Methods for establishing the material basis of Sanhua Decoction

Abstract

This invention discloses a method for establishing the material basis of Sanhua Decoction, relating to the field of traditional Chinese medicine quality analysis and testing technology. Specifically, the method for establishing the material basis of Sanhua Decoction includes: using thin-layer chromatography to identify rhubarb, magnolia bark, immature bitter orange, and / or notopterygium root. The method in this invention can provide a data foundation for the quality control of Sanhua Decoction, effectively ensuring the stability and controllability of the product quality.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine quality analysis and testing technology, and in particular to a method for establishing a material standard for Sanhua Decoction. Background Technology

[0002] Sanhua Decoction originates from Zhang Zhongjing's *Treatise on Cold Damage* (Shanghan Lun) of the Han Dynasty: "If the Taiyang disease is unresolved, heat accumulates in the bladder, causing the person to become delirious, and blood will flow from the lower abdomen; the person will recover from this. If the external symptoms are not resolved, it is not advisable to attack the disease; the external symptoms should be resolved first. Once the external symptoms are resolved, but there is still lower abdominal distension and tightness, then it is appropriate to attack the disease with Sanhua Decoction. Ingredients: 50 peach kernels (skin and tips removed), 4 liang of rhubarb, 2 liang of cinnamon twig (skin removed), 2 liang of licorice root (roasted), 2 liang of Glauber's salt. Preparation: Boil the above five ingredients in 7 liters of water until 2.5 liters remain. Remove the dregs, add Glauber's salt, bring to a boil again, and remove from heat when it is just about to boil. Take 5 he (approximately 150 ml) warm before meals, three times a day." Sanhua Decoction has a wide range of clinical applications, not only limited to symptoms of Taiyang blood stasis such as lower abdominal distension and pain, black stools, normal urination, delirium, and mania, but also widely used for sequelae of brain injury, dysmenorrhea and amenorrhea in gynecology, urinary retention due to kidney diseases, and urinary retention caused by prostatitis or benign prostatic hyperplasia. Sanhua Decoction has not yet been developed into a traditional Chinese medicine in China, but it has been developed into a Kampo preparation for widespread clinical use in Japan. As a Kampo medicine, it ranks among the top sellers in pharmacies. This formula has great development value in my country.

[0003] Currently, research on Sanhua Decoction mainly focuses on pharmacological studies and clinical applications. However, it lacks systematic and comprehensive quality control measures regarding its material basis, extraction process, multi-index component content determination, and characteristic chromatographic studies. Furthermore, there is limited research on how to measure the consistency between the formulation and traditional decoction quality. According to the "Simplified Registration and Approval Management Regulations for Compound Preparations of Classic Chinese Medicine Formulas (Draft for Comments)," the material reference for classic formulas, except for the molding process, should have preparation methods that are basically consistent with those recorded in ancient medical texts. Patent document CN108956845B discloses a thin-layer chromatography method for identifying four medicinal materials on a single blister pack of Sanhua Decoction freeze-dried powder. However, the prescription dosage and water addition amount are not verified, the preparation method is unclear, and it is difficult to verify the authenticity of the data and images. Patent CN114636779A discloses a method for constructing fingerprint spectra of freeze-dried Sanhua Decoction reference samples and its fingerprint spectra, constructing two sets of fingerprint spectra. However, this method has a long analysis time; for rhubarb, immature bitter orange, and magnolia bark, only three chromatographic peaks were observed after 50 minutes, and the separation of some chromatographic peaks was not ideal. Patent CN116818951A discloses a quality control method for Sanhua Decoction, which establishes a fingerprint spectrum of Sanhua Decoction and a quantitative detection method for 21 components using dual wavelengths. However, this method also suffers from problems such as excessively long analysis time, concentrated chromatographic peaks with poor separation in the 0–30 min and 52–85 min ranges, fewer peaks in the 30–52 min range, unstable baseline with inverted peaks, and insufficient separation for some components to meet quantitative determination requirements. The method is relatively crude. Patent CN114577574A discloses a freeze-dried powder of Sanhua Decoction reference sample, its preparation method, and quality detection method. Its thin-layer identification method is based on the 2020 edition of the Chinese Pharmacopoeia for rhubarb, magnolia bark, immature bitter orange, and notopterygium root, lacking innovation. The quantitative indicators are limited to magnolol and honokiol from magnolia bark, indicating a single indicator selection, and no fingerprint / characteristic chromatographic method has been established. The material reference for classic prescriptions should be established based on systematic research, with comprehensive testing items (including identification, extractives, content determination, fingerprinting, etc.) reflecting the quality of the reference sample. In principle, the content determination or fingerprinting should reflect information about each ingredient in the prescription, and reasonable quality requirement limits for the relevant testing items should be determined. The quality control methods for Sanhua Decoction established in the above four patent documents are relatively simple and cannot fully characterize the quality attributes of the Sanhua Decoction material reference. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for establishing the material basis of Sanhuatang, which can provide a data basis for the quality control of Sanhuatang in large-scale production and ensure the stability and controllability of Sanhuatang product quality.

[0005] To address the aforementioned technical problems, this invention provides a method for establishing the material basis of Sanhua Decoction, characterized by comprising:

[0006] Thin-layer chromatography was used to identify rhubarb, magnolia bark, immature bitter orange and / or notopterygium root.

[0007] Specifically, in some embodiments of the present invention, identification can be performed using thin-layer chromatography methods under the various medicinal materials in the Chinese Pharmacopoeia (2020 edition), but is not limited thereto.

[0008] Preferably, in some embodiments of the present invention, the following are included:

[0009] Thin-layer chromatography was used to identify rhubarb and magnolia bark respectively;

[0010] Thin-layer chromatography was used to simultaneously identify Citrus aurantium and Notopterygium incisum.

[0011] Specifically, in some embodiments of the present invention, the thin-layer chromatography identification method for rhubarb includes:

[0012] The rhubarb test solution was obtained by sequentially extracting Sanhua Decoction or its preparations with a mixed solution of ethanol aqueous solution, hydrochloric acid, chloroform and a mixed solution of anhydrous ethanol and ethyl acetate.

[0013] The rhubarb reference material was extracted sequentially with a mixed solution of ethanol aqueous solution and hydrochloric acid, and a mixed solution of chloroform and anhydrous ethanol and ethyl acetate to obtain a rhubarb reference material solution; and / or the rhein reference standard was dissolved in methanol to obtain a rhubarb reference standard solution.

[0014] The rhubarb test solution, rhubarb reference solution, and / or rhubarb reference herb solution were spotted onto the same silica gel G thin-layer plate and developed using a mixed solution of n-hexane, ethyl acetate, and formic acid as the developing solvent for examination.

[0015] In the mixed solution of ethanol aqueous solution and hydrochloric acid, the concentration of the ethanol aqueous solution is 10 vol% to 50 vol%, exemplary values ​​are 10 vol%, 15 vol%, 20 vol%, 25 vol%, 30 vol%, 35 vol%, 40 vol%, or 45 vol%, but not limited thereto. Preferably, it is 10 vol% to 40 vol%. The hydrochloric acid is an aqueous HCl solution, specifically, the concentration of the hydrochloric acid is 20 vol% to 38 vol%, exemplary values ​​are 22 vol%, 26 vol%, 30 vol%, 34 vol%, or 37 vol%, but not limited thereto. Preferably, it is 30 vol% to 38 vol%.

[0016] In the mixed solution of ethanol-water solution and hydrochloric acid, the volume ratio of ethanol-water solution to hydrochloric acid is 8 to 12:1, with exemplary ratios of 8.4:1, 8.9:1, 9.5:1, 10.2:1, 10.9:1, 11.3:1, or 11.8:1, but not limited thereto. Preferably, it is 9 to 11:1, and more preferably, it is 9.5 to 10.5:1.

[0017] In the mixed solution of anhydrous ethanol and ethyl acetate, the volume ratio of anhydrous ethanol to ethyl acetate is 1 to 4:1; exemplary ratios are 1.4:1, 1.7:1, 2.1:1, 2.5:1, 3.1:1, 3.3:1, or 3.8:1, but are not limited thereto. Preferably, the ratio is 1 to 3:1, and more preferably, it is 1.5 to 2.5:1.

[0018] In a mixed solution of hexane, ethyl acetate, and formic acid, the volume ratio of hexane, ethyl acetate, and formic acid is 20–40:5–20:0.1–1. Exemplary ratios are 22:7:0.2, 27:11:0.3, 33:13:0.4, 37:15:0.7, and 39:17:0.9, but are not limited thereto. A preferred ratio is 25–35:5–15:0.1–0.8, and a more preferred ratio is 28–32:8–13:0.2–0.6.

[0019] Preferably, in some embodiments of the present invention, the concentration of the ethanol-water solution is 30 vol%, the concentration of the hydrochloric acid is 36 vol%, and the volume ratio of the ethanol-water solution to the hydrochloric acid is 10:1; and / or

[0020] The volume ratio of anhydrous ethanol to ethyl acetate in the mixed solution of anhydrous ethanol and ethyl acetate is 2:1; and / or

[0021] In a mixed solution of n-hexane, ethyl acetate, and formic acid, the volume ratio of n-hexane, ethyl acetate, and formic acid is 30:10:0.5.

[0022] Specifically, in some embodiments of the present invention, any one of the rhubarb reference solution and the rhubarb reference herb solution can be sampled and measured together with the rhubarb test solution. In other embodiments of the present invention, all three can be sampled and measured simultaneously.

[0023] Specifically, in some embodiments of the present invention, in the step of extracting Sanhua Decoction or its preparation sequentially with a mixed solution of ethanol aqueous solution and hydrochloric acid, and a mixed solution of chloroform and anhydrous ethanol and ethyl acetate to obtain rhubarb test solution, Sanhua Decoction or its preparation is dissolved in a mixed solution of ethanol aqueous solution and hydrochloric acid, heated under reflux for 0.5 h to 2 h, cooled, and then extracted with chloroform by shaking 2 to 4 times. The resulting extract is dried, and the solid phase obtained after drying is dissolved in a mixed solution of anhydrous ethanol and ethyl acetate to obtain rhubarb test solution.

[0024] The dosage form of Sanhua Decoction can be a decoction, powder, pill, tablet, etc., but is not limited to these. Preferably, the dosage form of Sanhua Decoction is a decoction, and the freeze-dried powder obtained by freeze-drying the decoction is tested.

[0025] The ratio of Sanhua Decoction or its preparation to the mixed solution of ethanol aqueous solution and hydrochloric acid is 0.1g-0.5g:10mL-30mL; exemplary ratios are 0.17g:13mL, 0.22g:17mL, 0.3g:22mL, 0.4g:25mL, or 0.43g:27mL, but not limited thereto. A preferred ratio is 0.2g-0.4g:15mL-30mL, and a more preferred ratio is 0.25g-0.35g:15mL-25mL.

[0026] The ratio of Sanhua Decoction or its preparation to chloroform used in each shaking extraction is 0.1g–0.5g:10mL–30mL; exemplary ratios are 0.17g:13mL, 0.22g:17mL, 0.3g:22mL, 0.4g:25mL, or 0.43g:27mL, but not limited thereto. A preferred ratio is 0.1g–0.4g:15mL–30mL, and a more preferred ratio is 0.25g–0.35g:15mL–25mL.

[0027] The ratio of Sanhua Decoction or its preparation to a mixed solution of anhydrous ethanol and ethyl acetate is 0.1g–0.5g:1mL–5mL, with exemplary ratios being 0.12g:1.3mL, 0.17g:1.5mL, 0.21g:2.2mL, 0.33g:3.7mL, or 0.43g:4.5mL, but not limited thereto. A preferred ratio is 0.1g–0.3g:1mL–3mL.

[0028] The extract obtained from the shaking extraction can be dried by evaporation or evaporation, but is not limited to these methods. Preferably, the extract obtained from the shaking extraction is dried by evaporation.

[0029] Specifically, in some embodiments of the present invention, in the step of spotting the rhubarb test solution, rhubarb reference solution, and / or rhubarb reference herb solution onto the same silica gel G thin-layer plate, developing the plate with a mixed solution of n-hexane, ethyl acetate, and formic acid as the developing solvent, and then examining the plate, the sample volume is 3 μL to 8 μL. After development, the plate is examined under ultraviolet light or sunlight. Exemplarily, the sample volume is 4 μL, 5 μL, 6 μL, or 7 μL, but is not limited thereto. Preferably, it is 3 μL to 5 μL.

[0030] Specifically, in some embodiments of the present invention, the thin-layer chromatography identification method for Magnolia officinalis includes:

[0031] The Sanhua Decoction or its preparations were extracted sequentially with methanol and chloroform to obtain the Magnolia officinalis test solution.

[0032] Magnolia officinalis reference material was extracted sequentially with water and chloroform to obtain a Magnolia officinalis reference material solution; and / or magnolol reference standard was dissolved in methanol to obtain a Magnolia officinalis reference standard solution;

[0033] Spot the Magnolia officinalis test solution, Magnolia officinalis reference solution and / or Magnolia officinalis reference medicinal material solution onto the same silica gel G thin layer plate, develop with a mixed solution of petroleum ether and ethyl acetate as the developing solvent, spray with vanillin sulfuric acid solution, develop color at 100℃~110℃, and examine.

[0034] In the mixed solution of petroleum ether and ethyl acetate, the volume ratio of petroleum ether to ethyl acetate is 2 to 5:1, with exemplary ratios of 2.3:1, 2.8:1, 3.4:1, 3.9:1, or 4.5:1, but not limited thereto. Preferably, it is 2 to 4:1, and more preferably, it is 2.5 to 3.5:1.

[0035] Specifically, in some embodiments of the present invention, any one of the Magnolia officinalis reference solution and the Magnolia officinalis reference herb solution can be sampled and measured along with the Magnolia officinalis test solution. In other embodiments of the present invention, all three can be sampled and measured simultaneously. Preferably, in one embodiment, the Magnolia officinalis test solution and the Magnolia officinalis reference herb solution are sampled and measured.

[0036] Preferably, in some embodiments of the present invention, in the mixed solution of petroleum ether and ethyl acetate, the volume ratio of petroleum ether to ethyl acetate is 3:1, and the boiling range of petroleum ether is 60°C to 90°C.

[0037] Specifically, in some embodiments of the present invention, in the step of extracting Sanhua Decoction or its preparation with methanol and chloroform in sequence to obtain the Magnolia officinalis test solution, Sanhua Decoction or its preparation is mixed with methanol and ultrasonically treated for 10 min to 40 min. After removing methanol from the obtained extract, it is dissolved in water and then extracted with chloroform by shaking 2 to 4 times. The obtained extract is dried, and the solid phase obtained after drying is dissolved in methanol to obtain the Magnolia officinalis test solution.

[0038] The dosage form of Sanhua Decoction can be a decoction, powder, pill, tablet, etc., but is not limited to these. Preferably, the dosage form of Sanhua Decoction is a decoction, and the freeze-dried powder obtained by freeze-drying the decoction is tested.

[0039] The ratio of Sanhua Decoction or its preparation to methanol used in extraction is 0.5g–2g:15mL–30mL, with exemplary ratios being 0.55g:17mL, 0.89g:22mL, 1.05g:23mL, 1.37g:26mL, or 1.88g:29mL, but not limited to these. A preferred ratio is 0.5g–1.5g:18mL–25mL.

[0040] The ratio of Sanhua Decoction or its preparation to water is 0.5g-2g:5mL-20mL; exemplary ratios are 0.55g:7mL, 0.89g:12mL, 1.05g:14mL, 1.37g:17mL, or 1.8g:19mL, but not limited thereto. A preferred ratio is 0.5g-1.5g:6mL-15mL.

[0041] The ratio of Sanhua Decoction or its preparation to chloroform used in each shaking extraction is 0.5g–2g: 10mL–30mL; exemplary ratios are 0.7g: 13mL, 1.3g: 16mL, 1.8g: 23mL, or 1.9g: 29mL, but not limited thereto. A preferred ratio is 0.5g–1.5g: 15mL–25mL.

[0042] The ratio of Sanhua Decoction or its preparation to methanol used for dissolution is 0.5g–2g:0.5mL–3mL. Exemplary ratios are 0.7g:0.9mL, 1.2g:1.3mL, 1.7g:1.9mL, or 1.9g:3.5mL, but are not limited thereto. A preferred ratio is 0.5g–1.5g:0.5mL–1.5mL.

[0043] The ultrasonic power is 200W to 400W, and the ultrasonic frequency is 30kHz to 50kHz, but not limited to these. Preferably, the ultrasonic power is 300W and the frequency is 40kHz.

[0044] The extract obtained from the shaking extraction can be dried by evaporation or evaporation, but is not limited to these methods. Preferably, the extract obtained from the shaking extraction is dried by evaporation.

[0045] Specifically, in some embodiments of the present invention, in the steps of extracting the Magnolia officinalis reference material sequentially with water and chloroform to obtain a Magnolia officinalis reference material solution; and / or dissolving the magnolol reference standard with methanol to obtain a Magnolia officinalis reference standard solution, the magnolol reference material is mixed with water, boiled for 40 min to 60 min, filtered, and the filtrate is extracted with chloroform by shaking 2 to 4 times. The resulting extract is dried, and the solid phase obtained after drying is dissolved in methanol to obtain a Magnolia officinalis reference material solution.

[0046] The ratio of Magnolia officinalis reference material to water is 0.5g-2g:80mL-150mL; exemplary ratios are 0.7g:87mL, 1.3g:109mL, 1.7g:128mL or 1.8g:143mL, but not limited to these.

[0047] The ratio of Magnolia officinalis reference material to chloroform used in each shaking extraction is 0.5g–2g: 10mL–30mL; exemplary ratios are 0.7g: 13mL, 1.3g: 16mL, 1.8g: 23mL, or 1.9g: 29mL, but not limited thereto. A preferred ratio is 0.5g–1.5g: 15mL–25mL.

[0048] The ratio of Magnolia officinalis reference material to methanol is 0.5g–2g:0.5mL–3mL. Exemplary ratios are 0.7g:0.9mL, 1.2g:1.3mL, 1.7g:1.9mL, or 1.9g:3.5mL, but are not limited thereto. A preferred ratio is 0.5g–1.5g:0.5mL–1.5mL.

[0049] The extract obtained from the shaking extraction can be dried by evaporation or evaporation, but is not limited to these methods. Preferably, the extract obtained from the shaking extraction is dried by evaporation.

[0050] Specifically, in some embodiments of the present invention, in the step of spotting the Magnolia officinalis test solution, Magnolia officinalis reference solution and / or Magnolia officinalis reference medicinal material solution onto the same silica gel G thin-layer plate, developing with a mixed solution of petroleum ether and ethyl acetate as the developing solvent, and spraying with vanillin sulfuric acid solution, developing color at 100℃~110℃, and examining, the sample volume is 3μL~12μL, and the concentration of vanillin sulfuric acid solution is 3wt%~10wt%.

[0051] For example, the sample loading volume is 4 μL, 5 μL, 7 μL, 10 μL, or 11 μL, but is not limited thereto. Preferably, it is 5 μL to 10 μL, and more preferably, it is 10 μL.

[0052] Specifically, in some embodiments of the present invention, the thin-layer chromatography identification method for Citrus aurantium and Notopterygium incisum includes:

[0053] The Sanhua Decoction or its preparations were extracted with methanol to obtain the Citrus aurantium test solution;

[0054] The reference herbs Citrus aurantium and Notopterygium incisum were extracted with methanol to obtain Citrus aurantium reference herb solution and Notopterygium incisum reference herb solution, respectively; and / or the reference standards of purslane, naringin, and neohesperidin were dissolved with methanol to obtain Citrus aurantium reference standard solution.

[0055] The test solution of Citrus aurantium, the reference solution of Citrus aurantium, the reference medicinal material of Citrus aurantium and / or the reference medicinal material solution of Notopterygium incisum were spotted on the same silica gel G thin layer plate. The plate was developed using the lower layer of a mixed solution of chloroform, methanol and water as the developing solvent, and then sprayed with aluminum chloride ethanol solution. The plate was then developed at 100℃~110℃ and examined.

[0056] In the mixed solution of chloroform, methanol, and water, the volume ratio of chloroform, methanol, and water is 10–15:5–10:1–4. Exemplary ratios are 11:6:1.5, 13:7:2, 14:8:3, or 14.5:9:3.5, but are not limited thereto. A preferred ratio is 12–14:6–9:1–3.

[0057] Specifically, in some embodiments of the present invention, the test solution of Citrus aurantium and the reference solution of Citrus aurantium can be sampled and measured, or the test solution of Citrus aurantium, the reference medicinal material solution of Citrus aurantium, and the reference medicinal material solution of Notopterygium incisum can be sampled and measured, or all four can be sampled and measured together. Preferably, in one embodiment, the test solution of Citrus aurantium, the reference medicinal material solution of Citrus aurantium, the reference medicinal material solution of Notopterygium incisum, and the reference solution of Citrus aurantium are sampled and measured together.

[0058] Preferably, in some embodiments of the present invention, the volume ratio of chloroform, methanol and water in the mixed solution of chloroform, methanol and water is 13:7:2.

[0059] Specifically, in some embodiments of the present invention, in the step of extracting Sanhua Decoction or its preparation with methanol to obtain a Citrus aurantium test solution, Sanhua Decoction or its preparation is mixed with methanol, ultrasonically treated for 10 min to 40 min, the resulting extract is dried, and the resulting solid phase is dissolved in methanol to obtain a Citrus aurantium test solution.

[0060] The dosage form of Sanhua Decoction can be a decoction, powder, pill, tablet, etc., but is not limited to these. Preferably, the dosage form of Sanhua Decoction is a decoction, and the freeze-dried powder obtained by freeze-drying the decoction is tested.

[0061] The ratio of Sanhua Decoction or its preparation to methanol used in extraction is 0.1g-1g:20mL-30mL; exemplary ratios are 0.2g:21mL, 0.3g:23mL, 0.5g:24mL, 0.7g:26mL, or 0.9g:29mL, but not limited thereto. A preferred ratio is 0.2g-0.8g:22mL-27mL.

[0062] The ratio of Sanhua Decoction or its preparation to methanol used for dissolution is 0.1g-1g:0.5mL-3mL. Exemplary ratios are 0.2g:0.9mL, 0.5g:1.3mL, 0.7g:1.9mL, or 0.9g:2.6mL, but are not limited thereto. A preferred ratio is 0.2g-0.8g:0.5mL-1.5mL.

[0063] The ultrasonic power is 200W to 400W, and the ultrasonic frequency is 30kHz to 50kHz, but not limited to these. Preferably, the ultrasonic power is 300W and the frequency is 40kHz.

[0064] The extract obtained from the shaking extraction can be dried by evaporation or evaporation, but is not limited to these methods. Preferably, the extract obtained from the shaking extraction is dried by evaporation.

[0065] Specifically, in some embodiments of the present invention, the steps of spotting the *Citrus aurantium* test solution, *Citrus aurantium* reference solution, *Citrus aurantium* reference medicinal material and / or *Notopterygium incisum* reference medicinal material solution onto the same silica gel G thin-layer plate, developing the plate with the lower layer of a mixed solution of chloroform, methanol, and water as the developing solvent, spraying with aluminum chloride ethanol solution, developing the color at 100℃~110℃, and examining the plate, are as follows: the sample volume is 1μL~3μL, the concentration of the aluminum chloride ethanol solution is 3wt%~10wt%, and the plate is examined under ultraviolet light after color development.

[0066] For example, the sample loading volume is 1 μL, 2 μL, 3 μL, 4 μL or 5 μL, but is not limited thereto. Preferably it is 1 μL to 2 μL, more preferably it is 2 μL.

[0067] Preferably, in some embodiments of the present invention, the method for establishing the material basis of Sanhua Decoction further includes: constructing a fingerprint spectrum to identify the components in Sanhua Decoction or its preparations.

[0068] Specifically, in some embodiments of the present invention, the method for constructing the fingerprint spectrum of Sanhua Decoction includes:

[0069] The Sanhua Decoction or its preparations were extracted using the ninth extraction solvent to obtain a fingerprint spectrum test solution.

[0070] The reference standard was dissolved or extracted using the tenth extraction solvent to obtain a fingerprint chromatogram reference standard solution;

[0071] The fingerprint spectrum of the test solution and the fingerprint spectrum of the reference solution were determined by liquid chromatography to obtain the fingerprint spectrum of Sanhua Decoction.

[0072] The liquid chromatograph uses an octadecylsilane-bonded silica gel column as the stationary phase, methanol as mobile phase A, and a 0.05 vol%–0.3 vol% aqueous phosphoric acid solution as mobile phase B for gradient elution. The gradient elution curve is as follows:

[0073] From 0 min to 5 min, mobile phase A decreased from 3% to 21%, and mobile phase B decreased from 97% to 79%.

[0074] From 5 min to 20 min, mobile phase A decreased from 21% to 36%, and mobile phase B decreased from 79% to 64%.

[0075] Between 20 and 32 minutes, mobile phase A decreased from 36% to 50%, and mobile phase B decreased from 64% to 50%.

[0076] Between 32 and 42 minutes, mobile phase A decreased from 50% to 62%, and mobile phase B decreased from 50% to 38%.

[0077] From 42 to 50 minutes, mobile phase A decreased from 62% to 85%, and mobile phase B decreased from 38% to 15%.

[0078] Over 50-60 minutes, mobile phase A decreased from 85% to 95%, and mobile phase B decreased from 15% to 5%.

[0079] The reference standards include aloe-emodin-8-O-glucoside reference standard, naringin reference standard, neohesperidin reference standard, rhein-1-O-β-D-glucoside reference standard, rhein-8-O-β-D-glucoside reference standard, aloe-emodin reference standard, nobiletin reference standard, rhein reference standard, magnolol reference standard, magnolol reference standard, rhein reference standard, rhein reference standard and rhein methyl ether reference standard;

[0080] The ninth extraction solvent is a methanol aqueous solution with a concentration of 50 vol% to 100 vol% or an ethanol aqueous solution with a concentration of 50 vol% to 100 vol%, and the tenth extraction solvent is a methanol aqueous solution with a concentration of 30 vol% to 100 vol%. For example, the ninth extraction solvent may be a 60 vol% methanol aqueous solution, a 70 vol% methanol aqueous solution, a 75 vol% methanol aqueous solution, an 85 vol% methanol aqueous solution, a 50 vol% ethanol aqueous solution, a 60 vol% ethanol aqueous solution, a 70 vol% ethanol aqueous solution, an 85 vol% ethanol aqueous solution, or a 95 vol% ethanol aqueous solution, but is not limited thereto.

