Fingerprint and multi-index content determination method of radix morindae officinalis-rougui radix sophorae tonkinensis medicinal pair

Abstract

The application provides a kind of cinnabar root-shan dougen medicine pair compatibility fingerprint and a kind of multi-index content determination method, and this application utilizes HPLC-DAD to establish "throat medicine" cinnabar root-shan dougen medicine pair compatibility fingerprint and gallic acid, sophoramine, oxidized sophoramine, rock white cabbage element, three leaf bean purple sandalwood glycoside multi-index content determination method, and methodological investigation is carried out, and the method is stable and reliable.Meanwhile, the fingerprint of cinnabar root, shandougen medicine single decoction and the change of combined decoction is qualitatively analyzed and the content change of index medicine is studied, and the change of chemical components before and after compatibility is discussed, which can be used for the quality control of cinnabar root-shan dougen medicine pair and lay a foundation for subsequent related research.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine quality control technology, and relates to a fingerprint spectrum of the cinnabar root and sophora root medicinal pair and a method for determining the content of multiple indicators. Background Technology

[0002] Ardisiae Crenatae Radix is ​​the dried root of Ardisia crenata Sims, a plant in the Myrsinaceae family. It has a slightly bitter and pungent taste, and is neutral in nature. It excels at detoxifying, reducing swelling, promoting blood circulation, relieving pain, and dispelling wind and dampness. Clinically, it is mainly used for sore throat, rheumatic pain, and traumatic injuries. Sophorae Tonkinensis Radix et Rhizoma is the dried root and rhizome of Sophora tonkinensis Gagnep., a plant in the Fabaceae family. It has a bitter taste and is cold in nature. It excels at clearing heat and detoxifying, reducing swelling, and relieving sore throat. Clinically, it is mainly used for heat toxin accumulation, tonsillitis, sore throat, swollen gums, and oral ulcers. Both Ardisiae Crenatae Radix and Sophorae Tonkinensis Radix are used in preparations such as Yunxiang Wind-Dispelling and Pain-Relieving Tincture and Kaihoujian Spray. Ardisiae Crenatae Radix is ​​revered as a sacred medicine for throat ailments by the Miao people; Sophorae Tonkinensis Radix is ​​a key medicine for relieving sore throat. The combination of the two enhances their throat-soothing effect. Modern pharmacological studies have shown that the main chemical components in *Ardisia crenata* root include coumarins, saponins, flavonoids, and volatile oils, which have pharmacological effects such as anti-inflammatory, anti-tumor, antioxidant, hypoglycemic, hepatoprotective, and antifertility effects. The main chemical components in *Sophora tonkinensis* root include alkaloids, flavonoids, and lignans, which have pharmacological activities in many aspects, such as anti-tumor and antiviral effects.

[0003] Fingerprint spectroscopy, as a comprehensive and quantifiable identification method, possesses characteristics such as large information content, strong features, integrity, and fuzziness, which can comprehensively reflect the types and quantities of chemical components contained in traditional Chinese medicine. It has become one of the quality control models that conforms to the characteristics of traditional Chinese medicine and its application in quality control is becoming increasingly widespread. Therefore, this invention qualitatively analyzes the changes in fingerprint spectroscopy and the content changes of indicator components in single and combined decoctions of *Ardisia crenata* and *Sophora tonkinensis*, exploring the changes in chemical components before and after compatibility. This can be used for the quality control of the *Ardisia crenata*-*Sophora tonkinensis* herb pair and lays the foundation for subsequent related research. Therefore, it is necessary to establish a quality control method for the *Ardisia crenata*-*Sophora tonkinensis* herb pair. Summary of the Invention

[0004] The purpose of this invention is to provide a method for establishing the fingerprint spectrum of the cinnabar root-sophora root pair.

[0005] Another objective of this invention is to provide a method for determining the content of multiple indicators of the cinnamon root-sophora root pair, so as to improve the level of quality control.

