution: Prepare a negative sample without Phellodendron bark according to the ratio of the modified Sanhuang compound, and then prepare a negative sample solution according to the test sample solution preparation method; Identification conditions: Ethyl acetate-butanone-water-glacial acetic acid (volume ratio 7:7:2:2) was used as the developing solvent. The sample was examined under a 365 nm UV lamp with a sample volume of 5 μL.

9. The detection method according to any one of claims 4, 6 to 8, characterized in that: The following conditions must be met: A. The thin-layer chromatography of the sample to be tested shows spots of the same color at the corresponding positions as those of the reference standards of emodin, baicalin, or berberine hydrochloride. B. The thin-layer chromatogram of the sample to be tested shows spots of the same color at the corresponding positions as those of the reference herbs rhubarb, scutellaria baicalensis, or phellodendron amurense. C. The total content of emodin and chrysophanol in the sample to be tested shall not be less than 0.11 mg / mL.

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