[0081] For example, the tenth extraction solvent is a 40 vol% methanol aqueous solution, a 50 vol% methanol aqueous solution, a 60 vol% methanol aqueous solution, a 70 vol% methanol aqueous solution, a 75 vol% methanol aqueous solution, or an 85 vol% methanol aqueous solution, but is not limited to these.

[0082] For example, the concentration of the phosphoric acid aqueous solution is 0.05 vol%, 0.10 vol%, 0.15 vol%, 0.20 vol%, 0.25 vol%, or 0.30 vol%, but is not limited thereto. Preferably, it is 0.05 vol% to 0.2 vol%, more preferably 0.05 vol% to 0.15 vol%.

[0083] Preferably, in some embodiments of the present invention, the mobile phase B is a 0.1 vol% aqueous solution of phosphoric acid.

[0084] Preferably, in some embodiments of the present invention, the ninth extraction solvent is an aqueous methanol solution with a concentration of 70 vol% to 100 vol% or an aqueous ethanol solution with a concentration of 70 vol% to 100 vol%; and / or

[0085] The tenth extraction solvent is a methanol aqueous solution with a concentration of 70 vol% to 100 vol%.

[0086] Preferably, in some embodiments of the present invention, the ninth extraction solvent is a 70 vol% methanol-water solution; and / or

[0087] The tenth extraction solvent is methanol (i.e., 100 vol% methanol).

[0088] Specifically, in some embodiments of the present invention, the column length of the chromatographic column is 100 mm to 250 mm, exemplarily 100 mm, 150 mm, 200 mm, or 250 mm, but not limited thereto. The column diameter is 2.1 mm to 5 mm, exemplarily 2.1 mm, 3.0 mm, or 4.6 mm, but not limited thereto. The particle size of the stationary phase is 1.6 μm to 1.8 μm; exemplarily 1.6 μm, 1.7 μm, or 1.8 μm, but not limited thereto.

[0089] The column temperature of the chromatographic column is 28℃~32℃; exemplary values ​​are 28℃, 29℃, 30℃, 31℃ or 32℃, but it is not limited thereto.

[0090] The flow rate of the liquid chromatograph is 0.18 mL / min to 0.22 mL / min, exemplarily 0.19 mL / min, 0.2 mL / min or 0.21 mL / min, but is not limited thereto.

[0091] The detection wavelength of the liquid chromatograph is 220nm to 300nm; exemplary wavelengths are 225nm, 240nm, 255nm, 270nm or 285nm, but not limited thereto.

[0092] The injection volume of the fingerprint chromatogram test solution is 0.5 μL to 1.5 μL, with examples of 0.5 μL, 1.0 μL or 1.5 μL, but not limited to these.

[0093] The injection volume of the fingerprint reference solution is 0.5 μL to 1.5 μL, with examples of 0.5 μL, 1.0 μL or 1.5 μL, but not limited to these.

[0094] Preferably, in some embodiments of the present invention, the chromatographic column has a length of 150 mm, a diameter of 2.1 mm, and a stationary phase particle size of 1.6 μm;

[0095] The column temperature of the chromatographic column is 30℃;

[0096] The liquid chromatograph has a flow rate of 0.2 mL / min, a detection wavelength of 260 nm, an injection volume of 1 μL for the fingerprint sample solution, and an injection volume of 1 μL for the fingerprint reference solution.

[0097] Specifically, in some embodiments of the present invention, in the step of extracting Sanhua Decoction or its preparation using the ninth extraction solvent to obtain a fingerprint spectrum test solution, Sanhua Decoction or its preparation is mixed with a methanol aqueous solution with a concentration of 70 vol% to 100 vol%, and then ultrasonically treated or heated under reflux for 15 min to 45 min.

[0098] The dosage form of Sanhua Decoction can be a decoction, powder, pill, tablet, etc., but is not limited to these. Preferably, the dosage form of Sanhua Decoction is a decoction, and the freeze-dried powder obtained by freeze-drying it is tested.

[0099] The ratio of Sanhua Decoction or its preparation to the methanol-water solution is 0.1g-1g:10mL-50mL, with exemplary ratios being 0.13g:15mL, 0.17g:15mL, 0.2g:20mL, 0.27g:20mL, 0.39g:25mL, 0.55g:25mL, 0.77g:50mL, or 0.93g:50mL, but not limited thereto. Preferably, the ratio of Sanhua Decoction or its preparation to the methanol-water solution is 0.3g-0.8g:20mL-30mL. More preferably, it is 0.4g-0.5g:22mL-28mL.

[0100] When using ultrasonic extraction, the ultrasonic power is 200W to 400W and the ultrasonic frequency is 30kHz to 50kHz.

[0101] For example, the extraction time is 18 min, 22 min, 27 min, 34 min, 38 min, 40 min, or 42 min, but is not limited thereto. Preferably, it is 20 min to 40 min.

[0102] Specifically, in some embodiments of the present invention, in the step of extracting Sanhua Decoction or its preparation with the ninth extraction solvent to obtain the fingerprint spectrum test solution, Sanhua Decoction or its preparation is mixed with a 70 vol% methanol aqueous solution and ultrasonically treated with a power of 250 W and a frequency of 40 kHz for 30 min.

[0103] The ratio of Sanhua Decoction or its preparation to methanol aqueous solution is 0.4g:25mL.

[0104] Specifically, in some embodiments of the present invention, the concentration of aloe-emodin-8-O-glucoside reference standard in the fingerprint chromatogram reference solution is 10 μg / mL to 20 μg / mL, the concentration of naringin reference standard is 120 μg / mL to 180 μg / mL, the concentration of neohesperidin reference standard is 180 μg / mL to 250 μg / mL, the concentration of rhein-1-O-β-D-glucoside reference standard is 10 μg / mL to 30 μg / mL, the concentration of rhein-8-O-β-D-glucoside reference standard is 20 μg / mL to 50 μg / mL, and the concentration of rhein-8-O-β-D-glucoside reference standard is... The concentrations of the following substances are as follows: aloe-emodin reference standard: 10 μg / mL to 40 μg / mL; nobiletin reference standard: 5 μg / mL to 20 μg / mL; rhein reference standard: 20 μg / mL to 50 μg / mL; magnolol reference standard: 5 μg / mL to 30 μg / mL; honokiol reference standard: 5 μg / mL to 30 μg / mL; emodin reference standard: 5 μg / mL to 30 μg / mL; chrysophanol reference standard: 10 μg / mL to 30 μg / mL; and emodin methyl ether reference standard: 2 μg / mL to 15 μg / mL.

[0105] Specifically, in some embodiments of the present invention, the fingerprint spectrum includes 16 common peaks, wherein peak 1 is aloe-emodin-8-O-glucoside, peak 2 is naringin, peak 3 is neohesperidin, peak 4 is rhein-1-O-β-D-glucoside, peak 5 is rhein-8-O-β-D-glucoside, peak 7 is rhein-8-O-β-D-glucoside, peak 8 is aloe-emodin, peak 9 is norihesperidin, peak 10 is rhein, peak 11 is honokiol, peak 13 is honokiol, peak 14 is rhein, peak 15 is rhein, and peak 16 is rhein methyl ether.

[0106] Preferably, in some embodiments of the present invention, the method for establishing the material basis of Sanhua Decoction further includes: using liquid chromatography to determine the content of free anthraquinones, total anthraquinones, magnolol, honokiol, naringin, neohesperidin, synephrine, and gentianol in Sanhua Decoction or its preparations.

[0107] More preferably, the method for establishing the material standard of Sanhua Decoction also includes: using liquid chromatography to determine the content of magnolol, free anthraquinone and total anthraquinone in Sanhua Decoction or its preparations;

[0108] The contents of naringin and neohesperidin in Sanhua Decoction or its preparations were simultaneously determined by liquid chromatography.

[0109] The content of synephrine in Sanhua Decoction or its preparations was determined by liquid chromatography.

[0110] The content of qianghuo alcohol in Sanhua Decoction or its preparations was determined by liquid chromatography.

[0111] Specifically, in some embodiments of the present invention, the method for determining the content of free anthraquinones, total anthraquinones, and magnolol includes:

[0112] The Sanhua Decoction or its preparations are extracted using a first extraction solvent to obtain a free anthraquinone test solution; the free anthraquinone test solution is used for the determination of free anthraquinone, magnolol, and the content of magnolol.

[0113] The free anthraquinone test solution was extracted sequentially with hydrochloric acid solution and chloroform, and then dissolved in the first solvent to obtain the total anthraquinone test solution; the total anthraquinone test solution was used for the determination of the total anthraquinone content;

[0114] The reference standard was dissolved or extracted using a second extraction solvent to obtain an anthraquinone reference standard solution;

[0115] The free anthraquinone test solution, total anthraquinone test solution, and / or anthraquinone reference solution were determined by liquid chromatography.

[0116] The liquid chromatograph uses an octadecylsilane-bonded silica gel column as the stationary phase, acetonitrile as mobile phase A, and a 0.05 vol%–0.3 vol% aqueous phosphoric acid solution as mobile phase B for gradient elution. The gradient elution curve is as follows:

[0117] From 0 min to 10 min, mobile phase A was 28% and mobile phase B was 72%.

[0118] From 10 min to 28 min, mobile phase A decreased from 28% to 40%, and mobile phase B decreased from 72% to 60%.

[0119] Between 28 and 35 minutes, mobile phase A decreased from 40% to 42%, and mobile phase B decreased from 60% to 58%.

[0120] Between 35 and 50 minutes, mobile phase A decreased from 42% to 54%, and mobile phase B decreased from 58% to 46%.

[0121] The reference standards include aloe-emodin reference standard, rhein reference standard, chrysophanol reference standard, emodin reference standard, emodin methyl ether reference standard, magnolol reference standard and magnolol reference standard.

[0122] Wherein, the first extraction solvent, the second extraction solvent, and the first solvent are methanol aqueous solutions with a concentration of 70 vol% to 100 vol%; for example, the concentration of the methanol aqueous solution is 75 vol%, 80 vol%, 85 vol%, or 90 vol%, but is not limited thereto. Preferably, it is 90 vol% to 100 vol%.

[0123] The hydrochloric acid solution is an aqueous solution of hydrochloric acid with a concentration of 5 wt% to 15 wt%, exemplarily 5.8 wt%, 6.4 wt%, 7.5 wt%, 8.9 wt%, 10.2 wt%, 11.4 wt%, 13.3 wt%, or 14.1 wt%, but is not limited thereto. Preferably, it is 5 wt% to 10 wt%.

[0124] For example, the concentration of the phosphoric acid aqueous solution is 0.08 vol%, 0.12 vol%, 0.14 vol%, 0.18 vol%, 0.22 vol%, 0.26 vol%, or 0.28 vol%, but is not limited thereto. Preferably, it is 0.05 vol% to 0.2 vol%, more preferably 0.08 vol% to 0.12 vol%.

[0125] Specifically, in some embodiments of the present invention, anthraquinone reference solution and free anthraquinone test solution are jointly determined to obtain the content of free anthraquinone, magnolol, and honokiol in Sanhua Decoction or its preparations. In other embodiments, anthraquinone reference solution and total anthraquinone test solution are jointly determined to obtain the content of total anthraquinone in Sanhua Decoction or its preparations. In still other embodiments, all three can be determined together.

[0126] Preferably, in some embodiments of the present invention, the mobile phase B is a 0.1 vol% aqueous solution of phosphoric acid; and / or

[0127] The first extraction solvent is methanol (i.e., 100 vol% methanol); and / or

[0128] The second extraction solvent is methanol (i.e., 100 vol% methanol); and / or

[0129] The first solvent is methanol (i.e., 100 vol% methanol); and / or

[0130] The concentration of the hydrochloric acid solution is 8 wt%.

[0131] Specifically, in some embodiments of the present invention, the column length of the chromatographic column is 100 mm to 250 mm, exemplarily 100 mm, 150 mm, 200 mm, or 250 mm, but not limited thereto. The column diameter is 2.1 mm to 5 mm, exemplarily 3.0 mm, 3.9 mm, or 4.6 mm, but not limited thereto. The particle size of the stationary phase is 1.6 μm to 1.8 μm; exemplarily 1.6 μm, 1.7 μm, or 1.8 μm, but not limited thereto.

[0132] The column temperature of the chromatographic column is 26℃~30℃; exemplary values ​​are 26℃, 27℃, 28℃ or 29℃, but it is not limited thereto.

[0133] The flow rate of the liquid chromatograph is 0.30 mL / min to 0.34 mL / min, exemplarily 0.31 mL / min, 0.32 mL / min or 0.33 mL / min, but is not limited thereto.

[0134] The detection wavelength of the liquid chromatograph is 240nm to 280nm; exemplary wavelengths are 225nm, 240nm, 255nm, 270nm or 275nm, but not limited thereto.

[0135] The injection volume of the free anthraquinone test solution is 0.5 μL to 2 μL, exemplarily 1 μL, 1.5 μL or 2 μL, but not limited thereto.

[0136] The injection volume of the total anthraquinone test solution is 0.5 μL to 2 μL, exemplarily 1 μL, 1.5 μL or 2 μL, but not limited thereto.

[0137] The injection volume of the anthraquinone reference solution is 0.5 μL to 2 μL, exemplarily 1 μL, 1.5 μL or 2 μL, but not limited thereto.

[0138] Preferably, in some embodiments of the present invention, the chromatographic column has a length of 100 mm, a diameter of 2.1 mm, and a stationary phase particle size of 1.6 μm;

[0139] The column temperature of the chromatographic column is 28℃;

[0140] The flow rate of the liquid chromatograph is 0.32 mL / min, the detection wavelength is 254 nm, the injection volume of the free anthraquinone test solution is 1 μL, the injection volume of the total anthraquinone test solution is 1 μL, and the injection volume of the anthraquinone reference solution is 1 μL.

[0141] Specifically, in some embodiments of the present invention, in the step of extracting Sanhua Decoction or its preparation with a first extraction solvent to obtain a free anthraquinone test solution, Sanhua Decoction or its preparation is mixed with a methanol aqueous solution with a concentration of 70 vol% to 100 vol% and ultrasonically treated for 15 min to 45 min.

[0142] The dosage form of Sanhua Decoction can be a decoction, powder, pill, tablet, etc., but is not limited to these. Preferably, the dosage form of Sanhua Decoction is a decoction, and the freeze-dried powder obtained by freeze-drying it is measured.

[0143] The ratio of Sanhua Decoction or its preparation to the methanol-water solution is 0.1g-1g:10mL-50mL. Exemplary ratios include 0.13g:14mL, 0.17g:17mL, 0.19g:23mL, 0.2g:25mL, 0.27g:29mL, 0.39g:32mL, 0.43g:37mL, 0.55g:39mL, 0.63g:43mL, 0.77g:45mL, 0.89g:47mL, or 0.93g:49mL, but are not limited to these. Preferably, the ratio of Sanhua Decoction or its preparation to the methanol-water solution is 0.3g-0.5g:20mL-30mL.

[0144] The ultrasonic power is 200W to 400W, and the ultrasonic frequency is 30kHz to 50kHz, but not limited to these. Preferably, the ultrasonic power is 300W and the frequency is 40kHz.

[0145] Specifically, in some embodiments of the present invention, in the step of extracting the free anthraquinone test solution sequentially with hydrochloric acid solution and chloroform, and then dissolving it with a first solvent to obtain a total anthraquinone test solution, the free anthraquinone test solution is dried, the resulting solid phase is mixed with hydrochloric acid solution, and ultrasonically treated for 1 min to 5 min to obtain a first intermediate solution; the first intermediate solution is heated under reflux with chloroform for 30 min to 90 min to obtain a second intermediate solution; the second intermediate solution is extracted with chloroform 2 to 6 times, the resulting extracts are combined, dried, and the resulting solid phase is dissolved with the first solvent;

[0146] The volume ratio of the free anthraquinone test solution to the hydrochloric acid solution is 1:0.8 to 1.5; exemplary ratios are 1:0.9, 1:1.1, 1:1.3, or 1:1.4, but are not limited thereto. A preferred ratio is 1:0.9 to 1.1.

[0147] The volume ratio of the free anthraquinone test solution to the chloroform used for heating and reflux is 1:0.8 to 1.5; exemplary ratios are 1:0.9, 1:1.1, 1:1.3, or 1:1.4, but are not limited thereto. A preferred ratio is 1:0.9 to 1.1.

[0148] The volume ratio of the free anthraquinone test solution to the chloroform used in each extraction is 1:0.8 to 1.5; exemplary ratios are 1:0.9, 1:1.1, 1:1.3, or 1:1.4, but are not limited thereto. A preferred ratio is 1:0.9 to 1.1.

[0149] The volume ratio of the free anthraquinone test solution to the first solvent is 1:0.7 to 1.1. Exemplary ratios are 1:0.8, 1:0.9, or 1:1.05, but are not limited thereto. A preferred ratio is 1:0.8 to 1.0.

[0150] During ultrasonic treatment, the ultrasonic power is 200W to 400W and the ultrasonic frequency is 30kHz to 50kHz, but not limited to these. Preferably, the ultrasonic power is 300W and the frequency is 40kHz.

[0151] The extraction time for heating and reflux extraction is 40 min to 80 min, preferably 50 min to 80 min, and more preferably 60 min.

[0152] The drying treatment of the free anthraquinone test solution and the extract obtained from chloroform extraction can be evaporation, reduced pressure recovery, or evaporation, but is not limited to these methods. Reduced pressure recovery of the solvent to dryness is preferred.

[0153] Specifically, in some embodiments of the present invention, the concentrations of the aloe-emodin reference standard, rhein reference standard, emodin reference standard, chrysophanol reference standard, and emodin methyl ether reference standard in the anthraquinone reference standard solution are 10 μg / mL to 50 μg / mL, and the concentrations of the magnolol reference standard and magnolol reference standard are 80 μg / mL to 120 μg / mL.

[0154] Furthermore, it should be noted that the anthraquinone reference solution in this invention can be a single reference solution or a mixed reference solution of two or more reference standards, preferably a mixed reference solution.

[0155] Specifically, in some embodiments of the present invention, the method for determining the content of naringin and neohesperidin includes:

[0156] The Sanhua Decoction or its preparations were extracted using a third extraction solvent to obtain a naringin test solution.

[0157] Naringin and neohesperidin reference standards were dissolved or extracted using a fourth extraction solvent to obtain naringin reference standard solution.

[0158] The naringin test solution and naringin reference solution were determined by liquid chromatography.

[0159] The liquid chromatograph uses an octadecylsilane-bonded silica gel column as the stationary phase, acetonitrile as mobile phase A, and a 0.05 vol%–0.3 vol% aqueous phosphoric acid solution as mobile phase B for gradient elution. The gradient elution curve is as follows:

[0160] From 0 min to 10 min, mobile phase A was 16% and mobile phase B was 84%.

[0161] From 10 to 18 minutes, mobile phase A decreased from 16% to 18%, and mobile phase B decreased from 84% to 82%.

[0162] From 18 min to 23 min, mobile phase A was 18% and mobile phase B was 82%.

[0163] Between 23 and 25 minutes, mobile phase A decreased from 18% to 20%, and mobile phase B decreased from 82% to 80%.

[0164] Between 25 and 40 minutes, mobile phase A decreased from 20% to 24%, and mobile phase B decreased from 80% to 76%.

[0165] The third extraction solvent is an aqueous ethanol solution with a concentration of 60 vol% to 90 vol%. For example, the concentration of the aqueous ethanol solution can be 65 vol%, 70 vol%, 75 vol%, 80 vol%, or 85 vol%, but is not limited thereto. Preferably, it is 60 vol% to 80 vol%.

[0166] The fourth extraction solvent is a methanol-water solution with a concentration of 70 vol% to 100 vol%. Exemplarily, the concentration of the methanol-water solution is 75 vol%, 80 vol%, 85 vol%, 90 vol%, or 95 vol%, but is not limited thereto. Preferably, it is 90 vol% to 100 vol%.

[0167] For example, the concentration of the phosphoric acid aqueous solution is 0.08 vol%, 0.12 vol%, 0.14 vol%, 0.18 vol%, 0.22 vol%, 0.26 vol%, or 0.28 vol%, but is not limited thereto. Preferably, it is 0.05 vol% to 0.2 vol%, more preferably 0.08 vol% to 0.12 vol%.

[0168] Preferably, in some embodiments of the present invention, the mobile phase B is a 0.1 vol% aqueous solution of phosphoric acid; and / or

[0169] The third extraction solvent is a 70 vol% aqueous ethanol solution; and / or

[0170] The fourth extraction solvent is methanol (i.e., 100 vol% methanol).

[0171] Specifically, in some embodiments of the present invention, the column length of the chromatographic column is 100 mm to 250 mm, exemplarily 100 mm, 150 mm, 200 mm, or 250 mm, but not limited thereto. The column diameter is 3 mm to 10 mm, exemplarily 3.9 mm, 4.6 mm, 5 mm, or 10 mm, but not limited thereto. The particle size of the stationary phase is 3 μm to 10 μm; exemplarily 3 μm, 5 μm, or 10 μm, but not limited thereto.

[0172] The column temperature of the chromatographic column is 28℃~32℃; exemplary values ​​are 28℃, 29℃, 30℃ or 31℃, but not limited thereto.

[0173] The flow rate of the liquid chromatograph is 0.8 mL / min to 1.2 mL / min, with exemplary values ​​of 0.85 mL / min, 0.92 mL / min, 1.05 mL / min, 1.13 mL / min or 1.18 mL / min, but is not limited thereto.

[0174] The detection wavelength of the liquid chromatograph is 250nm to 300nm; exemplary wavelengths are 265nm, 274nm, 288nm or 296nm, but not limited thereto.

[0175] The injection volume of the naringin test solution is 5 μL to 15 μL, with examples of 7 μL, 9 μL, 11 μL, 12 μL or 14 μL, but not limited thereto.

[0176] The injection volume of the naringin reference solution is 5 μL to 15 μL, with examples of 7 μL, 9 μL, 11 μL, 12 μL or 14 μL, but not limited to these.

[0177] Preferably, in some embodiments of the present invention, the chromatographic column has a length of 150 mm, a diameter of 4.6 mm, and a stationary phase particle size of 5 μm;

[0178] The column temperature of the chromatographic column is 30℃;

[0179] The flow rate of the liquid chromatograph is 1.0 mL / min, the detection wavelength is 280 nm, the injection volume of the naringin test solution is 10 μL, and the injection volume of the naringin reference solution is 10 μL.

[0180] Specifically, in some embodiments of the present invention, in the step of extracting Sanhua Decoction or its preparation with a third extraction solvent to obtain naringin test solution, Sanhua Decoction or its preparation is mixed with an ethanol aqueous solution with a concentration of 60 vol% to 90 vol% and ultrasonically treated for 15 min to 45 min.

[0181] The dosage form of Sanhua Decoction can be a decoction, powder, pill, tablet, etc., but is not limited to these. Preferably, the dosage form of Sanhua Decoction is a decoction, and the freeze-dried powder obtained by freeze-drying it is measured.

[0182] The ratio of Sanhua Decoction or its preparation to an aqueous ethanol solution is 0.1g-1g:30mL-90mL. Exemplary ratios include 0.13g:34mL, 0.17g:37mL, 0.19g:43mL, 0.2g:45mL, 0.27g:51mL, 0.39g:59mL, 0.43g:67mL, 0.55g:69mL, 0.63g:73mL, 0.77g:79mL, 0.89g:83mL, or 0.93g:89mL, but are not limited to these. Preferably, the ratio of Sanhua Decoction or its preparation to an aqueous methanol solution is 0.1g-0.3g:40mL-60mL.

[0183] The ultrasonic power is 200W to 400W, and the ultrasonic frequency is 30kHz to 50kHz, but not limited to these. Preferably, the ultrasonic power is 250W and the frequency is 40kHz.

[0184] Specifically, in some embodiments of the present invention, the concentration of the naringin reference standard in the naringin reference solution is 100 μg / mL to 150 μg / mL, and the concentration of the neohesperidin reference standard is 100 μg / mL to 150 μg / mL.

[0185] Furthermore, it should be noted that the naringin reference solution in this invention can be a single reference solution or a mixed reference solution of two reference standards, with a mixed reference solution being preferred.

[0186] Specifically, in some embodiments of the present invention, the method for determining the synephrine content includes:

[0187] The Sanhua Decoction or its preparations were extracted using the fifth extraction solvent to obtain a synephrine test solution;

[0188] Synephrine reference standard was dissolved or extracted using the sixth extraction solvent to obtain a synephrine reference standard solution;

[0189] The synephrine test solution and synephrine reference solution were determined by liquid chromatography; the chromatographic column of the liquid chromatograph used octadecylsilane-bonded silica gel as the stationary phase, methanol as mobile phase A, and water as mobile phase B for isocratic elution.

[0190] The mobile phase B comprises 0.05 vol% to 0.3 vol% phosphoric acid and 0.05 wt% to 0.3 wt% sodium dodecyl sulfonate. Exemplarily, the concentration of phosphoric acid in mobile phase B is 0.07 vol%, 0.11 vol%, 0.15 vol%, 0.19 vol%, 0.22 vol%, 0.25 vol%, or 0.28 vol%, but is not limited thereto; preferably, it is 0.05 vol% to 0.15 vol%. Exemplarily, the concentration of sodium dodecyl sulfonate in mobile phase B is 0.07 wt%, 0.11 wt%, 0.15 wt%, 0.19 wt%, 0.22 wt%, 0.25 wt%, or 0.28 wt%, but is not limited thereto; preferably, it is 0.05 wt% to 0.15 wt%.

[0191] For example, the volume ratio of mobile phase A to mobile phase B is 43:57, 45:55, 47:53, or 49:51, but is not limited thereto. Preferably, it is 42-48:52-58.

[0192] The fifth extraction solvent is a methanol-water solution with a concentration of 60 vol% to 90 vol%, exemplarily a methanol-water solution with a concentration of 65 vol%, 70 vol%, 80 vol%, or 85 vol%, but not limited thereto. Preferably, the fifth extraction solvent is a methanol-water solution with a concentration of 65 vol% to 75 vol%.

[0193] The sixth extraction solvent is a methanol-water solution with a concentration of 5 vol% to 40 vol%. Examples include, but are not limited to, methanol-water solutions with a concentration of 10 vol%, 20 vol%, 30 vol%, or 35 vol%. Preferably, the sixth extraction solvent is a methanol-water solution with a concentration of 5 vol% to 15 vol%.