[0006] To achieve the objectives of this invention, the following technical solutions and steps are employed:

[0007] A method for establishing the fingerprint spectrum of the medicinal pair *Ardisia crenata* and *Sophora tonkinensis*, wherein the method is based on HPLC-DAD fingerprint detection, and the specific steps are as follows:

[0008] (1) Preparation of reference solution: Accurately weigh appropriate amounts of gallic acid, matrine, oxymatrine, bergenin, and pterostilbene, and place them in 10 mL volumetric flasks respectively. Add solvent to dissolve them completely and then dilute to volume to prepare a solution with a mass concentration of 1-2 mg / mL. -1 Prepare stock solutions of each reference standard; accurately measure an appropriate amount of each stock solution of the reference standard and dilute it with a solvent to prepare a mixed reference standard solution;

[0009] (2) Preparation of the test solution:

[0010] Preparation of freeze-dried powder by decoction: Accurately weigh 10g each of 10 batches of powdered cinnabar root and sophora root, place them in round-bottom flasks, add 6-10 times the amount of water and soak for 3-5 hours, decoct twice, the first decoction for 2-3 hours and the second decoction for 1-2 hours, combine the two decoction liquids, filter with gauze, concentrate, and freeze-dry to obtain freeze-dried powder of cinnabar root and sophora root;

[0011] Preparation of combined decoction freeze-dried powder: Accurately weigh 10g each of 10 batches of cinnabar root and sophora root powder, mix them, and operate according to the preparation method of single decoction freeze-dried powder to obtain combined decoction freeze-dried powder of cinnabar root and sophora root.

[0012] Take 0.05g of single-decoction lyophilized powder (equivalent to 0.325g of raw medicinal material) and about 0.1g of combined-decoction lyophilized powder (equivalent to 0.75g of raw medicinal material), accurately weigh them, place them in a stoppered conical flask, accurately add 20mL of methanol solution, stopper tightly, weigh the mass, sonicate for 30min, cool, replenish the lost mass with methanol solution, shake well, take the supernatant and filter it through a 0.22μm microporous membrane, take the filtrate, and obtain the test solution;

[0013] (3) Establishment of fingerprint chromatogram: The reference solution and the test solution were detected by high performance liquid chromatography, and the chromatograms were recorded to establish the fingerprint chromatogram of the cinnabar root and sophora root pair; the chromatographic conditions are as follows:

[0014] Chromatographic column: Agilent InfinityLab Poroshell HPH C18 column, 250 nm × 4.6 nm, 4 μm; mobile phase A: acetonitrile, mobile phase B: 0.2% phosphoric acid solution; gradient elution; column temperature: 30℃; flow rate: 0.7 mL / min. -1 The detection wavelength was 210 nm; the column temperature was 30 °C; and the injection volume was 10 μL.

[0015] The methanol solution mentioned in steps (1) and (2) of this invention is 10% methanol.

[0016] Preferably, in step (2) of the present invention, the ingredients are soaked in 8 times the amount of water for 4 hours and decocted twice, the first time for 2 hours and the second time for 1 hour.

[0017] In step (2) of this invention, the powder is made by crushing medicinal slices and passing them through a No. 2 sieve; the gauze is an 8-layer gauze.

[0018] The concentration described in step (2) of this invention is as follows: the concentrate is concentrated in a 70°C water bath until it is nearly dry to obtain the concentrate.

[0019] The freeze-drying conditions described in step (2) of this invention are as follows: the concentrated liquid that is nearly dry is pre-frozen at -80℃ for 8 hours and then freeze-dried to obtain freeze-dried powder of cinnamon root and sophora root.

[0020] The conditions for ultrasonic treatment in step (2) of this invention are: power 500W, frequency 40kHz.

[0021] The gradient elution procedure in step (3) of this invention is as follows: 0-14 min, 5% A; 14-35 min, 5%-30% A; 35-40 min, 30%-50% A; 40-50 min, 50%-60% A.

[0022] A method for determining the content of multiple indicators in a herbal pair of *Ardisia crenata* and *Sophora tonkinensis* includes the following steps:

[0023] (1) Preparation of reference solution: Accurately weigh appropriate amounts of gallic acid, matrine, oxymatrine, bergenin, and pterostilbene, and place them in 10 mL volumetric flasks respectively. Add solvent to dissolve them completely and then dilute to volume to prepare a solution with a mass concentration of 1-2 mg / mL. -1 Prepare stock solutions of each reference standard; accurately measure an appropriate amount of each stock solution of the reference standard and dilute it with a solvent to prepare a mixed reference standard solution;

[0024] (2) Preparation of the test solution:

[0025] Preparation of freeze-dried powder by decoction: Accurately weigh 10g each of 10 batches of powdered cinnabar root and sophora root, place them in round-bottom flasks, add 6-10 times the amount of water and soak for 3-5 hours, decoct twice, the first decoction for 2-3 hours and the second decoction for 1-2 hours, combine the two decoction liquids, filter with gauze, concentrate, and freeze-dry to obtain freeze-dried powder of cinnabar root and sophora root;

[0026] Preparation of combined decoction freeze-dried powder: Accurately weigh 10g each of 10 batches of cinnabar root and sophora root powder, mix them, and operate according to the preparation method of single decoction freeze-dried powder to obtain combined decoction freeze-dried powder of cinnabar root and sophora root.