[0194] Preferably, in some embodiments of the present invention, the volume ratio of mobile phase A to mobile phase B is 45:55; and / or

[0195] The mobile phase B comprises 0.1 vol% phosphoric acid and 0.1 wt% sodium dodecyl sulfate; and / or

[0196] The fifth extraction solvent is a 70 vol% methanol aqueous solution; and / or

[0197] The sixth extraction solvent is a 10 vol% methanol aqueous solution.

[0198] Specifically, in some embodiments of the present invention, the column length of the chromatographic column is 100 mm to 250 mm, exemplarily 100 mm, 150 mm, 200 mm, or 250 mm, but not limited thereto. The column diameter is 3 mm to 10 mm, exemplarily 3.9 mm, 4.6 mm, 5 mm, or 10 mm, but not limited thereto. The particle size of the stationary phase is 3 μm to 10 μm; exemplarily 3 μm, 5 μm, or 10 μm, but not limited thereto.

[0199] The column temperature of the chromatographic column is 28℃~32℃; exemplary values ​​are 29℃, 30℃ or 31℃, but not limited thereto.

[0200] The flow rate of the liquid chromatograph is 0.8 mL / min to 1.2 mL / min, with exemplary values ​​of 0.85 mL / min, 0.92 mL / min, 1.05 mL / min, 1.13 mL / min or 1.18 mL / min, but is not limited thereto.

[0201] The detection wavelength of the liquid chromatograph is 200nm to 250nm; exemplary wavelengths are 209nm, 220nm, 224nm, 235nm or 247nm, but not limited thereto.

[0202] The injection volume of the synephrine test solution is 5 μL to 15 μL, with examples of 7 μL, 9 μL, 11 μL, 12 μL or 14 μL, but not limited to these.

[0203] The injection volume of the synephrine reference solution is 5 μL to 15 μL, with examples of 7 μL, 9 μL, 11 μL, 12 μL or 14 μL, but not limited to these.

[0204] Preferably, in some embodiments of the present invention, the chromatographic column has a length of 150 mm, a diameter of 4.6 mm, and a stationary phase particle size of 5 μm;

[0205] The column temperature of the chromatographic column is 30℃;

[0206] The flow rate of the liquid chromatograph is 1.0 mL / min, the detection wavelength is 224 nm, the injection volume of the synephrine test solution is 10 μL, and the injection volume of the synephrine reference solution is 10 μL.

[0207] Specifically, in some embodiments of the present invention, in the step of extracting Sanhua Decoction or its preparation with a fifth extraction solvent to obtain a synephrine test solution, Sanhua Decoction or its preparation is mixed with a methanol aqueous solution with a concentration of 60 vol% to 90 vol% and ultrasonically treated for 15 min to 45 min.

[0208] The dosage form of Sanhua Decoction can be a decoction, powder, pill, tablet, etc., but is not limited to these. Preferably, the dosage form of Sanhua Decoction is a decoction, and the freeze-dried powder obtained by freeze-drying it is measured.

[0209] The ratio of Sanhua Decoction or its preparation to the methanol-water solution is 0.1g-1g:10mL-50mL. Exemplary ratios include 0.13g:15mL, 0.24g:23mL, 0.35g:29mL, 0.47g:35mL, 0.6g:39mL, 0.76g:43mL, 0.83g:45mL, or 0.92g:49mL, but are not limited thereto. Preferably, the ratio of Sanhua Decoction or its preparation to the methanol-water solution is 0.3g-0.6g:20mL-30mL.

[0210] The ultrasonic power is 200W to 400W, and the ultrasonic frequency is 30kHz to 50kHz, but not limited to these. Preferably, the ultrasonic power is 300W and the frequency is 40kHz.

[0211] Specifically, in some embodiments of the present invention, the method for determining the content of notopterygium alcohol includes:

[0212] The Sanhua Decoction or its preparations were extracted using the seventh extraction solvent to obtain the Qianghuo alcohol test solution;

[0213] The reference standard of notopterygium alcohol was dissolved or extracted using the eighth extraction solvent to obtain the reference standard solution of notopterygium alcohol;

[0214] The test solution and reference solution of notopterygium alcohol were determined by liquid chromatography.

[0215] The liquid chromatograph uses an octadecylsilane-bonded silica gel column as the stationary phase, methanol as mobile phase A, and a 0.1 vol%–0.4 vol% aqueous phosphoric acid solution as mobile phase B for gradient elution. The gradient elution curve is as follows:

[0216] From 0 min to 10 min, mobile phase A was 60% and mobile phase B was 40%.

[0217] Between 10 and 30 minutes, mobile phase A decreased from 60% to 72%, and mobile phase B decreased from 40% to 28%.

[0218] For example, the concentration of the phosphoric acid aqueous solution is 0.12 vol%, 0.17 vol%, 0.21 vol%, 0.28 vol%, 0.33 vol%, or 0.37 vol%, but is not limited thereto. Preferably, it is 0.1 vol% to 0.3 vol%.

[0219] The seventh and eighth extraction solvents are methanol-water solutions with a concentration of 60 vol% to 100 vol%. For example, the concentration of the methanol-water solution may be 65 vol%, 70 vol%, 75 vol%, 80 vol%, or 90 vol%, but is not limited thereto. Preferably, it is 70 vol% to 100 vol%.

[0220] Preferably, in some embodiments of the present invention, the mobile phase B is a 0.2 vol% aqueous solution of phosphoric acid; and / or

[0221] The seventh extraction solvent is a 70 vol% methanol aqueous solution; and / or

[0222] The eighth extraction solvent is methanol (i.e., 100 vol% methanol).

[0223] Specifically, in some embodiments of the present invention, the column length of the chromatographic column is 100 mm to 250 mm, exemplarily 100 mm, 150 mm, 200 mm, or 250 mm, but not limited thereto. The column diameter is 2.1 mm to 5.0 mm, exemplarily 2.1 mm, 3.9 mm, 4.6 mm, or 5 mm, but not limited thereto. The particle size of the stationary phase is 1.6 μm to 1.8 μm; exemplarily 1.6 μm, 1.7 μm, or 1.8 μm, but not limited thereto.

[0224] The column temperature of the chromatographic column is 28℃~32℃; exemplary values ​​are 28℃, 29℃, 30℃ or 31℃, but not limited thereto.

[0225] The flow rate of the liquid chromatograph is 0.23 mL / min to 0.27 mL / min, exemplarily 0.24 mL / min, 0.25 mL / min or 0.26 mL / min, but is not limited thereto.

[0226] The detection wavelength of the liquid chromatograph is 300nm to 350nm; exemplary wavelengths are 310nm, 320nm, 335nm, 340nm or 347nm, but not limited thereto.

[0227] The injection volume of the Qianghuo alcohol test solution is 1 μL to 5 μL, with examples being 1.5 μL, 2 μL, 2.5 μL, 3 μL or 4 μL, but not limited to these.

[0228] The injection volume of the notopterygium alcohol reference solution is 1 μL to 5 μL, with examples being 1.5 μL, 2 μL, 2.5 μL, 3 μL or 4 μL, but not limited to these.

[0229] Preferably, in some embodiments of the present invention, the chromatographic column has a length of 150 mm, a diameter of 2.1 mm, and a stationary phase particle size of 1.8 μm;

[0230] The column temperature of the chromatographic column is 30℃;

[0231] The flow rate of the liquid chromatograph is 0.25 mL / min, the detection wavelength is 310 nm, the injection volume of the notopterygium alcohol test solution is 2 μL, and the injection volume of the notopterygium alcohol reference solution is 2 μL.

[0232] Specifically, in some embodiments of the present invention, in the step of extracting Sanhua Decoction or its preparation using the seventh extraction solvent to obtain the Qianghuo alcohol test solution, Sanhua Decoction or its preparation is mixed with a methanol aqueous solution with a concentration of 60 vol% to 90 vol% and ultrasonically treated for 15 min to 45 min.

[0233] The dosage form of Sanhua Decoction can be a decoction, powder, pill, tablet, etc., but is not limited to these. Preferably, the dosage form of Sanhua Decoction is a decoction, and the freeze-dried powder obtained by freeze-drying it is measured.

[0234] The ultrasonic power is 200W to 400W, and the ultrasonic frequency is 30kHz to 50kHz, but not limited to these. Preferably, the ultrasonic power is 300W and the frequency is 40kHz.

[0235] The ratio of Sanhua Decoction or its preparation to the methanol-water solution is 0.1g-1g:10mL-50mL. Exemplary ratios include 0.13g:15mL, 0.24g:23mL, 0.35g:29mL, 0.47g:35mL, 0.6g:39mL, 0.76g:43mL, 0.83g:45mL, or 0.92g:49mL, but are not limited thereto. Preferably, the ratio of Sanhua Decoction or its preparation to the methanol-water solution is 0.3g-0.6g:20mL-30mL.

[0236] Specifically, in some embodiments of the invention, Sanhua Decoction comprises the following components in parts by weight: 30-32 parts rhubarb, 30-32 parts magnolia bark, 30-32 parts immature bitter orange, and 30-32 parts notopterygium root; preferably, Sanhua Decoction comprises the following components in parts by weight: 30.98 parts rhubarb, 30.98 parts magnolia bark, 30.98 parts immature bitter orange, and 30.98 parts notopterygium root. Specifically, when the dosage form of Sanhua Decoction is pills, tablets, etc., the Sanhua Decoction preparation also includes necessary excipients.

[0237] Specifically, in some embodiments of the present invention, the preparation method of Sanhua Decoction is as follows:

[0238] Take rhubarb, magnolia bark, immature bitter orange, and notopterygium root, crush them to 4-10 mesh, soak them in water for 10-40 minutes, bring to a boil over high heat, and simmer over low heat until the liquid is 40%-60% of the initial amount of water added.

[0239] The ratio of the total weight of rhubarb, magnolia bark, immature bitter orange, and notopterygium root to the weight of water used for soaking is 120-140:1600-2000.

[0240] Preferably, in some embodiments of the present invention, the preparation method of the Sanhua Decoction includes: taking rhubarb, magnolia bark, immature bitter orange, and notopterygium root, crushing them to 4-10 mesh, soaking them in water for 10-40 minutes, boiling them over high heat, simmering them over low heat until the decoction is 40%-60% of the initial water volume, filtering to obtain the Sanhua Decoction decoction, concentrating the Sanhua Decoction decoction and then freeze-drying it to obtain freeze-dried powder.

[0241] Implementing this invention has the following beneficial effects:

[0242] 1. In the method for establishing the material reference of Sanhua Decoction of the present invention, thin-layer chromatography is used to perform qualitative analysis on the reference sample of Sanhua Decoction. The analysis is accurate, rapid, highly sensitive, and low in cost, and all indicators can meet the needs of actual detection.

[0243] 2. In the method for establishing the material standard of Sanhua Decoction of the present invention, thin-layer identification and liquid chromatography are used to perform qualitative and quantitative analysis on the Sanhua Decoction standard sample. The analysis is accurate, rapid, highly sensitive and low cost, and all indicators can meet the needs of actual detection. Moreover, the two technical means complement each other and constitute a complete quality control method, which can more comprehensively and accurately characterize the key quality attributes of the material standard of Sanhua Decoction.

[0244] 3. This invention employs high-performance liquid chromatography (HPLC) with gradient elution to simultaneously determine the content of seven active ingredients in rhubarb and magnolia bark in Sanhua Decoction under the same chromatographic conditions. The established chromatographic method can effectively separate and determine the content of active ingredients such as aloe-emodin, rhein, emodin, chrysophanol, emodin methyl ether, magnolol, and honokiol in the reference sample. The analysis time is short, the chromatographic peak shape is good, the resolution is high, and the method is practical.

[0245] 4. This invention uses thin-layer chromatography to identify all medicinal materials in the formula. By optimizing the preparation method of the test sample and the developing system, a thin-layer identification method different from that of single medicinal materials in the Chinese Pharmacopoeia is established. It can identify Notopterygium incisum and Citrus aurantium using the same developing system and test sample preparation method. Furthermore, the introduction of reference medicinal materials and reference standards as references results in richer spot information and specificity. Attached Figure Description

[0246] Figure 1The images show thin-layer chromatograms (365 nm) of rhubarb at different spotting amounts in Example 1. 1–3 are rhubarb reference standards, with spotting amounts of 3 μL, 5 μL, and 10 μL, respectively; 4–6 are rhubarb reference medicinal materials, with spotting amounts of 3 μL, 5 μL, and 10 μL, respectively; and 7–9 are rhubarb test samples, with spotting amounts of 3 μL, 5 μL, and 10 μL, respectively.

[0247] Figure 2 These are thin-layer chromatograms (under sunlight) of rhubarb at different spotting amounts in Example 1. Among them, 1-3 are rhubarb reference standards, with spotting amounts of 3 μL, 5 μL and 10 μL, respectively; 4-6 are rhubarb reference medicinal materials, with spotting amounts of 3 μL, 5 μL and 10 μL, respectively; and 7-9 are rhubarb test samples, with spotting amounts of 3 μL, 5 μL and 10 μL, respectively.

[0248] Figure 3 The image shows the results of the rhubarb thin-layer chromatography specific detection (365nm) in Example 1, where 1 is a negative sample lacking rhubarb, 2 is a rhubarb reference standard, 3 is a rhubarb reference medicinal material, and 4-5 are rhubarb test samples.

[0249] Figure 4 The image shows the results of the rhubarb thin-layer chromatography specificity detection in Example 1 (under sunlight). In the image, 1 is a negative sample lacking rhubarb, 2 is a rhubarb reference standard, 3 is a rhubarb reference medicinal material, and 4-5 are rhubarb test samples.

[0250] Figure 5 The image shows a thin-layer chromatogram (365 nm) of rhubarb at 25°C in Example 1, where 1 is rhubarb reference standard, 2 is rhubarb reference medicinal material, and 3-5 are rhubarb test samples.

[0251] Figure 6 This is a thin-layer chromatogram of rhubarb at 25°C (under sunlight) in Example 1, where 1 is rhubarb reference standard, 2 is rhubarb reference medicinal material, and 3-5 are rhubarb test samples;

[0252] Figure 7 The image shows a thin-layer chromatogram (365 nm) of rhubarb at 4°C in Example 1, where 1 is rhubarb reference standard, 2 is rhubarb medicinal material, and 3-5 are rhubarb test samples.

[0253] Figure 8 This is a thin-layer chromatogram of rhubarb at 4°C (under sunlight) in Example 1, where 1 is rhubarb reference standard, 2 is rhubarb reference medicinal material, and 3-5 are rhubarb test samples;

[0254] Figure 9 The image shows a thin-layer chromatogram (365 nm) of rhubarb at a relative humidity of 31% in Example 1, where 1 is rhubarb reference standard, 2 is rhubarb reference medicinal material, and 3-5 are rhubarb test samples.

[0255] Figure 10 This is a thin-layer chromatogram of rhubarb (under sunlight) at a relative humidity of 31% in Example 1, where 1 is rhubarb reference standard, 2 is rhubarb reference medicinal material, and 3-5 are rhubarb test samples;

[0256] Figure 11 The image shows a thin-layer chromatogram (365 nm) of rhubarb at a relative humidity of 87% in Example 1, where 1 is rhubarb reference standard, 2 is rhubarb reference medicinal material, and 3-5 are rhubarb test samples.

[0257] Figure 12 This is a thin-layer chromatogram of rhubarb at a relative humidity of 87% (under sunlight) in Example 1, where 1 is rhubarb reference standard, 2 is rhubarb reference medicinal material, and 3-5 are rhubarb test samples;

[0258] Figure 13 The image shows a thin-layer chromatogram (365nm) of rhubarb obtained using Yantai Yinlong silica gel G plate in Example 1, where 1 is rhubarb reference standard, 2 is rhubarb reference medicinal material, and 3-5 are rhubarb test samples.

[0259] Figure 14 This is a thin-layer chromatogram of rhubarb (under sunlight) when using Yantai Yinlong silica gel G plate in Example 1. In this chromatogram, 1 is rhubarb reference standard, 2 is rhubarb reference medicinal material, and 3-5 are rhubarb test samples.

[0260] Figure 15 The image shows a thin-layer chromatogram (365 nm) of rhubarb obtained using Qingdao marine silica gel G plate in Example 1, where 1 is rhubarb reference standard, 2 is rhubarb reference medicinal material, and 3-5 are rhubarb test samples.

[0261] Figure 16 This is a thin-layer chromatogram (under sunlight) of rhubarb obtained using Qingdao marine silica gel G plate in Example 1, where 1 is rhubarb reference standard, 2 is rhubarb reference medicinal material, and 3-5 are rhubarb test samples;

[0262] Figure 17 The image shows a thin-layer chromatogram (365 nm) of rhubarb obtained using a spectrosilica gel G plate in Example 1, where 1 is rhubarb reference standard, 2 is rhubarb reference medicinal material, and 3-5 are rhubarb test samples.

[0263] Figure 18 This is a thin-layer chromatogram (under sunlight) of rhubarb obtained using a spectrosilica gel G plate in Example 1, where 1 is rhubarb reference standard, 2 is rhubarb raw material, and 3-5 are rhubarb test samples;

[0264] Figure 19 The image shows a thin-layer chromatogram (365 nm) of rhubarb when using Merck silica gel G plates in Example 1, where 1 is rhubarb reference standard, 2 is rhubarb reference medicinal material, and 3-5 are rhubarb test samples.

[0265] Figure 20 This is a thin-layer chromatogram (under sunlight) of rhubarb using Merck silica gel G plates in Example 1, where 1 is rhubarb reference standard, 2 is rhubarb raw material, and 3-5 are rhubarb test samples;

[0266] Figure 21 This refers to the thin-layer chromatography of rhubarb in multiple batches of Sanhua Decoction samples in Example 1. Figure 1 (365nm), where 1 is rhubarb reference standard, 2 is rhubarb reference medicinal material, and 3 to 10 are rhubarb thin-layer test samples from batches S1 to S8 respectively;

[0267] Figure 22 This refers to the thin-layer chromatography of rhubarb in multiple batches of Sanhua Decoction samples in Example 1. Figure 1 (Under sunlight), where 1 is rhubarb reference standard, 2 is rhubarb reference medicinal material, and 3 to 10 are rhubarb thin-layer test samples from batches S1 to S8 respectively;

[0268] Figure 23 This refers to the thin-layer chromatography of rhubarb in multiple batches of Sanhua Decoction samples in Example 1. Figure 2 (365nm), where 1 is rhubarb reference standard, 2 is rhubarb reference medicinal material, and 3 to 10 are rhubarb thin-layer test samples from batches S9 to S16 respectively;

[0269] Figure 24 This refers to the thin-layer chromatography of rhubarb in multiple batches of Sanhua Decoction samples in Example 1. Figure 2 (Under sunlight), where 1 is rhubarb reference standard, 2 is rhubarb reference medicinal material, and 3-10 are rhubarb thin-layer test samples from batches S9-S16 respectively;

[0270] Figure 25 These are thin-layer chromatograms of Magnolia officinalis under different spotting amounts in Example 1. Among them, 1 to 3 are Magnolia officinalis reference materials, with spotting amounts of 5 μL, 10 μL and 15 μL respectively, and 4 to 6 are Magnolia officinalis test samples, with spotting amounts of 5 μL, 10 μL and 15 μL respectively.

[0271] Figure 26 This is a thin-layer chromatography specific detection result diagram of Magnolia officinalis in Example 1, where 1 is the negative sample of Magnolia officinalis, 2 is the reference material of Magnolia officinalis, and 3-4 are the test samples of Magnolia officinalis.

[0272] Figure 27 This is a thin-layer chromatogram of Magnolia officinalis at 25°C in Example 1, where 1 is Magnolia officinalis reference material and 2-4 are Magnolia officinalis test samples;

[0273] Figure 28 This is a thin-layer chromatogram of Magnolia officinalis at 4°C in Example 1, where 1 is the reference material of Magnolia officinalis and 2-4 are the test samples of Magnolia officinalis.

[0274] Figure 29This is a thin-layer chromatogram of Magnolia officinalis at a relative humidity of 31% in Example 1, where 1 is Magnolia officinalis reference material and 2-4 are Magnolia officinalis test samples;

[0275] Figure 30 This is a thin-layer chromatogram of Magnolia officinalis at a relative humidity of 87% in Example 1, where 1 is Magnolia officinalis reference material and 2-4 are Magnolia officinalis test samples;

[0276] Figure 31 This is a thin-layer chromatogram of Magnolia officinalis when using Yantai Yinlong silica gel G plate in Example 1, where 1 is Magnolia officinalis reference material and 2-4 are Magnolia officinalis test samples.

[0277] Figure 32 This is a thin-layer chromatogram of Magnolia officinalis when using Qingdao marine silica gel G plate in Example 1, where 1 is Magnolia officinalis reference material and 2-4 are Magnolia officinalis test samples.

[0278] Figure 33 This is a thin-layer chromatogram of Magnolia officinalis when using a spectrosilica G plate in Example 1, where 1 is Magnolia officinalis reference material and 2-4 are Magnolia officinalis test samples.

[0279] Figure 34 This is a thin-layer chromatogram of Magnolia officinalis when Merck silica gel G plate was used in Example 1, where 1 is Magnolia officinalis reference material and 2-4 are Magnolia officinalis test samples.

[0280] Figure 35 This refers to the thin-layer chromatography of Magnolia officinalis in multiple batches of Sanhua Decoction samples in Example 1. Figure 1 Among them, 1 is the reference material of Magnolia officinalis, and 2 to 9 are the thin-layer test samples of Magnolia officinalis from batches S1 to S8, respectively;

[0281] Figure 36 This refers to the thin-layer chromatography of Magnolia officinalis in multiple batches of Sanhua Decoction samples in Example 1. Figure 2 Among them, 1 is the reference material of Magnolia officinalis, and 2 to 9 are the thin-layer test samples of Magnolia officinalis from batches S9 to S16 respectively;

[0282] Figure 37 These are thin-layer chromatograms of Citrus aurantium and Notopterygium incisum at different spotting volumes in Example 1. 1-3 are Citrus aurantium reference standards, with spotting volumes of 1 μL, 2 μL, and 3 μL, respectively; 4-6 are Notopterygium incisum reference materials, with spotting volumes of 1 μL, 2 μL, and 3 μL, respectively; 7-9 are Citrus aurantium reference materials, with spotting volumes of 1 μL, 2 μL, and 3 μL, respectively; and 10-12 are Citrus aurantium test samples, with spotting volumes of 1 μL, 2 μL, and 3 μL, respectively.

[0283] Figure 38 The results of thin-layer chromatography specific detection of Citrus aurantium and Notopterygium incisum in Example 1 are shown in the figure. Among them, 1 is a negative sample of Notopterygium incisum, 2 is a negative sample of Citrus aurantium, 3 is a reference standard of Citrus aurantium, 4 is a reference medicinal material of Notopterygium incisum, 5 is a reference medicinal material of Citrus aurantium, and 6 is a test sample of Citrus aurantium.

[0284] Figure 39 The following is a thin-layer chromatogram of Citrus aurantium and Notopterygium incisum at 25°C in Example 1, wherein 1 is Citrus aurantium reference standard, 2 is Notopterygium incisum reference material, 3 is Citrus aurantium reference material, and 4-5 are Citrus aurantium test sample.

[0285] Figure 40 The following is a thin-layer chromatogram of Citrus aurantium and Notopterygium incisum at 4°C in Example 1, wherein 1 is Citrus aurantium reference standard, 2 is Notopterygium incisum reference material, 3 is Citrus aurantium reference material, and 4-5 are Citrus aurantium test sample.

[0286] Figure 41 The image shows the thin-layer chromatograms of Citrus aurantium and Notopterygium incisum at a relative humidity of 31% in Example 1. In the image, 1 is Citrus aurantium reference standard, 2 is Notopterygium incisum reference material, 3 is Citrus aurantium reference material, and 4-5 are Citrus aurantium test sample.

[0287] Figure 42 The image shows the thin-layer chromatograms of Citrus aurantium and Notopterygium incisum at a relative humidity of 87% in Example 1. In the image, 1 is Citrus aurantium reference standard, 2 is Notopterygium incisum reference material, 3 is Citrus aurantium reference material, and 4-5 are Citrus aurantium test sample.

[0288] Figure 43 The following is a thin-layer chromatogram of Citrus aurantium and Notopterygium incisum when using Yantai Yinlong silica gel G plate in Example 1. In this chromatogram, 1 is Citrus aurantium reference standard, 2 is Notopterygium incisum reference material, 3 is Citrus aurantium reference material, and 4-5 are Citrus aurantium test sample.

[0289] Figure 44 This is a thin-layer chromatogram of Citrus aurantium and Notopterygium incisum when Qingdao marine silica gel G plate was used in Example 1. In this chromatogram, 1 is Citrus aurantium reference standard, 2 is Notopterygium incisum reference material, 3 is Citrus aurantium reference material, and 4-5 are Citrus aurantium test sample.

[0290] Figure 45 This is a thin-layer chromatogram of Citrus aurantium and Notopterygium incisum when using a spectrosilica gel G plate in Example 1. In this chromatogram, 1 is Citrus aurantium reference standard, 2 is Notopterygium incisum reference material, 3 is Citrus aurantium reference material, and 4-5 are Citrus aurantium test sample.

[0291] Figure 46 This is a thin-layer chromatogram of Citrus aurantium and Notopterygium incisum when Merck silica gel G plate was used in Example 1. In this chromatogram, 1 is Citrus aurantium reference standard, 2 is Notopterygium incisum reference material, 3 is Citrus aurantium reference material, and 4-5 are Citrus aurantium test sample.

[0292] Figure 47 Thin-layer chromatography of Fructus Aurantii Immaturus and Radix Notopterygii in multiple batches of Sanhua Decoction samples in Example 1 Figure 1 Among them, 1 is the reference standard of Citrus aurantium, 2 is the reference medicinal material of Notopterygium incisum, 3 is the reference medicinal material of Citrus aurantium, and 4 to 11 are the thin-layer test samples of Citrus aurantium from batches S1 to S8 respectively;

[0293] Figure 48Thin-layer chromatography of Fructus Aurantii Immaturus and Radix Notopterygii in multiple batches of Sanhua Decoction samples in Example 1 Figure 2 Among them, 1 is the reference standard of Citrus aurantium, 2 is the reference medicinal material of Notopterygium incisum, 3 is the reference medicinal material of Citrus aurantium, and 4 to 11 are the thin-layer test samples of Citrus aurantium from batches S9 to S16 respectively;

[0294] Figure 49 Figure 2 shows the results of the total anthraquinone specificity investigation in Sanhua Decoction in Example 2; where peak 1 is aloe-emodin peak, peak 2 is rhein peak, peak 3 is emodin peak, peak 4 is chrysophanol peak, and peak 5 is emodin methyl ether peak.

[0295] Figure 50 This is a graph showing the results of the investigation on the specificity of free anthraquinones in the Sanhua Decoction in Example 2; among them, peak 1 is the aloe-emodin peak, peak 2 is the rhein peak, peak 3 is the emodin peak, peak 4 is the chrysophanol peak, and peak 5 is the emodin methyl ether peak.

[0296] Figure 51 This is a graph showing the results of the specificity investigation of magnolol and honokiol in the Sanhua decoction in Example 2; where peak 1 is the honokiol peak and peak 2 is the honokiol peak.

[0297] Figure 52 This is the chromatogram of the specificity investigation of naringin and neohesperidin in the Sanhua decoction in Example 2; wherein, peak 1 is the naringin peak and peak 2 is the neohesperidin peak;

[0298] Figure 53 This is the chromatogram of the specificity investigation of synephrine in Example 2; where peak 1 is the synephrine peak;

[0299] Figure 54 This is the chromatogram of the specificity investigation of notopterygol in Example 2; wherein, peak 1 is notopterygol;

[0300] Figure 55 These are the fingerprint spectra of Sanhua Decoction when different detection wavelengths are used in Example 3;

[0301] Figure 56 This is the fingerprint spectrum of the Sanhua decoction when different combinations of mobile phases are used in Example 3;

[0302] Figure 57 This is the fingerprint spectrum of Sanhua Decoction when using mobile phase B with different concentrations in Example 3;

[0303] Figure 58 This is the fingerprint spectrum of the Sanhua decoction when gradient 1 is used in Example 3;

[0304] Figure 59 This is the fingerprint spectrum of the Sanhua decoction when gradient 2 is used in Example 3;

[0305] Figure 60 This is the fingerprint spectrum of the Sanhua decoction when gradient 3 is used in Example 3;

[0306] Figure 61 This is the fingerprint spectrum of the Sanhua Decoction when gradient 4 is used in Example 3;

[0307] Figure 62 These are the fingerprint spectra of the Sanhua Decoction when different flow rates were used in Example 3;

[0308] Figure 63 This is the fingerprint spectrum of the Sanhua Decoction when using different column temperatures in Example 3;

[0309] Figure 64 This is the specificity test result of the fingerprint spectrum of Sanhua Decoction in Example 3; among them, peak 1 is aloe-emodin-8-O-glucoside, peak 2 is naringin, peak 3 is neohesperidin, peak 4 is rhein-1-O-β-D-glucoside, peak 5 is rhein-8-O-β-D-glucoside, peak 7 is rhein-8-O-β-D-glucoside, peak 8 is aloe-emodin, peak 9 is norihesperidin, peak 10 is rhein, peak 11 is honokiol, peak 13 is honokiol, peak 14 is rhein, peak 15 is rhein, and peak 16 is rhein methyl ether.

[0310] Figure 65 It is a superimposed fingerprint spectrum of 16 batches of Sanhua Decoction in Example 3;

[0311] Figure 66 This is the reference fingerprint spectrum of Sanhua Decoction; among them, peak 1 is aloe-emodin-8-O-glucoside, peak 2 is naringin, peak 3 is neohesperidin, peak 4 is rhein-1-O-β-D-glucoside, peak 5 is rhein-8-O-β-D-glucoside, peak 7 is rhein-8-O-β-D-glucoside, peak 8 is aloe-emodin, peak 9 is norihesperidin, peak 10 is rhein, peak 11 is honokiol, peak 13 is honokiol, peak 14 is rhein, peak 15 is rhein, and peak 16 is rhein methyl ether. Detailed Implementation

[0312] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0313] Based on detailed textual research of ancient books and documents, this invention determines the formula and preparation method of Sanhua Decoction as follows: Take appropriate amounts of rhubarb, magnolia bark, immature bitter orange, and notopterygium root slices, crush them into coarse particles the size of hemp seeds (passing through a 4-mesh sieve but not exceeding 10 mesh), weigh 30.98g of each, place them in a 3L electric ceramic decoction pot, add 1800mL of water, soak for 30 minutes, cover and decoct over high heat (500W) until about 900mL of liquid remains, discard the dregs; filter the decoction through a 350-mesh sieve while hot to obtain the Sanhua Decoction decoction. Transfer the decoction to a 2000mL round-bottom flask, concentrate it to 300mL under reduced pressure at 65℃ using a rotary evaporator, and then transfer it to a vacuum freeze dryer to dry, thus obtaining the freeze-dried powder of the Sanhua Decoction reference sample.

[0314] The freeze-dried powder of the Sanhuatang reference sample prepared by the above method was used in all the following embodiments of the present invention.

[0315] To comprehensively reflect the quality information of Sanhua Decoction and achieve comprehensive and effective control of Sanhua Decoction product quality, this invention provides a method for establishing a material standard for Sanhua Decoction, which is described in detail below: Example 1: Thin-layer chromatography identification method for Sanhua Decoction

[0316] I. Thin-layer chromatographic identification method of rhubarb in Sanhua Decoction

[0317] 1.1 Identification Methods

[0318] (1) Preparation of reference solution: Take 0.2g of rhubarb reference material, add 100mL of water, heat to boiling for 45 minutes, filter, evaporate the filtrate to dryness, add 20mL of a 30% ethanol-hydrochloric acid (10:1) mixed solution to dissolve, heat under reflux in a water bath for 1 hour, cool immediately, extract twice with 20mL of chloroform each time, combine the chloroform extracts, evaporate to dryness, add 2mL of anhydrous ethanol-ethyl acetate (2:1) mixed solution to dissolve the residue, filter, and use as the rhubarb reference material solution. Take rhein reference standard, add methanol to prepare a solution containing 0.5mg per 1mL, and use as the rhubarb reference standard solution.

[0319] (2) Preparation of test solution: Take the freeze-dried powder of Sanhuatang reference sample, grind it into a fine powder, take about 0.3g, add 20mL of 30% ethanol-hydrochloric acid (10:1) mixed solution to dissolve it, heat it in a water bath under reflux for 1 hour, cool it immediately, extract it twice with chloroform, 20mL each time, combine the chloroform liquids, evaporate to dryness, add 2mL of anhydrous ethanol-ethyl acetate (2:1) mixed solution to dissolve the residue, filter it, and use it as the rhubarb test solution.

[0320] (3) Take 5 μL each of the rhubarb test solution, rhubarb reference material solution, and rhubarb reference standard solution and spot them separately on the same silica gel G thin-layer plate. Use n-hexane-ethyl acetate-formic acid (30:10:0.5) solution as the developing solvent, develop, remove, air dry, and examine under ultraviolet light (365 nm) or develop color in ammonia vapor and examine under sunlight. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatograms of the rhubarb reference material and the rhein reference standard; after being exposed to ammonia vapor, the spots turn red.

[0321] 1.2 Methodological Validation

[0322] 1.2.1 Comparison of different sampling amounts

[0323] Rhubarb test solution, rhubarb reference solution, and rhubarb reference herb solution were spotted separately onto the same silica gel G thin-layer plate (Yinlong silica gel G plate). The plate was developed using n-hexane-ethyl acetate-formic acid (30:10:0.5) as the developing solvent. The plates were then removed, dried, and examined under ultraviolet light (365 nm). Color development was then performed in ammonia vapor and examined under sunlight. The experimental results are shown below. Figures 1-2 .

[0324] Depend on Figures 1-2 It is evident that when the sample volumes of rhubarb test solution, rhubarb reference solution, and rhubarb reference herb solution are 3 μL and 5 μL, the main spot in the chromatogram of the test sample is clear at the corresponding positions as in the chromatograms of the reference solution and the reference herb, with good separation, no tailing, and no background interference. The clarity of the main spot is even higher when the sample volume is 5 μL. Therefore, the optimal sample volumes for the rhubarb identification method of this invention are 5 μL for the rhubarb test solution, 5 μL for the rhubarb reference solution, and 5 μL for the rhubarb reference herb solution.

[0325] 1.2.2 Specificity

[0326] Prepare a rhubarb-deficient negative sample solution according to the preparation method of the rhubarb test sample solution. Spot 5 μL of the rhubarb test sample solution, 5 μL of the rhubarb-deficient negative sample solution, 5 μL of the rhubarb reference solution, and 5 μL of the rhubarb reference herb solution onto the same silica gel G thin-layer plate (Yinlong silica gel G plate). Develop the plate using n-hexane-ethyl acetate-formic acid (30:10:0.5) as the developing solvent. Remove the plate, air dry, and examine under ultraviolet light (365 nm). Develop the color in ammonia vapor and examine under sunlight. Experimental results are shown below. Figures 3-4 As can be seen from the figure, the rhubarb thin-layer chromatography identification method of the present invention has no negative interference and good method specificity.

[0327] 1.2.3 Durability assessment:

[0328] (1) Comparison of different temperatures

[0329] 5 μL of rhubarb test solution, 5 μL of rhubarb reference solution, and 5 μL of rhubarb reference herb solution were spotted onto the same silica gel G thin-layer plate (Yinlong silica gel G plate). Hexane-ethyl acetate-formic acid (30:10:0.5) was used as the developing solvent. The plates were developed at room temperature (25℃) and low temperature (4℃), respectively. After development, the plates were removed, air-dried, and examined under ultraviolet light (365 nm). Color development was also performed in ammonia vapor and examined under sunlight. The experimental results are shown below. Figures 5-8 .

[0330] Depend on Figures 5-8 It is evident that the separation effect is good under both room temperature and low temperature conditions, and the chromatograms of the Sanhua Decoction reference sample show the same main spot color at the corresponding positions as the chromatograms of the reference substance and the reference medicinal material. The experimental results indicate that temperature has little effect on the thin-layer identification of rhubarb in Sanhua Decoction, suggesting that this thin-layer identification method is robust to different temperatures.

[0331] (2) Comparison of different humidity levels

[0332] 5 μL of rhubarb test solution, 5 μL of rhubarb reference solution, and 5 μL of rhubarb reference herb solution were spotted onto the same silica gel G thin-layer plate (Yinlong silica gel G plate). Hexane-ethyl acetate-formic acid (30:10:0.5) was used as the developing solvent. The plates were developed under low humidity (T: 25℃, RH: 31%) and high humidity (T: 25℃, RH: 87%) conditions, respectively. The plates were then removed, air-dried, and examined under ultraviolet light (365 nm), developed in ammonia vapor, and examined under sunlight. The experimental results are shown below. Figures 9-12 .

[0333] Depend on Figures 9-12 It is evident that the separation effect is good under both low and high humidity conditions, and the chromatogram of the Sanhua Decoction reference sample shows the same main spot of the same color at the corresponding positions as the chromatogram of the reference substance and the reference medicinal material. The experimental results indicate that humidity has little effect on the thin-layer identification of rhubarb in Sanhua Decoction, suggesting that this thin-layer identification method is robust to different humidity levels.

[0334] (3) Comparison of thin-layer boards from different manufacturers

[0335] 5 μL of rhubarb test solution, 5 μL of rhubarb reference solution, and 5 μL of rhubarb reference herb solution were spotted onto silica gel G thin-layer plates from different manufacturers (Yinlong silica gel G plate, Haiyang silica gel G plate, Spectro silica gel G plate, and Merck silica gel G plate). Hexane-ethyl acetate-formic acid (30:10:0.5) was used as the developing solvent, and the plates were developed under the same temperature and humidity conditions. The plates were then removed, air-dried, and examined under ultraviolet light (365 nm), developed in ammonia vapor, and examined under sunlight. The experimental results are shown in […]. Figures 13-20 .

[0336] The results showed that different manufacturers' silicone G thin-layer plates (Yinlong silicone G plate, Haiyang silicone G plate, Puke silicone G plate, and Merck silicone G plate) had no significant effect on the thin-layer identification of rhubarb in Sanhua Decoction, indicating that the thin-layer identification method has good durability with silicone G thin-layer plates from different manufacturers.

[0337] 1.3 Identification of different batches of samples

[0338] Sixteen batches of freeze-dried powder of Sanhua Decoction reference samples were taken and identified according to the above identification method. The results are as follows. Figures 21-24 As shown in the figure, the chromatograms of the 16 batches of Sanhua Decoction reference samples, rhubarb reference material, and rhubarb reference standard all show fluorescent spots of the same color at the corresponding positions; and after being exposed to ammonia vapor, the spots turn red.

[0339] II. Thin-layer chromatographic identification method for Magnolia officinalis in Sanhua Decoction

[0340] 2.1 Identification Methods

[0341] (1) Preparation of reference solution: Take 1.0g of Magnolia officinalis reference material, add 100mL of water, heat to boiling for 45 minutes, filter, evaporate the filtrate to about 10mL, extract with chloroform twice by shaking, 20mL each time, combine the chloroform extracts, evaporate to dryness, add 1mL of methanol to dissolve the residue, and use it as Magnolia officinalis reference material solution.

[0342] (2) Preparation of test solution: Take the freeze-dried powder of Sanhuatang reference sample, grind it finely, take about 1.0g, add 20mL of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 10mL of water to dissolve the residue, extract with chloroform twice by shaking, 20mL each time, combine the chloroform extracts, evaporate to dryness, add 1mL of methanol to dissolve the residue, and use it as the test solution of Magnolia officinalis.

[0343] (3) Take 10 μL each of the Magnolia officinalis test solution and the Magnolia officinalis reference solution and spot them separately on the same silica gel G thin-layer plate. Develop the plate using petroleum ether (60-90℃)-ethyl acetate (3:1) solution as the developing solvent. Remove the plate, air dry it, spray it with 5% vanillin-sulfuric acid solution, and heat it at 105℃ until the spots are clearly visible. Examine the plate under sunlight. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the Magnolia officinalis reference solution.

[0344] 2.2 Methodological Validation

[0345] 2.2.1 Comparison of different sampling amounts

[0346] The test solution and the reference solution of Magnolia officinalis were spotted separately onto the same silica gel G thin-layer plate (Yinlong silica gel G plate). Petroleum ether (60–90℃)-ethyl acetate (3:1) was used as the developing solvent. After development, the plate was removed, dried, sprayed with 5% vanillin-sulfuric acid solution, and heated at 105℃ until the spots were clearly visible. The results were then examined under sunlight. The experimental results are shown below. Figure 25 .

[0347] Depend on Figure 25 It is evident that when the sample volume of the Magnolia officinalis test solution is 5 μL and 10 μL, and the sample volume of the Magnolia officinalis reference material solution is 5 μL and 10 μL, the main spot in the chromatogram of the test sample is clear at the corresponding position as that of the reference material, with good separation, no tailing phenomenon, and no background interference. Especially when the sample volume is 10 μL, the clarity of the main spot is even higher. Therefore, the optimal sample volume for the identification method of Magnolia officinalis in this invention is 10 μL for both the Magnolia officinalis test solution and the Magnolia officinalis reference material solution.

[0348] 2.2.2 Specificity

[0349] A negative sample solution for Magnolia officinalis was prepared according to the method for preparing the test sample solution. 10 μL of the test sample solution, 10 μL of the negative sample solution, and 10 μL of the reference herb solution were spotted onto the same silica gel G thin-layer plate (Yinlong silica gel G plate). The plate was developed using petroleum ether (60–90℃)-ethyl acetate (3:1) as the developing solvent. The plate was then removed, dried, sprayed with 5% vanillin-sulfuric acid solution, and heated at 105℃ until the spots were clearly visible. The results were examined under sunlight. The experimental results are shown below. Figure 26 As can be seen from the figure, the thin-layer chromatography identification method for Magnolia officinalis of the present invention exhibits no negative interference and good method specificity.

[0350] 2.2.3 Durability assessment:

[0351] (1) Comparison of different temperatures

[0352] 10 μL of Magnolia officinalis test solution and 10 μL of Magnolia officinalis reference solution were spotted onto the same silica gel G thin-layer plate (Yinlong silica gel G plate). Petroleum ether (60–90℃)-ethyl acetate (3:1) was used as the developing solvent. The plates were developed at room temperature (25℃) and low temperature (4℃), respectively. After development, the plates were removed, air-dried, sprayed with 5% vanillin-sulfuric acid solution, and heated at 105℃ until the spots were clearly visible. The results were then examined under sunlight. The experimental results are shown below. Figures 27-28 .

[0353] Depend on Figures 27-28It is evident that the separation effect is good under both room temperature and low temperature conditions, and the chromatograms of the Sanhua Decoction reference sample show the same main spot color at the corresponding positions as the control herb. The experimental results indicate that temperature has little effect on the thin-layer identification of Magnolia officinalis in Sanhua Decoction, suggesting that this thin-layer identification method is robust to different temperatures.

[0354] (2) Comparison of different humidity levels

[0355] 10 μL of Magnolia officinalis test solution and 10 μL of Magnolia officinalis reference solution were spotted onto the same silica gel G thin-layer plate (Yinlong silica gel G plate). Petroleum ether (60–90℃)-ethyl acetate (3:1) was used as the developing solvent. The plates were developed under low humidity (T: 25℃, RH: 31%) and high humidity (T: 25℃, RH: 87%) conditions, respectively. The plates were then removed, air-dried, sprayed with 5% vanillin-sulfuric acid solution, and heated at 105℃ until the spots were clearly visible. The results were examined under sunlight. The experimental results are shown below. Figures 29-30 .

[0356] Depend on Figures 29-30 It is evident that the separation effect is good under both low and high humidity conditions, and the chromatogram of the Sanhua Decoction reference sample shows the same main spot of the same color at the corresponding position as the control herb. The experimental results indicate that humidity has little effect on the thin-layer identification of Magnolia officinalis in Sanhua Decoction, suggesting that this thin-layer identification method is robust to different humidity levels.

[0357] (3) Comparison of thin-layer boards from different manufacturers

[0358] 10 μL of Magnolia officinalis test solution and 10 μL of Magnolia officinalis reference solution were spotted onto silica gel G thin-layer plates from different manufacturers (Yinlong silica gel G plate, Haiyang silica gel G plate, Puke silica gel G plate, and Merck silica gel G plate). Petroleum ether (60–90℃)-ethyl acetate (3:1) was used as the developing solvent, and the plates were developed under the same temperature and humidity conditions. After development, the plates were removed, air-dried, sprayed with 5% vanillin-sulfuric acid solution, and heated at 105℃ until the spots were clearly visible. The results were then examined under sunlight. The experimental results are shown in the table below. Figures 31-34 .

[0359] The results showed that different manufacturers' silicone G thin-layer plates (Yinlong silicone G plate, Haiyang silicone G plate, Puke silicone G plate, Merck silicone G plate) had no significant effect on the thin-layer identification of Magnolia officinalis in Sanhua Decoction, indicating that the thin-layer identification method has good durability with silicone G thin-layer plates from different manufacturers.

[0360] 2.3 Identification of different batches of samples

[0361] Sixteen batches of Sanhuatang freeze-dried powder were taken and identified according to the above identification method. The results are as follows. Figures 35-36 As shown in the figure, the chromatograms of the 16 batches of Sanhua Decoction reference samples and the Magnolia officinalis reference material all show fluorescent spots of the same color at the corresponding positions.

[0362] III. Thin-layer chromatographic identification method for Fructus Aurantii Immaturus and Radix Notopterygii in Sanhua Decoction

[0363] 3.1 Identification Methods

[0364] (1) Preparation of reference solutions: Take 0.3g of Citrus aurantium reference material and 1.0g of Notopterygium incisum reference material, add 100mL of water respectively, heat to boiling for 45 minutes, filter, evaporate to dryness, add 25mL of methanol respectively, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 1mL of methanol to dissolve the residue respectively, and use them as Citrus aurantium reference material solutions and Notopterygium incisum reference material solutions. Take appropriate amounts of purslane, naringin, and neonaringin reference standards, add methanol to prepare Citrus aurantium reference standard solutions containing 0.2mg of each per 1mL.

[0365] (2) Preparation of test solution: Take the freeze-dried powder of Sanhuatang reference sample, grind it finely, take an appropriate amount of this product, grind it finely, take about 0.5g, add 25mL of methanol, sonicate for 30 minutes, filter, evaporate to dryness, add 1mL of methanol to dissolve the residue, and use it as the test solution of Citrus aurantium.

[0366] (3) Take 2 μL each of the *Fructus Aurantii Immaturus* test solution, *Fructus Aurantii Immaturus* reference material solution, *Notopterygium incisum* reference material solution, and *Fructus Aurantii Immaturus* reference standard solution, and spot them separately on the same silica gel G thin-layer plate. Use the lower layer of chloroform-methanol-water (13:7:2) solution as the developing solvent, develop, remove, air dry, spray with 5% aluminum trichloride ethanol solution, and heat at 105℃ until the spots are clearly visible. Examine under ultraviolet light (365 nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatograms of the *Fructus Aurantii Immaturus* reference material and the *Fructus Aurantii Immaturus* reference standard.

[0367] 3.2 Methodological Validation

[0368] 3.2.1 Comparison of different sampling amounts

[0369] The solutions of Citrus aurantium (test sample), Citrus aurantium (reference material), Notopterygium incisum (reference material), and Citrus aurantium (reference standard) were spotted separately onto the same silica gel G thin-layer plate (Yinlong silica gel G plate). The lower layer of a chloroform-methanol-water (13:7:2) solution was used as the developing solvent. After development, the plate was removed, dried, and sprayed with 5% aluminum trichloride ethanol solution. The plate was then heated at 105℃ until the spots were clearly visible and examined under a UV lamp (365nm). The experimental results are shown below. Figure 37 .

[0370] Depend on Figure 37It is evident that when the sample volume of the *Citrus aurantium* test solution is 1 μL, 2 μL, or 3 μL, the sample volume of the *Citrus aurantium* reference material solution is 1 μL, 2 μL, or 3 μL, the sample volume of the *Notopterygium incisum* reference material solution is 1 μL, 2 μL, or 3 μL, and the sample volume of the *Citrus aurantium* reference standard solution is 1 μL, 2 μL, or 3 μL, the main spot in the chromatogram of the test sample is clear at the corresponding position as the chromatograms of the reference materials and the reference standard, with good separation, no tailing phenomenon, and no background interference. Especially when the sample volume is 2 μL, the clarity of the main spot is even higher. Therefore, the optimal sample volume for identifying *Citrus aurantium* and *Notopterygium incisum* in this invention is 2 μL for each of the following: *Citrus aurantium* test solution, *Citrus aurantium* reference material solution, *Notopterygium incisum* reference material solution, and *Citrus aurantium* reference standard solution.

[0371] 3.2.2 Specificity

[0372] Negative samples of Citrus aurantium and Notopterygium incisum were prepared according to the same method as for the Citrus aurantium test sample solution. Two μL each of the following solutions were spotted onto the same silica gel G thin-layer plate (Yinlong silica gel G plate). The plate was developed using a chloroform-methanol-water (13:7:2) lower layer solution as the developing solvent. The plates were then removed, dried, sprayed with 5% aluminum trichloride ethanol solution, and heated at 105℃ until the spots were clearly visible. The results were examined under a UV lamp (365 nm). The experimental results are shown in the table below. Figure 38 As can be seen from the figure, the thin-layer chromatography identification method for Citrus aurantium and Notopterygium incisum of the present invention has no negative interference and good method specificity.

[0373] 3.2.3 Durability assessment:

[0374] (1) Comparison of different temperatures

[0375] Two μL each of the following solutions were spotted onto the same silica gel G thin-layer plate (Yinlong silica gel G plate): 2 μL of the test solution of Citrus aurantium, 2 μL of the reference herb solution of Citrus aurantium, 2 μL of the reference herb solution of Notopterygium incisum, and 2 μL of the reference standard solution of Citrus aurantium. The plates were developed using a chloroform-methanol-water (13:7:2) solution as the developing solvent at room temperature (25℃) and low temperature (4℃), respectively. The plates were then removed, dried, sprayed with 5% aluminum trichloride ethanol solution, and heated at 105℃ until the spots were clearly visible. The spots were then examined under a UV lamp (365nm). The experimental results are shown below. Figures 39-40 .

[0376] Depend on Figures 39-40It is evident that the separation effect is good under both room temperature and low temperature conditions, and the chromatograms of the Sanhua Decoction reference sample show the same main spot color at the corresponding positions as the chromatograms of the reference medicinal materials and the reference substance. The experimental results indicate that temperature has little effect on the thin-layer identification of Citrus aurantium and Notopterygium in Sanhua Decoction, suggesting that this thin-layer identification method is robust to different temperatures.

[0377] (2) Comparison of different humidity levels

[0378] Two μL each of the following solutions were spotted onto the same silica gel G thin-layer plate (Yinlong silica gel G plate): 2 μL of the test solution of Citrus aurantium, 2 μL of the reference herb solution of Citrus aurantium, 2 μL of the reference herb solution of Notopterygium incisum, and 2 μL of the reference standard solution of Citrus aurantium. The plates were developed using a chloroform-methanol-water (13:7:2) lower layer solution as the developing solvent under both low humidity (T: 25℃, RH: 31%) and high humidity (T: 25℃, RH: 87%) conditions. The plates were then removed, air-dried, sprayed with 5% aluminum trichloride ethanol solution, and heated at 105℃ until the spots were clearly visible. The spots were then examined under a UV lamp (365nm). The experimental results are shown below. Figures 41-42 .

[0379] Depend on Figures 41-42 It is evident that the separation effect is good under both low and high humidity conditions, and the chromatogram of the Sanhua Decoction reference sample shows the same main spot of the same color at the corresponding positions as the chromatograms of the reference medicinal materials and the reference substance. The experimental results indicate that humidity has little effect on the thin-layer identification of Citrus aurantium and Notopterygium in Sanhua Decoction, suggesting that this thin-layer identification method is robust to different humidity levels.

[0380] (3) Comparison of thin-layer boards from different manufacturers

[0381] 2 μL of *Citrus aurantium* test solution, 2 μL of *Citrus aurantium* reference material solution, 2 μL of *Notopterygium incisum* reference material solution, and 2 μL of *Citrus aurantium* reference standard solution were spotted onto silica gel G thin-layer plates from different manufacturers (Yinlong silica gel G plate, Haiyang silica gel G plate, Spectro silica gel G plate, and Merck silica gel G plate). The lower layer of a chloroform-methanol-water (13:7:2) solution was used as the developing solvent. The plates were developed under the same temperature and humidity conditions. After development, the plates were removed, air-dried, sprayed with 5% aluminum trichloride ethanol solution, and heated at 105℃ until the spots were clearly visible. The spots were then examined under a UV lamp (365 nm). The experimental results are shown below. Figures 43-46 .

[0382] The results showed that different manufacturers' silicone G thin-layer plates (Yinlong silicone G plate, Haiyang silicone G plate, Puke silicone G plate, Merck silicone G plate) had no significant effect on the thin-layer identification of Citrus aurantium and Notopterygium in Sanhua Decoction, indicating that the thin-layer identification method has good durability with silicone G thin-layer plates from different manufacturers.

[0383] 3.3 Identification of different batches of samples

[0384] Sixteen batches of freeze-dried powder of Sanhua Decoction reference samples were taken and identified according to the above identification method. The results are as follows. Figures 47-48 As shown in the figure, the chromatograms of the 16 batches of Sanhua Decoction reference samples, the chromatograms of Citrus aurantium reference materials, Notopterygium incisum reference materials, and Citrus aurantium reference standard all show fluorescent spots of the same color at the corresponding positions.

[0385] Example 2: Method for determining the content of Sanhua Decoction in reference samples

[0386] I. Determination of free anthraquinones, total anthraquinones, magnolol, and honokiol content in Sanhua Decoction

[0387] 1.1 Determination Method

[0388] 1.1.1 Chromatographic conditions

[0389] The column was packed with octadecylsilane-bonded silica gel (100 mm column length, 2.1 mm inner diameter, 1.6 μm particle size); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B. Gradient elution was performed according to the specifications in Table 1, with a flow rate of 0.32 mL / min; the column temperature was 28 °C; and the detection wavelength was 254 nm. The theoretical plate number, calculated based on the aloe-emodin peak, should be no less than 10,000.

[0390] Table 1. Gradient elution method for determining the content of free anthraquinones, total anthraquinones, magnolol, and honokiol.

[0391]

[0392] 1.1.2 Preparation of reference solution

[0393] Take appropriate amounts of aloe-emodin reference standard, rhein reference standard, emodin reference standard, chrysophanol reference standard, emodin methyl ether reference standard, magnolol reference standard, and honokiol reference standard, accurately weigh them, and add methanol to prepare mixed solutions containing 20 μg of aloe-emodin, emodin, emodin methyl ether, and chrysophanol per 1 mL, and 90 μg of magnolol and honokiol, to obtain anthraquinone reference standard solutions.

[0394] 1.1.3 Preparation of the test solution

[0395] Take the freeze-dried powder of the Sanhua Decoction reference sample, grind it into a fine powder, take about 0.4g, weigh it accurately, place it in a stoppered conical flask, add 25mL of methanol accurately, stopper tightly, weigh it, sonicate for 30min, cool it, weigh it again, make up the weight loss with methanol, shake well, filter it, and use it as a free anthraquinone test solution for the determination of free anthraquinone, magnolol and honokiol content. Accurately measure 10 mL of the filtrate and place it in a flask. Evaporate the solvent, add 10 mL of 8% hydrochloric acid solution, sonicate for 2 minutes, then add 10 mL of chloroform, heat under reflux for 1 hour, cool, place in a separatory funnel, wash the container with a small amount of chloroform and add it to the separatory funnel, separate the chloroform layer, extract the acid solution with chloroform 4 times, 10 mL each time, combine the chloroform solutions, recover the solvent under reduced pressure to dryness, dissolve the residue in methanol, transfer to a 10 mL volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate as the anthraquinone test solution for the determination of total anthraquinone content.

[0396] 1.1.4 Determination Method

[0397] Accurately pipette 1 μL each of the anthraquinone reference solution, free anthraquinone test solution, and total anthraquinone test solution into the liquid chromatograph and determine the results.

[0398] 1.2 Methodological Validation

[0399] 1.2.1 Specificity Examination

[0400] Take a rhubarb negative sample and prepare a rhubarb-total anthraquinone negative solution according to the preparation method of the total anthraquinone test solution. Accurately weigh appropriate amounts of aloe-emodin reference standards, rhein reference standards, emodin reference standards, chrysophanol reference standards, and emodin methyl ether reference standards. Add methanol to prepare mixed solutions containing 20 μg of each of the following: aloe-emodin, rhein reference standards, emodin, emodin methyl ether, and chrysophanol per 1 mL. Inject 1 μL each of the total anthraquinone test solution, the rhubarb mixed standard solution, the blank solvent, and the rhubarb-total anthraquinone negative solution into the liquid chromatograph for determination. The results are shown in the figure. Figure 49 As can be seen from the figure, no chromatographic peaks were observed at the corresponding retention times of the negative sample and the rhubarb mixed standard reference, indicating that other components in Sanhua Decoction did not interfere with the total anthraquinones.

[0401] Take a rhubarb negative sample and prepare a rhubarb-free anthraquinone negative solution according to the preparation method of the free anthraquinone test solution. Inject 1 μL each of the free anthraquinone test solution, rhubarb mixed standard solution, blank solvent, and rhubarb-free anthraquinone negative solution into the liquid chromatograph for determination. The results are shown in the figure. Figure 50 As can be seen from the figure, no chromatographic peaks were observed at the corresponding retention times of the negative sample and the rhubarb mixed standard reference, indicating that other components in Sanhua Decoction do not interfere with free anthraquinones.

[0402] Rhubarb, Magnolia officinalis double-negative samples, Magnolia officinalis negative sample, Notopterygium incisum slices, Rhubarb slices, and Citrus aurantium samples were taken separately and prepared according to the preparation method of free anthraquinone test solution to obtain rhubarb and Magnolia officinalis double-negative solution, Magnolia officinalis negative solution, Notopterygium incisum solution, Rhubarb solution, and Citrus aurantium solution. Appropriate amounts of magnolol reference standard and honokiol reference standard were accurately weighed and added to methanol to prepare mixed solutions containing 90 μg each of magnolol and honokiol, respectively, to obtain the Magnolia officinalis mixed standard reference solution. 1 μL of blank solvent from each of the free anthraquinone test solution, rhubarb and Magnolia officinalis double-negative solution, Magnolia officinalis negative solution, Magnolia officinalis mixed standard reference solution, Notopterygium incisum solution, Rhubarb solution, and Citrus aurantium solution were injected into the liquid chromatograph for determination. The results are shown in the figure. Figure 51 As can be seen from the figure, the negative sample showed interference from impurities at the corresponding retention times of the mixed standard of Magnolia officinalis and the magnolol reference standard. Compared with the single herbs of Citrus aurantium and Notopterygium incisum, the impurities originated from Notopterygium incisum. Through the determination of multiple batches of samples, the content of impurities accounted for less than 5% of the content limit of magnolol, which is within the acceptable range. This indicates that the interference of other components in Sanhua Decoction on Magnolia officinalis components does not affect the accuracy of the final result. The determination method of this invention has good specificity.

[0403] 1.2.2 Examination of Linear Relationships

[0404] (1) Linear relationship between total anthraquinones and free anthraquinones

[0405] Accurately weigh 2.005 mg of aloe-emodin reference standard, 5.089 mg of rhein reference standard, 4.158 mg of emodin reference standard, 4.414 mg of chrysophanol reference standard, and 0.807 mg of emodin methyl ether reference standard. Place them in a 20 mL volumetric flask and add methanol to prepare a solution containing 97.744 μg of aloe-emodin, 252.669 μg of rhein, 199.584 μg of emodin, 219.376 μg of chrysophanol, and 40.0 μg of emodin methyl ether per mL. A 27 μg mixed reference solution was used as the rhubarb mixed linear stock solution. This stock solution was diluted 2, 5, 10, 20, 50, and 100 times to obtain seven different concentrations of reference solutions. These solutions yielded aloe-emodin reference solutions containing 48.872 μg / mL, 19.549 μg / mL, 9.774 μg / mL, 4.887 μg / mL, 1.955 μg / mL, and 0.977 μg / mL per mL, respectively. Each mL contained 126.334 μg / mL of aloe-emodin. Rhein reference solutions containing 50.534 μg / mL, 25.267 μg / mL, 12.633 μg / mL, 5.053 μg / mL, and 2.527 μg / mL per mL; and emodin reference solutions containing 99.792 μg / mL, 39.917 μg / mL, 19.958 μg / mL, 9.979 μg / mL, 3.992 μg / mL, and 1.996 μg / mL per mL per mL; and 10 μg / mL of emodin reference solutions per mL per mL. Rhein reference solutions with concentrations of 9.688 μg / mL, 43.875 μg / mL, 21.938 μg / mL, 10.969 μg / mL, 4.388 μg / mL, and 2.194 μg / mL, and rhein methyl ether reference solutions with concentrations of 20.027 μg / mL, 8.011 μg / mL, 4.006 μg / mL, 2.003 μg / mL, 0.801 μg / mL, and 0.401 μg / mL, respectively, per mL.

[0406] Accurately pipette 1 μL of the above-mentioned reference solutions of different concentrations and determine them under the established chromatographic conditions. Plot the peak area as the ordinate (y) and the amount of reference standard as the abscissa (x), and calculate the linear regression equation. The results are as follows:

[0407] The regression equation for aloe-emodin is y = 15.887x - 1.591, with a correlation coefficient r = 0.9999, indicating that aloe-emodin has a good linear relationship between injection quality and peak area within the injection concentration range of 0.977 to 97.744 μg / mL.

[0408] The regression equation for rhein is y = 13.610x - 0.946, with a correlation coefficient r = 0.9999, indicating that the injection mass and peak area of ​​rhein have a good linear relationship within the injection concentration range of 2.527 to 252.669 μg / mL.

[0409] The regression equation for emodin is y = 11.995x - 4.308, with a correlation coefficient r = 0.9999, indicating that the injection mass and peak area of ​​emodin have a good linear relationship within the injection concentration range of 1.996 to 199.584 μg / mL.

[0410] The regression equation for rhein is y = 16.063x - 4.928, with a correlation coefficient r = 0.9999, indicating that the injection mass and peak area of ​​rhein have a good linear relationship within the injection concentration range of 2.194 to 219.376 μg / mL.

[0411] The regression equation for emodin methyl ether is y = 11.481x - 1.102, with a correlation coefficient r = 0.9999, indicating that the injection mass and peak area of ​​emodin methyl ether have a good linear relationship within the injection concentration range of 0.401 to 40.027 μg / mL.

[0412] (2) Linear relationship between magnolol and honokiol

[0413] Accurately weigh 2.534 mg of magnolol reference standard and 1.971 mg of honokiol reference standard, place them together in a 20 mL volumetric flask, add methanol to prepare a mixed reference solution containing 125.433 μg of honokiol and 98.353 μg of honokiol per mL, and use it as the mixed linear mother liquor. Pipette 4 mL, 3 mL, 2 mL, 1 mL, 0.1 mL, and 0.05 mL of the mother liquor into 5 mL volumetric flasks, respectively, and add methanol to the mark to obtain seven reference solutions of different concentrations. Prepare magnolol reference solutions containing 100.346 μg / mL, 75.260 μg / mL, 50.173 μg / mL, 25.087 μg / mL, 2.509 μg / mL, and 1.254 μg / mL per mL, and magnolol reference solutions containing 78.682 μg / mL, 59.012 μg / mL, 39.341 μg / mL, 19.671 μg / mL, 1.967 μg / mL, and 0.984 μg / mL per mL.

[0414] Accurately pipette 1 μL of the above-mentioned reference solutions of different concentrations and determine them under the established chromatographic conditions. Plot the peak area as the ordinate (y) and the amount of reference standard as the abscissa (x), and calculate the linear regression equation. The results are as follows:

[0415] The regression equation for magnolol was y = 3.473x + 2.837, with a correlation coefficient r = 0.9999, indicating a good linear relationship between injection mass and peak area for magnolol within the injection concentration range of 1.254–125.433 μg / mL. The regression equation for glutaraldehyde was y = 7.419x + 4.420, with a correlation coefficient r = 0.9999, indicating a good linear relationship between injection mass and peak area for glutaraldehyde within the injection concentration range of 0.984–98.353 μg / mL.

[0416] 1.2.3 Precision Examination

[0417] (1) Instrument precision

[0418] Accurately pipette 1 μL of the anthraquinone reference standard mixed solution and inject it six times under the determined chromatographic conditions. Analyze the results, calculating the RSD values ​​based on the peak areas of aloe-emodin, rhein, emodin, chrysophanol, emodin methyl ether, and magnolol and honokiol. The results show that with six consecutive injections of the same reference solution, the RSD values ​​of all peak areas were less than 3.0%, indicating good instrument precision.

[0419] (2) Intermediate precision

[0420] Different researchers were selected to conduct the tests at different times and using different high-performance liquid chromatographs. An appropriate amount of lyophilized powder of the Sanhuatang reference sample was taken, finely ground, and approximately 0.4 g was accurately weighed. Six parallel samples were prepared, and free anthraquinone test solutions and total anthraquinone test solutions were prepared separately. 1 μL of each of the anthraquinone reference solution, free anthraquinone test solution, and total anthraquinone test solution was accurately pipetted and analyzed. The contents of free anthraquinone, total anthraquinone, magnolol, and honokiol, as well as the RSD values, were calculated and compared with the results of the repeatability test.

[0421] The results showed that when the same batch of samples was operated by different personnel at different times on different instruments, and the determination was repeated 6 times, the RSD value of free anthraquinone content was 1.13%, and the RSD value of the 6 repeatability test data was 3.12%; the RSD value of total anthraquinone content was 2.34%, and the RSD value of the 6 repeatability test data was 1.98%; the RSD value of magnolol content was 1.29%, and the RSD value of the 6 repeatability test data was 2.04%; the RSD value of magnolol content was 1.48%, and the RSD value of the 6 repeatability test data was 2.20%. According to the "Guiding Principles for Validation of Analytical Methods for Drug Quality Standards" in the 2020 edition of the Chinese Pharmacopoeia, when the content of the analyte in the sample is between 0.01% and 0.1%, the intermediate precision RSD limit is <6%. Therefore, the intermediate precision of this method is good when different analysts operate on different dates and on different chromatographs.

[0422] 1.2.4 Stability Assessment

[0423] Accurately pipette 1 μL each of the free anthraquinone test solution and the total anthraquinone test solution, and inject them at 0, 3, 6, 9, 16, and 24 hours, respectively. Measure the peak areas of aloe-emodin, rhein, emodin, chrysophanol, emodin methyl ether, honokiol, and magnolol in the test solutions, and calculate the peak area RSD values. The results show that the peak area RSD values ​​of all indicators are less than 3.0%, indicating that the test solutions have good stability within 24 hours.

[0424] 1.2.5 Repeatability Test

[0425] Take an appropriate amount of freeze-dried powder of the Sanhua Decoction reference sample, grind it into a fine powder, take about 0.4g, accurately weigh it, and prepare 6 parallel portions. Prepare free anthraquinone test solution and total anthraquinone test solution respectively. Accurately pipette 1μL of each of the anthraquinone reference solution, free anthraquinone test solution, and total anthraquinone test solution, test and analyze, and calculate the content of free anthraquinone, total anthraquinone, magnolol, and honokiol, as well as the RSD value.

[0426] The results showed that when the same batch of samples was repeatedly measured 6 times, the RSD values ​​of the contents of free anthraquinone, total anthraquinone, magnolol, and honokiol were all less than 4%. According to the "Guidelines for Validation of Analytical Methods" in the 2020 edition of the Chinese Pharmacopoeia, when the content of the analyte in the sample is between 0.01% and 0.1%, the repeatability RSD limit is <4.0%, indicating that the repeatability of this analytical method is good.

[0427] 1.2.6 Accuracy Examination

[0428] Nine lyophilized samples of the Sanhua Decoction reference sample with known content were accurately weighed. Single-standard solutions of each reference standard were added at ratios of 1:0.5, 1:1, and 1:1.5 between the total anthraquinone test solution, free anthraquinone test solution, and reference standard. Three replicates of each ratio were prepared to generate nine test solutions of total anthraquinone and free anthraquinone. The solutions were then analyzed under the determined chromatographic conditions. The average recovery rate and RSD of each indicator were calculated. According to the "Guidelines for Analytical Method Validation" in the 2020 edition of the Chinese Pharmacopoeia, the recovery limit is 85%–110% when the content of the analyte in the sample is between 0.01% and 0.1%. The recoveries of all the above indicators were within this range, and the RSDs were all less than 3%, indicating that the accuracy of this assay method is good.

[0429] 1.2.7 Durability Assessment

[0430] (1) Investigation of different chromatographic columns

[0431] Three types of chromatographic columns were selected: Waters Cortecs T3 column (2.1 mm × 100 mm, 1.6 μm), ACEEXCEL Super C18 column (2.1 mm × 100 mm, 1.7 μm), and Agilent SB C18 column (2.1 mm × 100 mm, 1.8 μm).

[0432] Take 1 μL each of the free anthraquinone test solution and the total anthraquinone test solution, and determine them using the three chromatographic columns mentioned above. Calculate the contents of free anthraquinone, total anthraquinone, magnolol, and honokiol, as well as their RSD values. The results show that the separation effect of each chromatographic column is good, and the RSD value is ≤5%, indicating that the analytical method has good robustness under different chromatographic columns.

[0433] (2) Investigation at different column temperatures

[0434] The column temperatures were set to 26℃, 28℃, and 30℃ respectively to investigate the effect of column temperature on the determination of free anthraquinones, total anthraquinones, magnolol, and honokiol content in the Sanhuatang reference sample.

[0435] Take 1 μL each of the free anthraquinone test solution and the total anthraquinone test solution, and determine them using the column temperatures mentioned above. Calculate the contents of free anthraquinone, total anthraquinone, magnolol, and honokiol, as well as the RSD values. The results show that the RSD values ​​of free anthraquinone, total anthraquinone, magnolol, and honokiol in the Sanhua Tang reference samples at different column temperatures are all less than 5%, indicating that the analytical method has good robustness within a column temperature range of ±2℃.

[0436] (3) Investigation of different flow velocities

[0437] The flow rates were set to 0.30 mL / min, 0.32 mL / min, and 0.34 mL / min, respectively, to investigate the effect of flow rate on the determination of free anthraquinones, total anthraquinones, magnolol, and honokiol content in the Sanhua decoction reference sample.

[0438] Take 1 μL each of the free anthraquinone test solution and the total anthraquinone test solution, and determine them at the above flow rates. Calculate the contents of free anthraquinone, total anthraquinone, magnolol, and honokiol, as well as the RSD values. The results show that the RSD values ​​of free anthraquinone, total anthraquinone, magnolol, and honokiol in the Sanhua Tang reference samples at different flow rates are all less than 5%, indicating that the analytical method has good robustness in the range of 0.32 ± 0.02 mL / min.

[0439] 1.3 Determination of the content of different batches of Sanhua Decoction reference samples

[0440] Sixteen batches of Sanhuatang reference samples were lyophilized, and free anthraquinone test solutions and total anthraquinone test solutions were prepared respectively. The contents of free anthraquinone, total anthraquinone, magnolol, and honokiol in the 16 batches of Sanhuatang reference samples were determined. The experimental results are shown in Table 2.

[0441] Table 2. Results of determination of free anthraquinones, total anthraquinones, magnolol, and honokiol content in the reference sample of Sanhua Decoction.

[0442]

[0443] Based on the above test results, the content of free anthraquinones in the 16 batches of Sanhua decoction fluctuated between 0.109% and 0.380%, with an average of 0.294% and a SD value of 0.09%. The range calculated based on ±3SD of the average content was 0.034% to 0.554%. The content of total anthraquinones in the 16 batches of Sanhua decoction fluctuated between 0.405% and 0.763%, with an average of 0.523% and a SD value of 0.11%. The range calculated based on ±3SD of the average content was 0.190% to 0.855%. The content of magnolol in the 16 batches of Sanhua decoction fluctuated between 0.029% and 0.240%, with an average of 0.105% and a SD value of 0.07%. The range calculated based on ±3SD of the average content was -0.099% to 0.309%, with the minimum value being negative, which does not conform to actual production. The range of magnolol content determined by -SD to +3SD of the mean is reasonable, and should be 0.037% to 0.309%. The magnolol content in 16 batches of Sanhua Decoction fluctuated between 0.054% and 0.204%, with a mean of 0.108% and an SD of 0.04%. The range calculated using ±3SD of the mean content is -0.016% to 0.231%. Similarly, the range of magnolol content determined by -2SD to +3SD of the mean should be 0.025% to 0.231%. Therefore, it can be determined that the free anthraquinone content in Sanhua Decoction should be controlled at 0.034% to 0.554%, the total anthraquinone content at 0.190% to 0.855%, the magnolol content at 0.037% to 0.309%, and the magnolol content at 0.025% to 0.231%.

[0444] II. Determination of Naringin and Neohesperidin Content in Sanhua Decoction

[0445] 2.1 Determination Method

[0446] 2.1.1 Chromatographic conditions

[0447] The column was packed with octadecylsilane-bonded silica gel (150 mm column length, 4.6 mm inner diameter, 5 μm particle size); acetonitrile mobile phase A and 0.1% phosphoric acid aqueous solution mobile phase B were used for gradient elution according to the specifications in Table 3; the column temperature was 30 °C; the flow rate was 1.0 mL per minute; and the detection wavelength was 280 nm. The theoretical plate number, calculated based on the naringin peak, should be no less than 3000.

[0448] Table 3 Gradient elution table for the determination of naringin and neohesperidin content.

[0449]

[0450] 2.1.2 Preparation of reference solution

[0451] Take appropriate amounts of naringin reference standard and neonaringin reference standard, accurately weigh them, and add methanol to prepare solutions containing 120 μg each of naringin and neohesperidin per 1 mL, thus obtaining the naringin reference standard solution.

[0452] 2.1.3 Preparation of the test solution

[0453] Take an appropriate amount of lyophilized powder of the Sanhua Decoction reference sample, grind it into a fine powder, take about 0.2g, weigh it accurately, place it in a stoppered conical flask, accurately add 50mL of 70% ethanol, weigh it, sonicate it (power 250W, frequency 40kHz) for 30 minutes, take it out and let it cool, weigh it again, make up the lost weight with 70% ethanol, shake it well, filter it, and take the filtrate to obtain the naringin test solution.

[0454] 2.1.4 Determination Method

[0455] Accurately pipette 10 μL each of the naringin reference solution and the naringin test solution into the liquid chromatograph and determine the result.

[0456] 2.2 Methodological Validation

[0457] 2.2.1 Specificity Examination

[0458] Take a negative sample of Citrus aurantium and prepare a negative solution for Citrus aurantium deficiency according to the preparation method of the naringin test solution. Inject 10 μL each of the naringin test solution, the negative sample solution for Citrus aurantium deficiency, and the naringin reference solution into the liquid chromatograph, and determine the chromatograms according to the determined chromatographic conditions. See the results below. Figure 52 The negative sample solution showed no chromatographic peaks at the corresponding retention times of the reference standard, indicating that other components in the formulation did not interfere with naringin and neohesperidin, demonstrating the good specificity of this method.

[0459] 2.2.2 Examination of Linear Relationships

[0460] Accurately weigh 22.478 mg of naringin reference standard and 22.255 mg of neonaringin reference standard, place them in a 25 mL volumetric flask, and add methanol to prepare a reference solution containing 824.483 μg of naringin and 884.859 μg of neohesperidin per mL. Use this as the mixed linear mother liquor. Transfer 3 mL of the mother liquor to a 5 mL volumetric flask, add methanol to the mark, and serially dilute according to the above steps to obtain five different concentrations of reference solutions, preparing a solution containing 494 μg of naringin per mL. The single-standard reference solutions of naringin were 0.690 μg / mL, 296.814 μg / mL, 178.088 μg / mL, 106.853 μg / mL, and 64.112 μg / mL, respectively. The single-standard reference solutions of neohesperidin contained 530.915 μg / mL, 318.549 μg / mL, 191.130 μg / mL, 114.678 μg / mL, and 68.807 μg / mL per mL.

[0461] Accurately pipette 1 μL of the above-mentioned reference solutions of different concentrations and determine them under the established chromatographic conditions. Plot the peak area as the ordinate (y) and the amount of reference standard as the abscissa (x), and calculate the linear regression equation. The results are as follows:

[0462] The regression equation for naringin was y = 0.1811x - 2.7661, with a correlation coefficient r = 0.9993, indicating that the injection mass and peak area of ​​naringin showed a good linear relationship within the injection concentration range of 64.112–824.876 μg / mL.

[0463] The regression equation for neohesperidin is y = 0.2023x + 2.8161, with a correlation coefficient r = 0.9995, indicating that neohesperidin has a good linear relationship between injection mass and peak area within the injection concentration range of 68.807–884.859 μg / mL.

[0464] 2.2.3 Precision Examination

[0465] (1) Instrument precision

[0466] Accurately pipette 10 μL of naringin reference solution and inject it six times under the determined chromatographic conditions. Analyze the results and calculate the RSD values ​​based on the peak areas of naringin and neohesperidin. The results showed that with six consecutive injections of the same reference solution, the RSD values ​​of all peak areas were less than 3.0%, indicating good instrument precision.

[0467] (2) Intermediate precision

[0468] Different researchers were selected to conduct the tests at different times and using different high-performance liquid chromatographs. An appropriate amount of lyophilized Sanhuatang reference sample powder was taken, finely ground, and approximately 0.2g was accurately weighed. Six parallel samples were prepared to prepare naringin test solution. 10μL of each of the naringin reference solution and naringin test solution were accurately pipetted and tested. The contents of naringin and neohesperidin and the RSD value were calculated and compared with the results of the repeatability test.

[0469] The results showed that when the same batch of samples was measured six times by different personnel on different instruments, the RSD value of naringin content was 1.18%, compared to an RSD value of 2.87% for the six data points in the repeatability test; the RSD value of neohesperidin content was 1.72%, compared to an RSD value of 1.49% for the six data points in the repeatability test. According to the "Guiding Principles for Validation of Analytical Methods for Drug Quality Standards" in the 2020 edition of the Chinese Pharmacopoeia, when the content of the analyte in the sample is 1%, the intermediate precision RSD limit is <4%. Therefore, the intermediate precision of this method is good even when different analysts operate on different dates and on different chromatographs.

[0470] 2.2.4 Stability Assessment

[0471] Accurately pipette 10 μL of the naringin test solution and inject it at 0, 3, 6, 9, 15, 21, and 24 hours. Measure the peak areas of naringin and neohesperidin in the test solution and calculate the peak area RSD value. The results show that the peak area RSD values ​​of both naringin and neohesperidin are less than 3.0%, indicating that the naringin test solution has good stability within 24 hours.

[0472] 2.2.5 Repeatability Test

[0473] An appropriate amount of lyophilized powder of the Sanhua Decoction reference sample was finely ground. Approximately 0.2 g was accurately weighed and prepared in six parallel assays. A naringin test solution was prepared, and 10 μL of the naringin reference solution was accurately pipetted for testing and analysis. The contents of naringin and neohesperidin, as well as their RSD values, were calculated. The results showed that in six repeated assays of the same batch of samples, the RSD values ​​of naringin and neohesperidin were all less than 2%. According to the "Guidelines for Validation of Analytical Methods" in the 2020 edition of the Chinese Pharmacopoeia, when the content of the analyte in the sample is 1%, the repeatability RSD limit is <2.0%, indicating that the analytical method has good repeatability.

[0474] 2.2.6 Accuracy Examination

[0475] Nine lyophilized samples of the Sanhua Decoction reference sample with known content were accurately weighed. The reference solution was added to the sample at ratios of naringin test solution to reference solution of 1:0.5, 1:1, and 1:1.5, with three replicates for each ratio, to prepare nine test solutions of Citrus aurantium. The solutions were then analyzed under the chromatographic conditions described in section 2.1, and the average recovery rate and RSD of each indicator were calculated. According to the "Guidelines for Validation of Analytical Methods" in the 2020 edition of the Chinese Pharmacopoeia, the recovery limit is 92%–105% when the content of the analyte in the sample is 1%. The recoveries of all the above indicators were within this range, and the RSDs were all less than 3%, indicating that the analytical method has good accuracy.

[0476] 2.2.7 Durability Test

[0477] (1) Investigation of different chromatographic columns

[0478] Three types of chromatographic columns were selected: Waters Xselect HSS T3 column (4.6 mm × 150 mm, 5 μm), Waters Xbridge C18 column (4.6 mm × 150 mm, 5 μm), and Agilent ZORBAX SB C18 column (4.6 mm × 150 mm, 5 μm).

[0479] Take 10 μL each of the naringin test solution and the naringin reference solution, and determine them using the three chromatographic columns mentioned above. Calculate the content and RSD values ​​of naringin and neohesperidin. The results show that the separation effect of each chromatographic column is good, and the RSD value is ≤3%, indicating that the analytical method has good robustness under different chromatographic columns.

[0480] (2) Investigation at different column temperatures

[0481] The column temperatures were set at 28℃, 30℃, and 32℃ respectively to investigate the effect of column temperature on the determination of naringin and neohesperidin content in the Sanhuatang reference sample.

[0482] Take 10 μL each of the naringin test solution and the naringin reference solution, and determine them using the column temperatures mentioned above. Calculate the contents of free anthraquinones, total anthraquinones, magnolol, and honokiol, as well as the RSD values. The results show that the RSD values ​​of naringin and neohesperidin in the Sanhuatang reference samples at different column temperatures are all less than 3%, indicating that the analytical method has good robustness within the range of 30±2℃.

[0483] (3) Investigation of different flow velocities

[0484] The flow rates were set to 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min, respectively, to investigate the effect of flow rate on the determination of naringin and neohesperidin content in the Sanhua decoction reference sample.

[0485] Take 10 μL each of the naringin test solution and the naringin reference solution, and determine them at the above flow rates. Calculate the content and RSD values ​​of naringin and neohesperidin. The results show that the RSD values ​​of naringin and neohesperidin in the Sanhua Tang reference samples at different flow rates are all less than 3%, indicating that the analytical method has good robustness in the range of 1.0 ± 0.2 mL / min.

[0486] 2.3 Determination of the content of different batches of Sanhua Decoction reference samples

[0487] Sixteen batches of Sanhuatang reference samples were freeze-dried to prepare naringin test solution, and the contents of naringin and neohesperidin in the sixteen batches of Sanhuatang reference samples were determined. The experimental results are shown in Table 4.

[0488] Table 4. Results of determination of naringin and neohesperidin content in the reference sample of Sanhua Decoction.

[0489]

[0490]

[0491] Based on the above test results, the content of naringin in 16 batches of Sanhua Decoction fluctuated from 5.431% to 9.413%, with an average of 6.817% and a SD value of 1.22%. The range calculated based on the mean content ±3SD was 3.167% to 10.466%. The content of neohesperidin in 16 batches of Sanhua Decoction fluctuated from 6.499% to 11.295%, with an average of 8.856% and a SD value of 1.53%. The range calculated based on the mean content ±3SD was 4.276% to 13.436%. Therefore, it can be determined that the content of naringin in Sanhua Decoction should be controlled at 3.167% to 10.466%, and the content of neohesperidin should be controlled at 4.276% to 13.436%.

[0492] III. Determination of Synephrine Content in Sanhua Decoction

[0493] 3.1 Determination Method

[0494] 3.1.1 Chromatographic conditions

[0495] The column was packed with octadecylsilane-bonded silica gel (150 mm column length, 4.6 mm inner diameter, 5 μm particle size); the mobile phase was methanol-water (containing 0.1% phosphoric acid and 0.1% sodium dodecyl sulfonate) (45:55); the column temperature was 30 °C; the flow rate was 1.0 mL / min; and the detection wavelength was 224 nm. The theoretical plate number, calculated based on the synephrine peak, should be no less than 3000.

[0496] 3.1.2 Preparation of reference solution

[0497] Weigh an appropriate amount of synephrine reference standard and add 10% methanol to prepare a reference standard solution containing 20 μg of synephrine reference standard per 1 mL.

[0498] 3.1.3 Preparation of the test solution

[0499] Take an appropriate amount of lyophilized powder of Sanhua Decoction reference sample, grind it into a fine powder, take about 0.4g, weigh it accurately, place it in a stoppered conical flask, accurately add 25mL of 70% methanol, weigh it, sonicate it (power 250W, frequency 40kHz) for 30 minutes, take it out and let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the synephrine test solution.

[0500] 3.1.4 Determination Method

[0501] Accurately pipette 10 μL each of the synephrine reference solution and the synephrine test solution into the liquid chromatograph and determine the result.

[0502] 3.2 Methodological Validation

[0503] 3.2.1 Specificity Examination

[0504] Take a negative sample of Citrus aurantium and prepare a synephrine-deficient negative solution according to the preparation method of the synephrine test solution. Inject 10 μL each of the synephrine test solution, the synephrine-deficient negative sample solution, and the synephrine reference solution into the liquid chromatograph, and determine the chromatograms according to the determined chromatographic conditions. See the results below. Figure 53 The negative sample solution showed no chromatographic peaks at the corresponding retention times of the reference standard, indicating that other components in the formulation did not interfere with synephrine, demonstrating the good specificity of this method.

[0505] 3.2.2 Examination of Linear Relationships

[0506] Accurately weigh 3.127 mg of synephrine reference standard and place it in a 25 mL volumetric flask. Add methanol to prepare a reference solution containing 124.830 μg of synephrine per mL. Use this solution as the linear mother liquor. Transfer 3 mL of the mother liquor to a 5 mL volumetric flask and add methanol to the mark. Dilute the solution stepwise according to the above steps to obtain five different concentrations of reference solution, preparing synephrine reference solutions containing 74.898 μg / mL, 44.939 μg / mL, 26.963 μg / mL, 16.178 μg / mL, and 9.707 μg / mL per mL.

[0507] Accurately pipette 10 μL of the above-mentioned reference solutions of different concentrations, and determine them under the established chromatographic conditions, recording the chromatograms. Plot the peak area as the ordinate (y) and the amount of reference standard as the abscissa (x), and calculate the linear regression equation. The results are as follows:

[0508] The regression equation for synephrine is y = 30267x - 15693, with a correlation coefficient r = 1.0000, indicating that synephrine has a good linear relationship between injection mass and peak area within the injection concentration range of 9.707 to 124.830 μg / mL.

[0509] 3.2.3 Precision Examination

[0510] (1) Instrument precision

[0511] Accurately pipette 10 μL of the synephrine reference standard mixture and inject it six times under the determined chromatographic conditions. Analyze the results, and calculate the RSD values ​​based on the peak areas of naringin and neohesperidin. The results show that with six consecutive injections of the same reference solution, the RSD values ​​of all peak areas were less than 3.0%, indicating good instrument precision.

[0512] (2) Intermediate precision

[0513] Different researchers were selected to conduct the tests at different times and using different high-performance liquid chromatographs. An appropriate amount of lyophilized Sanhuatang reference sample powder was taken, finely ground, and approximately 0.4g was accurately weighed. Six parallel samples were prepared to prepare synephrine test solutions. 10μL of each of the synephrine reference solution and synephrine test solution were accurately pipetted and tested. The synephrine content and RSD value were calculated and compared with the results of the repeatability test.

[0514] The results showed that when the same batch of samples was measured six times by different personnel on different instruments, the RSD value for synephrine content was 1.99%, compared to an RSD value of 2.06% for the six data points in the repeatability test. According to the "Guiding Principles for Validation of Analytical Methods for Drug Quality Standards" in the 2020 edition of the Chinese Pharmacopoeia, the content of the analyte in the sample should be within the specified range. At that time, the intermediate precision RSD limit was <6%, therefore, the method showed good intermediate precision when operated by different analysts on different dates and under different chromatographs.

[0515] 3.2.4 Stability Test

[0516] Accurately pipette 10 μL of the synephrine test solution and inject it at 0, 3, 6, 9, 12, 15, 18, and 25 hours. Measure the peak area of ​​synephrine in the test solution and calculate the peak area RSD value. The results show that the peak area RSD value of synephrine is less than 1.79%, indicating that the test solution has good stability within 25 hours.

[0517] 3.2.5 Repeatability Test

[0518] An appropriate amount of lyophilized powder of the Sanhua Decoction reference sample was finely ground. Approximately 0.4 g was accurately weighed and used in six parallel assays to prepare synephrine test solutions. 10 μL of the synephrine reference solution was accurately pipetted for testing and analysis. The synephrine content and RSD value were calculated. The results showed that in six repeated assays of the same batch of samples, the RSD value of the synephrine content was less than 4%. According to the "Guidelines for Validation of Analytical Methods" in the 2020 edition of the Chinese Pharmacopoeia, when the content of the analyte in the sample is between 0.01% and 0.1%, the repeatability RSD limit is <4.0%, indicating that the repeatability of this analytical method is good.

[0519] 2.2.6 Accuracy Examination

[0520] Nine lyophilized powders of the Sanhua Decoction reference sample with known content were accurately weighed. Synephrine reference solution was added to the sample at ratios of 1:0.5, 1:1, and 1:1.5, with three replicates for each ratio, to prepare nine synephrine test solutions. The solutions were then analyzed under the determined chromatographic conditions. The average recovery rate and RSD of each indicator were calculated. According to the "Guidelines for Analytical Method Validation" in the 2020 edition of the Chinese Pharmacopoeia, the recovery limit for the analyte content in the sample is 90%–108% when it is 0.1%. The recoveries of all the above indicators were within this range, and the RSDs were all less than 3%, indicating that the analytical method has good accuracy.

[0521] 3.2.7 Durability Test

[0522] (1) Investigation of different chromatographic columns

[0523] Three types of chromatographic columns were selected: Waters Xselect HSS T3 column (4.6 mm × 150 mm, 5 μm), Waters Xbridge C18 column (4.6 mm × 150 mm, 5 μm), and Agilent ZORBAX SB C18 column (4.6 mm × 150 mm, 5 μm).

[0524] Take 10 μL each of the synephrine test solution and the synephrine reference solution, and determine them using the three chromatographic columns mentioned above. Calculate the synephrine content and RSD value. The results show that the separation effect of each chromatographic column is good, and the RSD value is ≤3%, indicating that the analytical method has good robustness under different chromatographic columns.

[0525] (2) Investigation at different column temperatures

[0526] The column temperatures were set to 28℃, 30℃ and 32℃ respectively to investigate the effect of column temperature on the determination of synephrine content in the Sanhuatang reference sample.

[0527] Take 10 μL each of the synephrine test solution and the synephrine reference solution, and determine them using the column temperatures mentioned above. Calculate the synephrine content and RSD value. The results show that the RSD values ​​of the synephrine content in the Sanhuatang reference samples at different column temperatures are all less than 3%, indicating that the analytical method has good robustness within the range of 30±2℃.

[0528] (3) Investigation of different flow velocities

[0529] The flow rates were set to 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min, respectively, to investigate the effect of flow rate on the determination of synephrine content in the Sanhua decoction reference sample.

[0530] Take 10 μL each of the synephrine test solution and the synephrine reference solution, and determine the content and RSD value using the above flow rates. The results show that the RSD value of the synephrine content in the Sanhua Tang reference samples at different flow rates is less than 3%, indicating that the analytical method has good robustness in the range of 1.0 ± 0.2 mL / min. 3.3 Content determination of different batches of Sanhua Tang reference samples

[0531] Sixteen batches of Sanhuatang reference samples were lyophilized to prepare naringin test solution, and the synephrine content in the sixteen batches of Sanhuatang reference samples was determined. The experimental results are shown in Table 5.

[0532] Table 5. Results of Synephrine Content Determination in Sanhua Decoction Reference Samples

[0533]

[0534] Based on the above test results, the synephrine content in the 16 batches of Sanhua Decoction fluctuated between 0.161% and 0.430%, with an average of 0.287% and a SD value of 0.07%. The range calculated based on the mean content ± 3SD was 0.076% to 0.498%. Therefore, it can be determined that the synephrine content in Sanhua Decoction should be controlled between 0.076% and 0.498%.

[0535] IV. Determination of Notopterygium alcohol content in Sanhua Decoction

[0536] 4.1 Determination Method

[0537] 4.1.1 Chromatographic conditions

[0538] The column was packed with octadecylsilane-bonded silica gel (150 mm column length, 2.1 mm inner diameter, 1.8 μm particle size); methanol was used as mobile phase A, and 0.2% phosphoric acid was used as mobile phase B, with gradient elution performed according to the specifications in Table 6; the column temperature was 30 °C; the flow rate was 0.25 mL per minute; and the detection wavelength was 310 nm. The theoretical plate number, calculated based on the notopterygium alcohol peak, should be no less than 10,000.

[0539] Table 6 Gradient elution table for determination of Qianghuo alcohol content

[0540]

[0541] 4.1.2 Preparation of reference solution

[0542] Weigh an appropriate amount of notopterygium alcohol reference standard and add methanol to prepare a reference solution containing 10 μg of notopterygium alcohol reference standard per 1 mL.

[0543] 4.1.3 Preparation of the test solution

[0544] Take an appropriate amount of lyophilized powder of Sanhua Decoction reference sample, grind it into a fine powder, take about 0.4g, weigh it accurately, place it in a stoppered conical flask, accurately add 25mL of 70% methanol, weigh it, sonicate it (power 250W, frequency 40kHz) for 30 minutes, take it out and let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the Qianghuo test sample solution.

[0545] 4.1.4 Determination Method

[0546] Accurately pipette 2 μL each of the notopterygium alcohol reference solution and the notopterygium alcohol test solution into the liquid chromatograph and determine the result.

[0547] 4.2 Methodological Validation

[0548] 4.2.1 Specificity Examination

[0549] Take a negative sample of Notopterygium incisum and prepare a negative solution lacking Notopterygium incisum according to the preparation method of Notopterygium incisum alcohol test solution. Inject 2 μL each of the Notopterygium incisum alcohol test solution, the negative solution lacking Notopterygium incisum, and the Notopterygium incisum alcohol reference solution into the liquid chromatograph, and determine the chromatograms according to the determined chromatographic conditions. See the results below. Figure 54 The negative sample solution showed no chromatographic peaks at the corresponding retention times of the reference standard, indicating that other components in the formulation did not interfere with the notopterygium alcohol, demonstrating the good specificity of this method.

[0550] 4.2.2 Examination of Linear Relationships

[0551] Accurately weigh 1.016 mg of notopterygium alcohol reference standard and place it in a 20 mL volumetric flask. Add methanol to prepare a reference solution containing 50.729 μg of notopterygium alcohol per mL, which will be used as the linear mother liquor. Accurately measure 1, 1, 1, 2, 3, and 4 mL of the solution into 100, 10, 1, 5, 5, and 5 mL volumetric flasks, respectively, and dilute to the mark with methanol to obtain six different concentrations of reference solution. Prepare notopterygium alcohol reference solutions containing 0.507 μg / mL, 5.073 μg / mL, 10.146 μg / mL, 20.292 μg / mL, 30.438 μg / mL, and 40.583 μg / mL per mL.

[0552] Accurately pipette 2 μL of the above-mentioned reference solutions at different concentrations, and determine the chromatograms under the established chromatographic conditions. Plot the peak area on the ordinate (y) and the amount of reference standard on the abscissa (x), and calculate the linear regression equation. The results are as follows:

[0553] The regression equation for Notopterygium glutamate is y = 18857x + 1936.8, with a correlation coefficient r = 0.9996, indicating that the injection mass and peak area of ​​Notopterygium glutamate have a good linear relationship within the injection concentration range of 0.507 to 50.729 μg / mL.

[0554] 4.2.3 Precision Test

[0555] (1) Instrument precision

[0556] Accurately pipette 2 μL of the notopterygium alcohol reference solution and inject it six times under the determined chromatographic conditions. Analyze the results and calculate the RSD value based on the peak area of ​​notopterygium alcohol. The results show that with six consecutive injections of the same notopterygium alcohol reference solution, the RSD values ​​of all peak areas were less than 3.0%, indicating good instrument precision.

[0557] (2) Intermediate precision

[0558] Different researchers were selected to conduct the tests at different times and using different high-performance liquid chromatographs. An appropriate amount of lyophilized powder of the Sanhua Decoction reference sample was taken, finely ground, and about 0.4g was accurately weighed. Six parallel samples were prepared to prepare the Qianghuo alcohol test solution. 2μL of each of the Qianghuo alcohol reference solution and the Qianghuo alcohol test solution were accurately pipetted and tested. The Qianghuo alcohol content and RSD value were calculated and compared with the results of the repeatability test.

[0559] The results showed that when the same batch of samples was measured six times by different personnel at different times on different instruments, the RSD value of the thymol content was 0.77%, which was 1.31% compared with the RSD value of the six data in the repeatability test. According to the "Guiding Principles for Validation of Analytical Methods for Drug Quality Standards" in the 2020 edition of the Chinese Pharmacopoeia, when the content of the analyte in the sample is 0.01%, the intermediate precision RSD limit is <8%. Therefore, the intermediate precision of this method is good when different analysts operate on different dates and under different chromatographs.

[0560] 4.2.4 Stability Assessment

[0561] Accurately pipette 2 μL of the notopterygium alcohol test solution and inject it at 0, 2, 4, 8, 12, 18, and 24 hours, respectively. Measure the peak area of ​​notopterygium alcohol in the test solution and calculate the RSD value of the peak area. The results show that the RSD value of the notopterygium alcohol peak area is less than 3.0%, indicating that the notopterygium alcohol test solution has good stability within 24 hours.

[0562] 4.2.5 Repeatability Test

[0563] An appropriate amount of lyophilized powder of the Sanhua Decoction reference sample was finely ground. Approximately 0.4 g was accurately weighed and used in six parallel assays to prepare the Qianghuo alcohol test solution. 2 μL of the Qianghuo alcohol reference solution was accurately pipetted into each solution for testing and analysis. The Qianghuo alcohol content and RSD value were calculated. The results showed that the RSD value of the Qianghuo alcohol content was 1.57% in six repeated assays of the same batch of samples. According to the "Guidelines for Validation of Analytical Methods" in the 2020 edition of the Chinese Pharmacopoeia, when the content of the analyte in the sample is 0.01%, the repeatability RSD limit is <4.0%, indicating that the repeatability of this analytical method is good.

[0564] 4.2.6 Accuracy Examination

[0565] Nine lyophilized powders of the Sanhua Decoction reference sample with known content were accurately weighed. The reference solution of Qianghuo alcohol was added in triplicate at ratios of 1:0.5, 1:1, and 1:1.5, and the ratio of the test solution to the reference standard was 1:0.5, 1:1, and 1:1.5, respectively. The test solutions were prepared under the determined chromatographic conditions, and the average recovery rate and RSD of each indicator were calculated. According to the "Guidelines for Validation of Analytical Methods" in the 2020 edition of the Chinese Pharmacopoeia, the recovery limit is 85%–110% when the content of the analyte in the sample is 0.01%. The recoveries of all the above indicators were within this range, and the RSDs were all less than 3%, indicating that the accuracy of this analytical method is good.

[0566] 4.2.7 Durability Test

[0567] (1) Investigation of different chromatographic columns

[0568] Three types of chromatographic columns were selected: Waters HSS T3 column (2.1 mm × 150 mm, 1.8 μm), Agilent ZORBAX SB Aq column (2.1 mm × 150 mm, 1.8 μm), and Agilent ZORBAX SB C18 column (2.1 mm × 150 mm, 1.8 μm).

[0569] Take 2 μL each of the notopterygium alcohol test solution and the notopterygium alcohol reference solution, and determine them using the three chromatographic columns mentioned above. Calculate the notopterygium alcohol content and RSD value. The results show that the separation effect of each chromatographic column is good, and the RSD value of the notopterygium alcohol content is ≤3%, indicating that the analytical method has good robustness under different chromatographic columns.

[0570] (2) Investigation at different column temperatures

[0571] The column temperatures were set to 28℃, 30℃ and 32℃ respectively to investigate the effect of column temperature on the determination of the content of Qianghuo alcohol in the Sanhua Decoction reference sample.

[0572] Take 2 μL each of the notopterygium alcohol test solution and the notopterygium alcohol reference solution, and determine them using the column temperatures mentioned above. Calculate the notopterygium alcohol content and RSD value. The results show that the RSD values ​​of the notopterygium alcohol content in the Sanhua Decoction reference samples at different column temperatures are all less than 3%, indicating that the analytical method has good robustness within the range of 30±2℃.

[0573] (3) Investigation of different flow velocities

[0574] The flow rates were set to 0.23 mL / min, 0.25 mL / min, and 0.27 mL / min, respectively, to investigate the effect of flow rate on the determination of qianghuo alcohol content in the Sanhua decoction reference sample.

[0575] Take 2 μL each of the notopterygium alcohol test solution and the notopterygium alcohol reference solution, and determine the notopterygium alcohol content and RSD value using the above flow rates. The results show that the RSD value of the notopterygium alcohol content in the Sanhua Decoction reference samples at different flow rates is less than 3%, indicating that the analytical method has good robustness in the range of 0.25±0.02 mL / min. 4.3 Determination of the content of notopterygium alcohol in different batches of Sanhua Decoction reference samples

[0576] Sixteen batches of Sanhua Decoction reference samples were lyophilized to prepare Qianghuo alcohol test solution, and the content of Qianghuo alcohol in the sixteen batches of Sanhua Decoction reference samples was determined. The experimental results are shown in Table 7.

[0577] Table 7. Results of determination of Qianghuo alcohol content in Sanhua Decoction reference samples

[0578]

[0579] Based on the above test results, the content of notopterygium alcohol in 16 batches of Sanhua Decoction fluctuated between 0.0242% and 0.0771%, with an average of 0.0445% and a SD value of 0.02%. The range calculated based on ±3SD of the average content was -0.011% to 0.100%, with a minimum value being negative, which does not conform to actual production. It is more reasonable to determine the content range of notopterygium alcohol based on -SD to +3SD of the average, which should be 0.026% to 0.100%. Therefore, it can be determined that the content of notopterygium alcohol in Sanhua Decoction should be controlled between 0.026% and 0.100%.

[0580] Example 3: Method for constructing the fingerprint spectrum of Sanhua Decoction

[0581] 1. Preparation of the test solution

[0582] Take 0.4g of Sanhuatang lyophilized powder, place it in an Erlenmeyer flask, accurately add 25mL of 70% methanol, seal tightly, weigh, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool, weigh again, replenish the lost weight with 70% methanol, shake well, filter, and collect the filtrate to obtain the product.

[0583] 2. Preparation of reference solution

[0584] Accurately weigh the following reference standards: aloe-emodin-8-O-glucoside, naringin, neohesperidin, chrysophanol-1-O-β-D-glucoside, chrysophanol-8-O-β-D-glucoside, aloe-emodin, nobiletin, rhein, magnolol, honokiol, emodin, chrysophanol, and emodin methyl ether. Dissolve each in methanol to prepare a solution containing 15 μg of aloe-emodin-8-O-glucoside and 150 μg of naringin per 1 mL. A mixed solution of 200 μg of neohesperidin reference standard, 20 μg of rhein-1-O-β-D-glucoside reference standard, 30 μg of rhein-8-O-β-D-glucoside reference standard, 20 μg of emodin-8-O-β-D-glucoside reference standard, 10 μg of aloe-emodin reference standard, 10 μg of nobiletin reference standard, 30 μg of rhein reference standard, 15 μg of magnolol reference standard, 15 μg of magnolol reference standard, 10 μg of emodin reference standard, 15 μg of rhein reference standard, and 5 μg of emodin methyl ether reference standard was used to obtain a mixed fingerprint chromatogram reference solution.

[0585] 3 Chromatographic conditions

[0586] Chromatographic column: Waters CORTECS T3 column (150 mm length, 2.1 mm inner diameter, 1.6 μm particle size); methanol as mobile phase A, 0.1% phosphoric acid aqueous solution as mobile phase B, gradient elution according to Table 8; column temperature 30℃; flow rate 0.2 mL / min; detection wavelength 260 nm. The theoretical plate number, calculated based on the naringin peak, should be no less than 10,000.

[0587] Table 8 Gradient elution procedure conditions

[0588]

[0589] 4. Measurement

[0590] Accurately pipette 1 μL each of the reference solution and the fingerprint sample solution, inject them into the liquid chromatograph, and determine the results. The fingerprint sample should show chromatographic peaks corresponding to the retention times of the reference peak. Using the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints and Mark peak matching, the similarity between the fingerprint sample and the reference fingerprint should not be less than 0.90.

[0591] 5. Investigation of chromatographic conditions

[0592] 5.1 Selection of detection wavelength

[0593] Different absorption wavelengths were investigated; the absorption wavelengths were 220 nm, 260 nm, 280 nm, and 290 nm. A Waters CORTECS T3 column (1.6 μm, 150 mm × 2.1 mm) was used as the chromatographic column; methanol was used as mobile phase A, and 0.1% phosphoric acid was used as mobile phase B, with gradient elution performed according to the specifications in Table 8; the flow rate was 0.2 mL per minute; the injection volume was 1 μL; the results are shown in [Table 8]. Figure 55 .

[0594] Comparison of fingerprint spectra at different wavelengths (220nm, 260nm, 280nm, and 290nm) shows that all chromatographic peaks can be obtained at all four detection wavelengths, and the separation degree of each peak is relatively high. However, when the detection wavelength is 260nm, the peak area of ​​the main chromatographic peak in the spectrum is larger, satisfying the principle of maximizing information content. Therefore, the optimal detection wavelength for the Sanhuatang fingerprint spectrum is determined to be 260nm.

[0595] 5.2 Selection of Mobile Phase

[0596] This section screened the mobile phase types, selecting acetonitrile-phosphoric acid, methanol-water, methanol-0.1% phosphoric acid, and methanol-0.2% phosphoric acid aqueous solution as mobile phases. A Waters CORTECS T3 column (1.6 μm, 150 mm × 2.1 mm) was used, and gradient elution was performed according to the specifications in Table 8. The flow rate was 0.2 mL / min, the detection wavelength was 260 nm, the injection volume was 1 μL, and the detection wavelength was 260 nm. The results are shown in [Table 8]. Figures 56-57 .

[0597] Figure 56 The results show the test results using different combinations of mobile phases. The figures indicate that: when acetonitrile-phosphoric acid is used as the mobile phase, many components of the sample elute earlier, resulting in poor peak separation; when methanol-water is used, fewer peaks are observed, and the resolution is also poor; when methanol-phosphoric acid is used, the chromatogram is richer, the peak resolution is better, the baseline is more stable, and the elution time is suitable. Therefore, methanol-phosphoric acid is the recommended mobile phase combination.

[0598] Figure 57 The results show the test results using mobile phase B (phosphoric acid aqueous solution) at different concentrations. The figure shows that different concentrations of phosphoric acid can achieve separation of all chromatographic peaks, and the baseline is relatively stable. However, when using 0.1% phosphoric acid, the separation of each chromatographic peak is better, and the response value is also relatively higher. Therefore, 0.1% phosphoric acid is the optimal choice for mobile phase B.

[0599] 5.3 Examination of different gradients

[0600] This section examines the effect of different gradient elution curves on the fingerprint chromatogram of Sanhua Decoction. The specific gradient conditions are as follows:

[0601] Gradient 1: Using a Waters CORTECS T3 C18 column (1.6 μm, 150 mm × 2.1 mm) as the column, with methanol as mobile phase A and 0.1% phosphoric acid as mobile phase B, gradient elution was performed according to the specifications in Table 9; the flow rate was 0.3 mL per minute; the injection volume was 1 μL; the results are shown in Table 9. Figure 58 .

[0602] Table 9 Fingerprint Spectrum of Sanhua Decoction - Elution Gradient 1

[0603]

[0604]

[0605] Gradient 2: A Waters CORTECS T3 C18 column (2.1 mm × 150 mm, 1.6 μm) was used as the chromatographic column; methanol was used as mobile phase A and 0.1% phosphoric acid was used as mobile phase B, with gradient elution performed according to Table 10; the detection wavelength was 260 nm, the column temperature was 30 °C, the flow rate was 0.3 mL / min, and the injection volume was 1 μL. The results are shown in Table 10. Figure 59 .

[0606] Table 10 Fingerprint Spectrum of Sanhua Decoction Elution Gradient 2

[0607]

[0608] Gradient 3: A Waters CORTECS T3 C18 column (2.1 mm × 150 mm, 1.6 μm) was used as the chromatographic column; methanol was used as mobile phase A and 0.1% phosphoric acid was used as mobile phase B, with gradient elution performed according to Table 11; the detection wavelength was 260 nm, the column temperature was 30 °C, the flow rate was 0.2 mL per minute, and the injection volume was 1 μL. The results are shown in Table 11. Figure 60 .

[0609] Table 11 Fingerprint Spectrum of Sanhua Decoction, Elution Gradient 3

[0610]

[0611] Gradient 4: A Waters CORTECS T3 C18 column (2.1 mm × 150 mm, 1.6 μm) was used as the chromatographic column; methanol was used as mobile phase A and 0.1% phosphoric acid was used as mobile phase B, with gradient elution performed according to Table 12; the detection wavelength was 260 nm, the column temperature was 30 °C, the flow rate was 0.2 mL / min, and the injection volume was 1 μL. The results are shown in Table 12. Figure 61 .

[0612] Table 12 Fingerprint spectrum of Sanhua Decoction, elution gradient 4

[0613]

[0614]

[0615] The results showed that, compared with gradient 1 and gradient 2, the peak positions of each chromatographic peak in gradient 1 were all later and the separation of each chromatographic peak was poor. The separation of the chromatographic peaks in the latter half of the fingerprint spectrum obtained by gradient 2 was still poor. Compared with gradient 2 and gradient 3, the chromatographic peak separation effect of gradient 3 was greatly improved, but there were still some peaks that were poorly separated. In contrast, the chromatographic peaks in the fingerprint spectrum obtained by gradient 4 all achieved good separation effect. Therefore, gradient 4 was selected as the elution gradient.

[0616] 5.4 Investigation of different flow velocities

[0617] This section investigates the effects of mobile phase flow rates of 0.18 mL / min, 0.2 mL / min, and 0.22 mL / min on the fingerprint chromatogram of Sanhuatang. Methanol was used as mobile phase A, and 0.1% phosphoric acid was used as mobile phase B. Gradient elution was performed according to the specifications in Table 8. The injection volume was 1 μL. The results are shown in [Table 8]. Figure 62 .

[0618] The results showed that all chromatographic peaks could be well separated at different flow rates. Furthermore, the separation effect and peak shape were better when the flow rate was 0.2 mL / min, so 0.2 mL / min was selected as the optimal flow rate.

[0619] 5.5 Investigation at different column temperatures

[0620] The column temperature was investigated, and the effects of column temperatures of 28℃, 30℃, and 32℃ on the chromatographic behavior of the fingerprint chromatogram of Sanhuatang were examined. Methanol was used as mobile phase A, and 0.1% phosphoric acid was used as mobile phase B. Gradient elution was performed according to the specifications in Table 8; the flow rate was 0.3 mL per minute; and the injection volume was 1 μL. The results are shown in [Table 8]. Figure 63 .

[0621] The results showed that different column temperatures had no significant effect on the fingerprint spectrum of Sanhuatang, indicating that the method can adapt to certain column temperature variations. However, considering factors such as peak shape, peak resolution, and baseline, the optimal column temperature for studying the fingerprint spectrum of Sanhuatang in this experiment was 30℃.

[0622] 5.6 Determination of Optimal Chromatographic Conditions

[0623] Based on the above experiments, the chromatographic conditions were determined as follows: octadecylsilane-bonded silica gel was used as the packing material (WatersCORTECS T3, column length 150 mm, inner diameter 2.1 mm, particle size 1.6 μm); methanol was used as mobile phase A, and 0.2% phosphoric acid aqueous solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 8; the flow rate was 0.2 mL per minute; the column temperature was 30℃; the detection wavelength was 260 nm; and the theoretical plate number, calculated based on the naringin peak, should not be less than 10,000.

[0624] Investigation of the preparation method of fingerprint spectrum test solution

[0625] 6.1 Investigation of Extraction Solvents

[0626] The effects of different solvents on the fingerprint chromatogram of Sanhuatang were investigated. 100% methanol, 70% methanol, 50% methanol, 70% ethanol, and 50% ethanol (all concentrations are volume concentrations) were used as extraction solvents. The peak shape and resolution of 16 chromatographic peaks in Sanhuatang were observed, and the "total peak area / sample weight" of the 16 characteristic peaks was calculated to compare the effects of different extraction solvents on the fingerprint chromatogram of Sanhuatang and select the optimal extraction solvent.

[0627] Take an appropriate amount of Sanhuatang lyophilized powder (batch number: SHT01), grind it finely, take about 0.4g, and accurately weigh it in 5 parallel groups. Place it in a stoppered conical flask, and accurately add 10mL each of methanol, 70% methanol, 50% methanol, 70% ethanol, and 50% ethanol. Sonicate the mixture (power 250W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and collect the filtrate. Accurately pipette 1μL of the fingerprint chromatogram test solution and inject it into an ultra-high performance liquid chromatograph for determination. The experimental results are shown in Table 13.

[0628] Table 13. Investigation of fingerprint chromatograms of Sanhua Decoction using different extraction solvents.

[0629]

[0630] Experimental results show that there is no significant difference in the total peak area / sample weight of the fingerprint spectrum of Sanhuatang with different extraction solvents. However, when using 50% methanol and 50% ethanol as extraction solvents, the filtration is more difficult. Considering the preparation methods of other indicators of Sanhuatang, 70% methanol was selected as the optimal extraction solvent for the fingerprint spectrum of Sanhuatang.

[0631] 6.2 Examination of Extraction Methods

[0632] Take an appropriate amount of this product (batch number: SHT01), grind it into a fine powder, take about 0.4g, and accurately weigh it in two parallel groups. Place the samples in a stoppered conical flask, accurately add 10mL of 70% methanol, weigh the samples, and sonicate them separately (power 250W, frequency 40kHz) for 30 minutes, then heat under reflux for 30 minutes, cool, and weigh them again. Make up the weight loss with 70% methanol, shake well, filter, and use the filtrate as the test solution. Accurately pipette 1μL of the fingerprint spectrum test solution and inject it into an ultra-high performance liquid chromatograph for determination. The experimental results are shown in Table 14.

[0633] Table 14. Investigation of different extraction methods for fingerprint spectroscopy of Sanhua Decoction

[0634]

[0635] Experimental results show that there is no significant difference in the total peak area / sample weight of the fingerprint spectrum of Sanhuatang under different extraction methods. Considering the overall energy consumption and the simplicity of experimental operation, ultrasonic extraction is selected as the optimal extraction method for the fingerprint spectrum of Sanhuatang.

[0636] 6.3 Examination of extraction time

[0637] Take an appropriate amount of this product (batch number: SHT01), grind it into a fine powder, accurately weigh about 0.4 g, and perform three parallel trials. Place the solutions in stoppered conical flasks, add 10 mL of 70% methanol, and sonicate for 15 minutes, 30 minutes, and 45 minutes respectively (power 250 W, frequency 40 kHz). Cool, shake well, filter, and collect the filtrate. Accurately pipette 1 μL of the fingerprint chromatogram test solution and inject it into an ultra-high performance liquid chromatograph for determination. The experimental results are shown in Table 15.

[0638] Table 15. Investigation of fingerprint chromatograms of Sanhua Decoction at different extraction times.

[0639]

[0640] Experimental results showed that there was no significant difference in the total peak area / sample weight of the Sanhuatang fingerprint spectrum at different extraction times. To ensure the robustness of the method, 30 minutes was selected as the extraction time for the Sanhuatang fingerprint spectrum.

[0641] 6.4 Investigation on the amount of extraction solvent used

[0642] Take an appropriate amount of this product (batch number: SHT01), grind it finely, and accurately weigh approximately 0.4 g in three parallel groups. Place each group in a stoppered conical flask, and accurately add 10 mL, 25 mL, and 50 mL of 70% methanol respectively. Sonicate the mixture (250 W power, 40 kHz frequency) for 30 minutes, cool, shake well, and filter. Use the filtrate as the test solution. Accurately pipette 1 μL of the fingerprint chromatogram test solution and inject it into an ultra-high performance liquid chromatograph for determination. The experimental results are shown in Table 16.

[0643] Table 16 Investigation of different extraction solvent dosages in the fingerprint spectrum of Sanhua Decoction

[0644]

[0645] Experimental results show that when the extraction solvent volume is 25 mL and 50 mL, there is no significant difference in the total peak area / sample volume of the Sanhuatang fingerprint spectrum, which is slightly higher than that when the solvent volume is 10 mL. This indicates that 25 mL of 70% methanol can completely extract the sample. To save solvent, 25 mL was selected as the extraction solvent volume for the Sanhuatang fingerprint spectrum.

[0646] 6.5 Determination of the optimal fingerprint spectrum test solution preparation method

[0647] Based on the above experimental results, the optimal method for preparing the fingerprint spectrum test solution was determined as follows:

[0648] Accurately weigh 0.4g of Sanhuatang lyophilized powder, place it in a stoppered conical flask, add 25mL of 70% methanol, seal tightly, weigh, sonicate (power 250W, frequency 40kHz) for 30 minutes, remove, cool, weigh again, replenish the lost weight with 70vol% methanol, shake well, filter, and obtain the fingerprint spectrum test solution.

[0649] 7. Methodological Validation

[0650] 7.1 Specificity

[0651] Take the freeze-dried powder of Sanhua Decoction missing each herb, and prepare it according to the preparation method of fingerprint spectrum test solution to obtain negative sample solution missing each herb.

[0652] Take the freeze-dried powders of rhubarb, magnolia bark, immature bitter orange, and notopterygium root, and prepare reference solutions of the single herbs according to the fingerprint spectrum test solution preparation method.

[0653] Inject 1 μL each of the fingerprint chromatogram test solution, the negative sample solution lacking each herb, and the reference solution of a single herb into the liquid chromatograph. Analyze according to the chromatographic conditions described in section 3. The results are as follows: Figure 64 As shown.

[0654] Depend on Figure 64It can be seen that a total of 16 common peaks were identified in the fingerprint spectrum of Sanhua Decoction, and 14 peaks were identified, namely, peak 1 (aloe-emodin-8-O-glucoside), peak 2 (naringin), peak 3 (neohesperidin) (S), peak 4 (chrysophanol-1-O-β-D-glucoside), peak 5 (chrysophanol-8-O-β-D-glucoside), peak 7 (chrysophanol-8-O-β-D-glucoside), peak 8 (aloe-emodin), peak 9 (hesperidin), peak 10 (rhein), peak 11 (honokiol), peak 13 (honokiol), peak 14 (emodin), peak 15 (chrysophanol), and peak 16 (emodin methyl ether). Three peaks (peaks 2, 3, and 9) originated from Citrus aurantium, two peaks (peaks 11 and 13) from Magnolia officinalis, and eleven peaks (peaks 1, 4, 5, 6, 7, 8, 10, 12, 14, 15, and 16) from Rheum palmatum. The chromatogram of the test sample showed the same chromatographic peaks at the corresponding retention times as the reference sample, with no negative interference. In summary, this indicates that the method has good specificity.

[0655] 7.2 Precision

[0656] Take an appropriate amount of Sanhuatang lyophilized powder, grind it finely, and accurately weigh 0.4g. Prepare the fingerprint chromatogram test solution according to the determined fingerprint chromatogram test solution preparation method. Under the determined chromatographic conditions, inject the sample six times repeatedly and analyze each time. Select neohesperidin with a moderate elution time and a high response factor as the S peak. Calculate the RSD values ​​of the relative retention time and relative peak area of ​​the 16 common peaks in the six determinations. The experimental results show that the RSD of the relative retention time of each common peak is 0.02%–0.14%, and the RSD of the relative peak area is 0.16%–5.14%. The similarity of the six consecutive injections is 1.000, indicating that the method has good precision.

[0657] 7.3 Intermediate Precision

[0658] Another analyst precisely weighed 0.4 g of the same batch of Sanhuatang lyophilized powder on different dates and prepared six parallel fingerprint sample solutions according to the established fingerprint sample solution preparation method. These solutions were then analyzed on different instruments under the established chromatographic conditions. Using neohesperidin as a reference peak, the RSD values ​​of the relative retention time and relative peak area of ​​each common peak were calculated. The results showed that the RSD of the relative retention time of each common peak was 0.01%–0.08%, and the RSD of the relative peak area was 0.10%–1.75%. Combined with the six repeatable parallel samples, the RSD of the relative retention time of each common peak was 0.62%–2.95%, and the RSD of the relative peak area was 0.51%–9.86%, with a similarity of 1.000 for all samples, indicating good intermediate precision of the method.

[0659] 7.4 Repeatability

[0660] Accurately weigh 0.4 g of the same batch of Sanhuatang lyophilized powder and prepare six parallel fingerprint sample solutions according to the established fingerprint sample solution preparation method. These solutions were then injected sequentially under the established chromatographic conditions. Neohesperidin, with a moderate elution time and a high response factor, was used as the S peak. The RSD values ​​of the relative retention times and relative peak areas of the 16 common peaks were calculated. The results showed that the RSD of the relative retention times of each common peak ranged from 0.07% to 0.36%, and the RSD of the relative peak areas ranged from 0.51% to 7.11%. The similarity of the six parallel samples was 1.000, indicating good repeatability of the method.

[0661] 7.5 Stability

[0662] An appropriate amount of lyophilized Sanhua Decoction powder was finely ground, and 0.4 g was accurately weighed. The fingerprint sample solution was prepared according to the established method for preparing the fingerprint sample solution. The sample was injected and detected at 0, 2, 4, 8, 12, and 24 hours under the established chromatographic conditions. Neohesperidin was used as the reference peak, and the RSD values ​​of the relative retention time and relative peak area of ​​each chromatographic peak were calculated. The results showed that the RSD of the relative retention time of each common peak was 0.11%–0.47%, and the RSD of the relative peak area was 0.31%–6.17%, with a similarity of 1.000 for all peaks. This indicates that the fingerprint sample solution has good stability within 24 hours.

[0663] Determination of common peaks in 8 different batches of samples

[0664] Sixteen batches of Sanhua Tang reference samples (numbered SHT1 to SHT16) were taken and fingerprint chromatogram test solutions were prepared according to the established method. The fingerprint chromatogram test solutions were accurately pipetted and injected under the established chromatographic conditions. The overlay chromatograms of the 16 batches of Sanhua Tang samples are shown in Figure 65. Sixteen common peaks with clear components and good reproducibility were selected from the 16 batches of Sanhua Tang reference samples as standard identification peaks. A control fingerprint chromatogram was generated using the averaging method. The control fingerprint chromatogram is shown below. Figure 66 As shown in Table 17, the similarity between the fingerprint chromatograms of 16 batches of Sanhuatang reference samples and the control fingerprint chromatograms was calculated.

[0665] Table 17. Similarity Evaluation Results of Sanhua Decoction Fingerprint Spectra

[0666]

[0667] Fingerprint similarity analysis: The similarity results of the fingerprint spectra of 16 batches ranged from 0.963 to 0.998, with all similarities greater than 0.960, indicating that the differences between different batches were small and the quality was relatively stable. Fourteen of the fingerprints were identified using reference standards, in the following order: Peak 1 (aloe-emodin-8-O-glucoside), Peak 2 (naringin), Peak 3 (neohesperidin) (S), Peak 4 (chrysophanol-1-O-β-D-glucoside), Peak 5 (chrysophanol-8-O-β-D-glucoside), Peak 7 (chrysophanol-8-O-β-D-glucoside), Peak 8 (aloe-emodin), Peak 9 (hesperidin), Peak 10 (rhein), Peak 11 (honokiol), Peak 13 (honokiol), Peak 14 (emodin), Peak 15 (chrysophanol), and Peak 16 (emodin methyl ether).

[0668] Based on the above results, the fingerprint standard for the Sanhua Decoction material reference is determined as follows: the fingerprint chromatogram of the test sample should show chromatographic peaks corresponding to the retention times of the reference chromatographic peaks. According to the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine, the similarity between the fingerprint chromatogram of the test sample and the reference fingerprint chromatogram should not be less than 0.90.

[0669] The above description is a preferred embodiment of the invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the invention, and these improvements and modifications are also considered to be within the scope of protection of the invention.

Claims

1. A method for establishing the material basis of a Sanhua decoction, characterized in that, include: Thin-layer chromatography was used to identify rhubarb and magnolia bark respectively; Thin-layer chromatography was used to simultaneously identify Citrus aurantium and Notopterygium incisum. The thin-layer chromatography identification method for Citrus aurantium and Notopterygium incisum includes: The Sanhua Decoction was extracted with methanol to obtain test solutions of Citrus aurantium and Notopterygium incisum. The reference herbs Citrus aurantium and Notopterygium incisum were extracted with methanol to obtain Citrus aurantium reference herb solution and Notopterygium incisum reference herb solution, respectively; the reference standards of purslane, naringin, and neohesperidin were dissolved with methanol to obtain Citrus aurantium reference standard solution. The test solutions of Citrus aurantium and Notopterygium incisum, the reference solution of Citrus aurantium, the reference medicinal material of Citrus aurantium, and the reference medicinal material of Notopterygium incisum were spotted on the same silica gel G thin-layer plate. The plate was developed using the lower layer of a mixed solution of chloroform, methanol, and water as the developing solvent, and then sprayed with aluminum trichloride ethanol solution. The plate was then developed at 100℃~110℃ and examined. In the mixed solution of chloroform, methanol, and water, the volume ratio of chloroform, methanol, and water is 13:7:

2.

2. The method for establishing the material basis of the Sanhua Decoction as described in claim 1, characterized in that, include: The thin-layer chromatography identification method for rhubarb includes: The rhubarb test solution was obtained by first extracting the Sanhua decoction with a mixed solution of ethanol aqueous solution and hydrochloric acid, then with chloroform, and finally with a mixed solution of anhydrous ethanol and ethyl acetate. The rhubarb reference material was first extracted with a mixed solution of ethanol aqueous solution and hydrochloric acid, then with chloroform, and finally with a mixed solution of anhydrous ethanol and ethyl acetate to obtain a rhubarb reference material solution; the rhein reference standard was dissolved in methanol to obtain a rhubarb reference standard solution. The rhubarb test solution, rhubarb reference solution, and rhubarb reference herb solution were spotted onto the same silica gel G thin-layer plate and developed using a mixed solution of n-hexane, ethyl acetate, and formic acid as the developing solvent for examination. In the mixed solution of ethanol aqueous solution and hydrochloric acid, the concentration of ethanol aqueous solution is 10 vol%~50 vol%, the concentration of hydrochloric acid is 20 vol%~38 vol%, and the volume ratio of ethanol aqueous solution to hydrochloric acid is 8~12:

1. In a mixed solution of anhydrous ethanol and ethyl acetate, the volume ratio of anhydrous ethanol to ethyl acetate is 1 to 4:

1. In a mixed solution of n-hexane, ethyl acetate, and formic acid, the volume ratio of n-hexane, ethyl acetate, and formic acid is 30:10:0.

5.

3. The method for establishing the material basis of the Sanhua Decoction as described in claim 2, characterized in that, The concentration of the ethanol-water solution is 30 vol%, the concentration of the hydrochloric acid is 36 vol%, and the volume ratio of the ethanol-water solution to the hydrochloric acid is 10:1; and / or The volume ratio of anhydrous ethanol to ethyl acetate in the mixed solution of anhydrous ethanol and ethyl acetate is 2:

1.

4. The method for establishing the material basis of the Sanhua Decoction as described in claim 2, characterized in that, In the step of first extracting Sanhua Decoction with a mixed solution of ethanol aqueous solution and hydrochloric acid, then extracting with chloroform, and finally extracting with a mixed solution of anhydrous ethanol and ethyl acetate to obtain rhubarb test solution, Sanhua Decoction is dissolved in a mixed solution of ethanol aqueous solution and hydrochloric acid, heated under reflux for 0.5h~2h, cooled, and then extracted with chloroform by shaking 2~4 times. The resulting extract is dried, and the solid phase obtained after drying is dissolved in a mixed solution of anhydrous ethanol and ethyl acetate to obtain rhubarb test solution. The ratio of the mixture of Sanhua Decoction with ethanol aqueous solution and hydrochloric acid is 0.1g~0.5g:10mL~30mL; The ratio of the three-component decoction to the chloroform used in each shaking extraction is 0.1g~0.5g: 10mL~30mL; The ratio of the mixture of Sanhua Decoction with anhydrous ethanol and ethyl acetate is 0.1g~0.5g:1mL~5mL.

5. The method for establishing the material basis of the Sanhua Decoction as described in claim 2, characterized in that, The step of spotting the rhubarb test solution, rhubarb reference solution, and rhubarb reference herb solution onto the same silica gel G thin-layer plate, developing the plate with a mixed solution of n-hexane, ethyl acetate, and formic acid as the developing solvent, and examining the plate, involves spotting a volume of 3 μL to 8 μL. After development, the plate is examined under ultraviolet light or sunlight.

6. The method for establishing the material basis of the Sanhua Decoction as described in claim 1, characterized in that, The thin-layer chromatography identification method for Magnolia officinalis includes: The Sanhua decoction was extracted sequentially with methanol and chloroform to obtain the Magnolia officinalis test solution; Magnolia officinalis reference material was extracted sequentially with water and chloroform to obtain a Magnolia officinalis reference material solution; magnolol reference standard was dissolved in methanol to obtain a Magnolia officinalis reference standard solution. The Magnolia officinalis test solution, Magnolia officinalis reference solution and Magnolia officinalis reference medicinal material solution were spotted on the same silica gel G thin layer plate, developed with a mixed solution of petroleum ether and ethyl acetate as the developing solvent, and sprayed with vanillin sulfuric acid solution. The color was developed at 100℃~110℃ and examined. In the mixed solution of petroleum ether and ethyl acetate, the volume ratio of petroleum ether to ethyl acetate is 3:

1.

7. The method for establishing the material basis of the Sanhua Decoction as described in claim 6, characterized in that, In a mixed solution of petroleum ether and ethyl acetate, the boiling range of petroleum ether is 60℃~90℃.

8. The method for establishing the material basis of the Sanhua Decoction as described in claim 6, characterized in that, In the step of extracting the Sanhua decoction sequentially with methanol and chloroform to obtain the Magnolia officinalis test solution, the Sanhua decoction is mixed with methanol and ultrasonically treated for 10 min to 40 min. After removing methanol from the obtained extract, it is dissolved in water and then extracted with chloroform by shaking 2 to 4 times. The obtained extract is dried, and the resulting solid phase is dissolved in methanol to obtain the Magnolia officinalis test solution. The ratio of the three-component decoction to the methanol used for extraction is 0.5g~2g:15mL~30mL; The ratio of Sanhua Decoction to water is 0.5g~2g:5mL~20mL; The ratio of the three-component decoction to the chloroform used in each shaking extraction is 0.5g~2g:10mL~30mL; The ratio of Sanhua Decoction to methanol used for dissolving is 0.5g~2g:0.5mL~3mL.

9. The method for establishing the material basis of the Sanhua Decoction as described in claim 6, characterized in that, In the step of spotting the Magnolia officinalis test solution, Magnolia officinalis reference solution, and Magnolia officinalis reference medicinal material solution onto the same silica gel G thin-layer plate, developing the plate with a mixture of petroleum ether and ethyl acetate as the developing solvent, and spraying it with vanillin sulfuric acid solution, the plate is developed at 100℃~110℃. In the inspection step, the sample volume is 3μL~12μL, and the concentration of vanillin sulfuric acid solution is 3wt%~10wt%.

10. The method for establishing the material basis of the Sanhua Decoction as described in claim 6, characterized in that, In the step of extracting the Magnolia officinalis reference material sequentially with water and chloroform to obtain a Magnolia officinalis reference material solution, and dissolving magnolol reference standard with methanol to obtain a Magnolia officinalis reference standard solution, the Magnolia officinalis reference material is mixed with water, boiled for 40 min to 60 min, filtered, and the filtrate is extracted with chloroform by shaking 2 to 4 times. The extract is dried, and the solid phase obtained after drying is dissolved in methanol to obtain a Magnolia officinalis reference material solution. Among them, the ratio of Magnolia officinalis reference material to water was 0.5g~2g:80mL~150mL; The ratio of Magnolia officinalis reference material to chloroform used in each shake extraction was 0.5g~2g:10mL~30mL; The ratio of Magnolia officinalis reference material to methanol was 0.5g~2g:0.5mL~3mL.

11. The method for establishing the material basis of the Sanhua Decoction as described in claim 1, characterized in that, In the step of extracting the Sanhua Decoction with methanol to obtain the test solution of Citrus aurantium and Notopterygium incisum, the Sanhua Decoction is mixed with methanol and ultrasonically treated for 10 min to 40 min. The resulting extract is dried, and the solid phase obtained after drying is dissolved in methanol to obtain the test solution of Citrus aurantium and Notopterygium incisum. The ratio of the three-component decoction to the methanol used in the extraction is 0.1g~1g:20mL~30mL; The ratio of Sanhua Decoction to methanol used for dissolving is 0.1g~1g:0.5mL~3mL.

12. The method for establishing the material basis of the Sanhua Decoction as described in claim 1, characterized in that, The test solutions of Citrus aurantium and Notopterygium incisum, the reference solution of Citrus aurantium, the reference medicinal material of Citrus aurantium, and the reference medicinal material of Notopterygium incisum were spotted on the same silica gel G thin-layer plate. The plate was developed using the lower layer of a mixed solution of chloroform, methanol, and water as the developing solvent, and then sprayed with aluminum chloride ethanol solution. The plate was then developed at 100℃~110℃. In the inspection step, the sample volume was 1μL~3μL, and the concentration of aluminum chloride ethanol solution was 3wt%~10wt%. After development, the plate was inspected under ultraviolet light.

13. The method for establishing the material basis of the Sanhua Decoction as described in claim 1, characterized in that, Also includes: Fingerprint mapping was constructed to identify the components in Sanhua Decoction.

14. The method for establishing the material basis of the Sanhua Decoction as described in claim 13, characterized in that, The method for constructing the fingerprint spectrum of the Sanhua Decoction includes: The Sanhua decoction was extracted with the ninth extraction solvent to obtain the fingerprint spectrum test solution; The reference standard was dissolved or extracted using the tenth extraction solvent to obtain a fingerprint chromatogram reference standard solution; The fingerprint spectrum of the test solution and the fingerprint spectrum of the reference solution were determined by liquid chromatography to obtain the fingerprint spectrum of Sanhua Decoction. The liquid chromatograph uses an octadecylsilane-bonded silica gel column as the stationary phase, methanol as mobile phase A, and a 0.05 vol%~0.3 vol% aqueous phosphoric acid solution as mobile phase B for gradient elution. The gradient elution curve is as follows: From 0 min to 5 min, mobile phase A decreased from 3% to 21%, and mobile phase B decreased from 97% to 79%. From 5 min to 20 min, mobile phase A decreased from 21% to 36%, and mobile phase B decreased from 79% to 64%. Between 20 and 32 minutes, mobile phase A decreased from 36% to 50%, and mobile phase B decreased from 64% to 50%. From 32 to 42 minutes, mobile phase A decreased from 50% to 62%, and mobile phase B decreased from 50% to 38%. From 42 to 50 minutes, mobile phase A decreased from 62% to 85%, and mobile phase B decreased from 38% to 15%. From 50 to 60 minutes, mobile phase A decreased from 85% to 95%, and mobile phase B decreased from 15% to 5%. The reference standard includes aloe-emodin-8- O - Glucoside reference standard, naringin reference standard, neohesperidin reference standard, rhein-1- O - β -D-glucoside reference standard, rhein-8- O - β -D-glucoside reference standard, emodin-8- O - β -D-glucoside reference standard, aloe-emodin reference standard, noriheptacortin reference standard, rhein reference standard, magnolol reference standard, magnolol reference standard, emodin reference standard, chrysophanol reference standard and emodin methyl ether reference standard. The ninth extraction solvent is a methanol aqueous solution with a concentration of 50 vol% to 100 vol% or an ethanol aqueous solution with a concentration of 50 vol% to 100 vol%, and the tenth extraction solvent is a methanol aqueous solution with a concentration of 30 vol% to 100 vol%.

15. The method for establishing the material basis of the Sanhua Decoction as described in claim 14, characterized in that, The mobile phase B is a 0.1 vol% aqueous solution of phosphoric acid.

16. The method for establishing the material basis of the Sanhua Decoction as described in claim 14, characterized in that, The ninth extraction solvent is a methanol aqueous solution with a concentration of 70 vol% to 100 vol% or an ethanol aqueous solution with a concentration of 70 vol% to 100 vol%; and / or The tenth extraction solvent is a methanol aqueous solution with a concentration of 70 vol% to 100 vol%.

17. The method for establishing the material basis of the Sanhua Decoction as described in claim 14 or 16, characterized in that, The ninth extraction solvent is a 70 vol% methanol-water solution; and / or The tenth extraction solvent is methanol.

18. The method for establishing the material basis of the Sanhua Decoction as described in claim 14, characterized in that, The chromatographic column has a length of 100 mm to 250 mm and a diameter of 2.1 mm to 5 mm, and the stationary phase has a particle size of 1.6 μm to 1.8 μm. The column temperature of the chromatographic column is 28℃~32℃; The flow rate of the liquid chromatograph is 0.18 mL / min to 0.22 mL / min, and the detection wavelength is 220 nm to 300 nm; the injection volume of the fingerprint chromatogram test solution is 0.5 μL to 1.5 μL, and the injection volume of the fingerprint chromatogram reference solution is 0.5 μL to 1.5 μL.

19. The method for establishing the material basis of the Sanhua Decoction as described in claim 14 or 18, characterized in that, The chromatographic column has a length of 150 mm, a diameter of 2.1 mm, and a stationary phase particle size of 1.6 μm. The column temperature of the chromatographic column is 30℃; The liquid chromatograph has a flow rate of 0.2 mL / min, a detection wavelength of 260 nm, an injection volume of 1 μL for the fingerprint sample solution, and an injection volume of 1 μL for the fingerprint reference solution.

20. The method for establishing the material basis of the Sanhua Decoction as described in claim 14, characterized in that, In the step of extracting the Sanhua decoction with the ninth extraction solvent to obtain the fingerprint spectrum test solution, the Sanhua decoction is mixed with a methanol aqueous solution with a concentration of 70 vol% to 100 vol%, and ultrasonically treated or heated under reflux for 15 min to 45 min. The ratio of Sanhua Decoction to methanol aqueous solution is 0.1g~1g:10mL~50mL.

21. The method for establishing the material basis of the Sanhua Decoction as described in claim 14 or 20, characterized in that, In the step of extracting the Sanhua Decoction with the ninth extraction solvent to obtain the fingerprint spectrum test solution, the Sanhua Decoction is mixed with a 70 vol% methanol aqueous solution and ultrasonically treated with a power of 250 W and a frequency of 40 kHz for 30 min. The ratio of Sanhua Decoction to methanol aqueous solution is 0.4g:25mL.

22. The method for establishing the material basis of the Sanhua Decoction as described in claim 14, characterized in that, The fingerprint reference solution contained aloe-emodin-8- O The concentration of the glucoside reference standard is 10 μg / mL to 20 μg / mL, the concentration of the naringin reference standard is 120 μg / mL to 180 μg / mL, the concentration of the neohesperidin reference standard is 180 μg / mL to 250 μg / mL, and the concentration of the rhein-1- O - β The concentration of the -D-glucoside reference standard is 10 μg / mL to 30 μg / mL, and the rhein-8- O - β The concentration of the -D-glucoside reference standard is 20 μg / mL to 50 μg / mL, and the rhein-8- O - β The concentrations of the D-glucoside reference standard are 10 μg / mL to 40 μg / mL, the aloe-emodin reference standard are 5 μg / mL to 20 μg / mL, the nobiletin reference standard are 5 μg / mL to 20 μg / mL, the rhein reference standard are 20 μg / mL to 50 μg / mL, the magnolol reference standard are 5 μg / mL to 30 μg / mL, the honokiol reference standard is 5 μg / mL to 30 μg / mL, the emodin reference standard is 5 μg / mL to 30 μg / mL, the chrysophanol reference standard is 10 μg / mL to 30 μg / mL, and the emodin methyl ether reference standard is 2 μg / mL to 15 μg / mL.

23. The method for establishing the material basis of the Sanhua Decoction as described in claim 13, characterized in that, The fingerprint spectrum includes 16 common peaks, among which peak 1 is aloe-emodin-8- O - Glucoside, peak 2 is naringin, peak 3 is neohesperidin, peak 4 is rhein-1- O - β -D-glucoside, peak 5 is rhein-8- O - β -D-glucoside, peak 7 is emodin-8- O - β -D-glucoside, peak 8 is aloe-emodin, peak 9 is noriheptacortin, peak 10 is rhein, peak 11 is honokiol, peak 13 is honokiol, peak 14 is emodin, peak 15 is rhein, and peak 16 is emodin methyl ether.

24. The method for establishing the material basis of the Sanhua Decoction as described in claim 13, characterized in that, Also includes: The contents of free anthraquinones, total anthraquinones, magnolol, honokiol, naringin, neohesperidin, synephrine, and gentianol in Sanhua decoction were determined by liquid chromatography.

25. The method for establishing the material basis of the Sanhua Decoction as described in claim 13, characterized in that, Also includes: The contents of magnolol, honokiol, free anthraquinones, and total anthraquinones in Sanhua Decoction were determined by liquid chromatography. The contents of naringin and neohesperidin in Sanhua decoction were simultaneously determined by liquid chromatography. The content of synephrine in Sanhua Decoction was determined by liquid chromatography. The content of qianghuo alcohol in Sanhua Decoction was determined by liquid chromatography.

26. The method for establishing the material basis of Sanhua Decoction as described in claim 25, characterized in that, The methods for determining the content of free anthraquinones, total anthraquinones, magnolol, and honokiol include: The Sanhua decoction was extracted with the first extraction solvent to obtain a free anthraquinone test solution; the free anthraquinone test solution was used for the determination of free anthraquinone, magnolol, and the content of magnolol. The free anthraquinone test solution was extracted sequentially with hydrochloric acid solution and chloroform, and then dissolved in the first solvent to obtain the total anthraquinone test solution; the total anthraquinone test solution was used for the determination of the total anthraquinone content; The reference standard was dissolved or extracted using a second extraction solvent to obtain an anthraquinone reference standard solution; The free anthraquinone test solution, total anthraquinone test solution, and anthraquinone reference solution were determined by liquid chromatography. The liquid chromatograph uses an octadecylsilane-bonded silica gel column as the stationary phase, acetonitrile as mobile phase A, and a 0.05 vol%~0.3 vol% aqueous phosphoric acid solution as mobile phase B for gradient elution. The gradient elution curve is as follows: From 0 min to 10 min, mobile phase A was 28% and mobile phase B was 72%. From 10 min to 28 min, mobile phase A decreased from 28% to 40%, and mobile phase B decreased from 72% to 60%. Between 28 and 35 minutes, mobile phase A decreased from 40% to 42%, and mobile phase B decreased from 60% to 58%. From 35 to 50 minutes, mobile phase A decreased from 42% to 54%, and mobile phase B decreased from 58% to 46%. The reference standards include aloe-emodin reference standard, rhein reference standard, chrysophanol reference standard, emodin reference standard, emodin methyl ether reference standard, magnolol reference standard and magnolol reference standard. The first extraction solvent, the second extraction solvent, and the first solvent are all methanol aqueous solutions with a concentration of 70 vol% to 100 vol%. The concentration of the hydrochloric acid solution is 5wt%~15wt%.

27. The method for establishing the material basis of the Sanhua Decoction as described in claim 26, characterized in that, The mobile phase B is a 0.1 vol% aqueous solution of phosphoric acid; and / or The first extraction solvent is methanol; and / or The second extraction solvent is methanol; and / or The first solvent is methanol; and / or The concentration of the hydrochloric acid solution is 8 wt%.

28. The method for establishing the material basis of Sanhua Decoction as described in claim 26, characterized in that, The chromatographic column has a length of 100 mm to 250 mm and a diameter of 2.1 mm to 5 mm, and the stationary phase has a particle size of 1.6 μm to 1.8 μm. The column temperature of the chromatographic column is 26℃~30℃; The flow rate of the liquid chromatograph is 0.30 mL / min to 0.34 mL / min, and the detection wavelength is 240 nm to 280 nm; the injection volume of the free anthraquinone test solution is 0.5 μL to 2 μL, the injection volume of the total anthraquinone test solution is 0.5 μL to 2 μL, and the injection volume of the anthraquinone reference solution is 0.5 μL to 2 μL.

29. The method for establishing the material basis of the Sanhua Decoction as described in claim 26 or 28, characterized in that, The chromatographic column has a length of 100 mm, a diameter of 2.1 mm, and a stationary phase particle size of 1.6 μm. The column temperature of the chromatographic column is 28℃; The flow rate of the liquid chromatograph is 0.32 mL / min, the detection wavelength is 254 nm, the injection volume of the free anthraquinone test solution is 1 μL, the injection volume of the total anthraquinone test solution is 1 μL, and the injection volume of the anthraquinone reference solution is 1 μL.

30. The method for establishing the material basis of Sanhua Decoction as described in claim 26, characterized in that, In the step of extracting Sanhua Decoction with the first extraction solvent to obtain a free anthraquinone test solution, Sanhua Decoction is mixed with a methanol aqueous solution with a concentration of 70 vol% to 100 vol% and ultrasonically treated for 15 min to 45 min. The ratio of Sanhua Decoction to methanol aqueous solution is 0.1g~1g:10mL~50mL.

31. The method for establishing the material basis of Sanhua Decoction as described in claim 26, characterized in that, In the step of extracting the free anthraquinone test solution sequentially with hydrochloric acid solution and chloroform, and then dissolving it with the first solvent to obtain the total anthraquinone test solution, the free anthraquinone test solution is dried, the resulting solid phase is mixed with hydrochloric acid solution, and ultrasonically treated for 1 min to 5 min to obtain the first intermediate solution. The first intermediate solution was heated under reflux with chloroform for 30 to 90 minutes to obtain the second intermediate solution; The second intermediate solution was extracted with chloroform 2 to 6 times. The resulting extracts were combined, dried, and the resulting solid phase was dissolved in the first solvent. The volume ratio of the free anthraquinone test solution to the hydrochloric acid solution is 1:0.8~1.

5. The volume ratio of the free anthraquinone test solution to the chloroform used for heating and reflux is 1:0.8~1.5; The volume ratio of the free anthraquinone test solution to the chloroform used in each extraction is 1:0.8~1.5; The volume ratio of the free anthraquinone test solution to the first solvent is 1:0.7~1.

1.

32. The method for establishing the material basis of the Sanhua Decoction as described in claim 26, characterized in that, In the anthraquinone reference solution, the concentrations of aloe-emodin reference standard, rhein reference standard, emodin reference standard, chrysophanol reference standard, and emodin methyl ether reference standard are 10 μg / mL to 50 μg / mL, and the concentrations of magnolol reference standard and magnolol reference standard are 80 μg / mL to 120 μg / mL.

33. The method for establishing the material basis of Sanhua Decoction as described in claim 25, characterized in that, The methods for determining the content of naringin and neohesperidin include: The three-component decoction was extracted using a third extraction solvent to obtain test solutions of naringin and neohesperidin; Naringin and neohesperidin reference standards were dissolved or extracted using a fourth extraction solvent to obtain reference solutions of naringin and neohesperidin. The test solutions of naringin and neohesperidin, and the reference solutions of naringin and neohesperidin were determined by liquid chromatography. The liquid chromatograph uses an octadecylsilane-bonded silica gel column as the stationary phase, acetonitrile as mobile phase A, and a 0.05 vol%~0.3 vol% aqueous phosphoric acid solution as mobile phase B for gradient elution. The gradient elution curve is as follows: From 0 min to 10 min, mobile phase A was 16% and mobile phase B was 84%. From 10 to 18 minutes, mobile phase A decreased from 16% to 18%, and mobile phase B decreased from 84% to 82%. From 18 to 23 minutes, mobile phase A was 18% and mobile phase B was 82%. Between 23 and 25 minutes, mobile phase A decreased from 18% to 20%, and mobile phase B decreased from 82% to 80%. Between 25 and 40 minutes, mobile phase A decreased from 20% to 24%, and mobile phase B decreased from 80% to 76%. The third extraction solvent is an aqueous ethanol solution with a concentration of 60 vol% to 90 vol%, and the fourth extraction solvent is an aqueous methanol solution with a concentration of 70 vol% to 100 vol%.

34. The method for establishing the material basis of the Sanhua Decoction as described in claim 33, characterized in that, The mobile phase B is a 0.1 vol% aqueous solution of phosphoric acid; and / or The third extraction solvent is a 70 vol% aqueous ethanol solution; and / or The fourth extraction solvent is methanol.

35. The method for establishing the material basis of Sanhua Decoction as described in claim 33, characterized in that, The chromatographic column has a length of 100 mm to 250 mm and a diameter of 3 mm to 4.6 mm, and the stationary phase has a particle size of 3 μm to 5 μm. The column temperature of the chromatographic column is 28℃~32℃; The flow rate of the liquid chromatograph is 0.8 mL / min to 1.0 mL / min, and the detection wavelength is 250 nm to 300 nm; the injection volume of the test solutions of naringin and neohesperidin is 5 μL to 15 μL, and the injection volume of the reference solutions of naringin and neohesperidin is 5 μL to 15 μL.

36. The method for establishing the material basis of the Sanhua Decoction as described in claim 33 or 35, characterized in that, The chromatographic column has a length of 150 mm, a diameter of 4.6 mm, and a stationary phase particle size of 5 μm. The column temperature of the chromatographic column is 30℃; The flow rate of the liquid chromatograph is 1.0 mL / min, the detection wavelength is 280 nm, the injection volume of the test solutions of naringin and neohesperidin is 10 μL, and the injection volume of the reference solutions of naringin and neohesperidin is 10 μL.

37. The method for establishing the material basis of the Sanhua Decoction as described in claim 33, characterized in that, In the step of extracting Sanhua Decoction with a third extraction solvent to obtain test solutions of naringin and neohesperidin, Sanhua Decoction is mixed with an ethanol aqueous solution with a concentration of 60 vol% to 90 vol% and ultrasonically treated for 15 min to 45 min. The ratio of Sanhua Decoction to ethanol-water solution is 0.1g~1g:30mL~90mL.

38. The method for establishing the material basis of the Sanhua Decoction as described in claim 33, characterized in that, In the reference solutions of naringin and neohesperidin, the concentration of the naringin reference standard is 100 μg / mL to 150 μg / mL, and the concentration of the neohesperidin reference standard is 100 μg / mL to 150 μg / mL.

39. The method for establishing the material basis of Sanhua Decoction as described in claim 25, characterized in that, The method for determining the synephrine content includes: The Sanhua decoction was extracted using the fifth extraction solvent to obtain a synephrine test solution; Synephrine reference standard was dissolved or extracted using the sixth extraction solvent to obtain a synephrine reference standard solution; The synephrine test solution and synephrine reference solution were determined by liquid chromatography; the chromatographic column of the liquid chromatograph used octadecylsilane-bonded silica gel as the stationary phase, methanol as mobile phase A, and water as mobile phase B for isocratic elution. The volume ratio of mobile phase A to mobile phase B is 40~50:50~60, and mobile phase B includes 0.05 vol%~0.3 vol% phosphoric acid and 0.05 wt%~0.3 wt% sodium dodecyl sulfonate. The fifth extraction solvent is a methanol aqueous solution with a concentration of 60 vol% to 90 vol%, and the sixth extraction solvent is a methanol aqueous solution with a concentration of 5 vol% to 40 vol%.

40. The method for establishing the material basis of the Sanhua Decoction as described in claim 39, characterized in that, The volume ratio of mobile phase A to mobile phase B is 45:55; and / or The mobile phase B comprises 0.1 vol% phosphoric acid and 0.1 wt% sodium dodecyl sulfonate; and / or The fifth extraction solvent is a 70 vol% methanol aqueous solution; and / or The sixth extraction solvent is a 10 vol% methanol aqueous solution.

41. The method for establishing the material basis of the Sanhua Decoction as described in claim 39, characterized in that, The chromatographic column has a length of 100 mm to 250 mm and a diameter of 3 mm to 4.6 mm, and the stationary phase has a particle size of 3 μm to 5 μm. The column temperature of the chromatographic column is 28℃~32℃; The flow rate of the liquid chromatograph is 0.8 mL / min to 1.0 mL / min, and the detection wavelength is 200 nm to 250 nm; the injection volume of the synephrine test solution is 5 μL to 15 μL, and the injection volume of the synephrine reference solution is 5 μL to 15 μL.

42. The method for establishing the material basis of the Sanhua Decoction as described in claim 39 or 41, characterized in that, The chromatographic column has a length of 150 mm, a diameter of 4.6 mm, and a stationary phase particle size of 5 μm. The column temperature of the chromatographic column is 30℃; The flow rate of the liquid chromatograph is 1.0 mL / min, the detection wavelength is 224 nm, the injection volume of the synephrine test solution is 10 μL, and the injection volume of the synephrine reference solution is 10 μL.

43. The method for establishing the material basis of the Sanhua Decoction as described in claim 39, characterized in that, In the step of extracting the Sanhua decoction with the fifth extraction solvent to obtain the synephrine test solution, the Sanhua decoction is mixed with a methanol aqueous solution with a concentration of 60 vol% to 90 vol% and ultrasonically treated for 15 min to 45 min. The ratio of Sanhua Decoction to methanol aqueous solution is 0.1g~1g:10mL~50mL.

44. The method for establishing the material basis of the Sanhua Decoction as described in claim 25, characterized in that, The method for determining the content of notopterygium alcohol includes: The Sanhua decoction was extracted using the seventh extraction solvent to obtain the Qianghuo alcohol test solution; The reference standard of notopterygium alcohol was dissolved or extracted using the eighth extraction solvent to obtain the reference standard solution of notopterygium alcohol; The test solution and reference solution of notopterygium alcohol were determined by liquid chromatography. The liquid chromatograph uses an octadecylsilane-bonded silica gel column as the stationary phase, methanol as mobile phase A, and a 0.1 vol%~0.4 vol% aqueous phosphoric acid solution as mobile phase B for gradient elution. The gradient elution curve is as follows: From 0 min to 10 min, mobile phase A was 60% and mobile phase B was 40%. From 10 to 30 minutes, mobile phase A decreased from 60% to 72%, and mobile phase B decreased from 40% to 28%. The seventh and eighth extraction solvents are methanol aqueous solutions with a concentration of 60 vol% to 100 vol%.

45. The method for establishing the material basis of the Sanhua Decoction as described in claim 44, characterized in that, The mobile phase B is a 0.2 vol% aqueous solution of phosphoric acid; and / or The seventh extraction solvent is a 70 vol% methanol aqueous solution; and / or The eighth extraction solvent is methanol.

46. ​​The method for establishing the material basis of the Sanhua Decoction as described in claim 44, characterized in that, The chromatographic column has a length of 100 mm to 250 mm and a diameter of 2.1 mm to 5 mm, and the stationary phase has a particle size of 1.6 μm to 1.8 μm. The column temperature of the chromatographic column is 28℃~32℃; The flow rate of the liquid chromatograph is 0.23 mL / min to 0.27 mL / min, and the detection wavelength is 300 nm to 350 nm; the injection volume of the notopterygium alcohol test solution is 1 μL to 5 μL, and the injection volume of the notopterygium alcohol reference solution is 1 μL to 5 μL.

47. The method for establishing the material basis of the Sanhua Decoction as described in claim 44 or 46, characterized in that, The chromatographic column has a length of 150 mm, a diameter of 2.1 mm, and a stationary phase particle size of 1.8 μm. The column temperature of the chromatographic column is 30℃; The flow rate of the liquid chromatograph is 0.25 mL / min, the detection wavelength is 310 nm, the injection volume of the notopterygium alcohol test solution is 2 μL, and the injection volume of the notopterygium alcohol reference solution is 2 μL.

48. The method for establishing the material basis of the Sanhua Decoction as described in claim 44, characterized in that, In the step of extracting Sanhua Decoction with the seventh extraction solvent to obtain the Qianghuo alcohol test solution, Sanhua Decoction is mixed with a methanol aqueous solution with a concentration of 60 vol% to 90 vol% and ultrasonically treated for 15 min to 45 min. The ratio of Sanhua Decoction to methanol aqueous solution is 0.1g~1g:10mL~50mL.

49. The method for establishing the material basis of the Sanhua Decoction as described in claim 1, characterized in that, Sanhua Decoction comprises the following components by weight: rhubarb 30-32 parts, magnolia bark 30-32 parts, immature bitter orange 30-32 parts, and notopterygium root 30-32 parts.

50. The method for establishing the material basis of the Sanhua Decoction as described in claim 49, characterized in that, The preparation method of Sanhua Decoction is as follows: Take rhubarb, magnolia bark, immature bitter orange, and notopterygium root, crush them to 4-10 mesh, soak them in water for 10-40 minutes, bring to a boil over high heat, and simmer over low heat until the liquid is 40%-60% of the initial amount of water added. The ratio of the total weight of rhubarb, magnolia bark, immature bitter orange, and notopterygium root to the weight of water used for soaking is 120-140:1600-2000.

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