[0027] (3) Content determination: The content of the reference solution and the test solution was determined by high performance liquid chromatography (HPLC); the chromatographic conditions are as follows:

[0028] Chromatographic column: Agilent InfinityLab Poroshell HPH C18 column, 250 nm × 4.6 nm, 4 μm; mobile phase A: acetonitrile, mobile phase B: 0.2% phosphoric acid solution; gradient elution; column temperature: 30℃; flow rate: 0.7 mL / min. -1 The detection wavelength was 210 nm; the column temperature was 30 °C; and the injection volume was 10 μL.

[0029] The methanol solution mentioned in steps (1) and (2) of this invention is 10% methanol.

[0030] Beneficial effects of this invention:

[0031] 1. This invention uses high performance liquid chromatography (HPLC) and selects gallic acid, matrine, oxymatrine, bergenin, and pterostilbene as reference standards to identify the fingerprint chromatographic peaks of the *Ardisia crenata*-*Sophora tonkinensis* herb pair. The results are as follows: (1) Five common peaks were identified in 10 batches of *Ardisia crenata* decoction, among which peak 4 is the chromatographic peak of bergenin and was selected as the reference peak (S); (2) Eleven common peaks were identified in 10 batches of *Sophora tonkinensis* decoction, among which peak 3 is the chromatographic peak of oxymatrine and was selected as the reference peak (S); (3) Ten batches of *Ardisia crenata*... Fourteen common peaks were identified in the combined decoction of Sophora tonkinensis, among which peak No. 8 was the chromatographic peak of Bergenin and was selected as the reference peak (S); all three were common peaks with a peak area of ​​more than 90% of the total peak area; (4) The difference in chromatographic peaks and the overall similarity were evaluated by using the Chinese medicine chromatographic fingerprint similarity evaluation system. The chromatograms of the tested samples of single decoction of Cinnamomum cassia, single decoction of Sophora tonkinensis, and combined decoction of Sophora tonkinensis were all more than 0.90 similar to their respective reference fingerprints, indicating that the drug was stable in quality for each batch of medicinal materials and freeze-dried powder, and the method was reasonable and stable.

[0032] 2. This invention uses comparative analysis of the spectra of single decoctions, combined decoctions, and mixed reference standards of the medicinal pair to preliminarily assign the common peaks in the combined decoction of *Ardisia crenata* and *Sophora tonkinensis*. It is basically determined that peaks 1-3, 8, and 12 originate from *Ardisia crenata*, and peaks 4-7 and 9-14 originate from *Sophora tonkinensis*. By comparing the retention time with the UV spectrum, it can be determined that peak 3 is gallic acid, peak 4 is matrine, peak 6 is oxymatrine, peak 8 is bergenin, and peak 14 is pterostilbene glycoside.

[0033] 3. The fingerprint chromatogram established by this invention has good specificity, integrity, instrument precision, repeatability and stability as shown by the methodological investigation; the common peaks in the chromatogram are sharp and symmetrical, with good separation and similarity of not less than 0.90.

[0034] 4. The fingerprint spectrum of the drug pair after compatibility in this invention shows significant changes, with the content of most chemical components increasing and the content of some components decreasing. Among them, oxymatrine undergoes conversion and is partially reduced after decoction, indicating that the dissolution of the main effective components of the cinnabar root-sophora root combination affects each other. This invention, through the study of fingerprint spectrum and content determination of the cinnabar root-sophora root combination before and after compatibility, can comprehensively and rapidly characterize the quality of the Chinese medicine combination and the differences before and after compatibility, which is beneficial for overall detection and quality control and has practical value in clinical application. Attached Figure Description

[0035] Figure 1 Chromatograms of gallic acid, matrine, oxymatrine, bergenin, and trifolin pterostilbene reference standards

[0036] Figure 2 Chromatogram of the test sample of the decoction of Aralia elata and Sophora tonkinensis.

[0037] Figure 3 Chromatogram of a negative sample lacking Cinnamomum cassia

[0038] Figure 4 Chromatogram of a negative sample of Sophora tonkinensis.

[0039] Figure 5 Chromatogram of the common peak fingerprint of Aralia elata and Sophora tonkinensis

[0040] Figure 6 Chromatogram of the common peak fingerprint of cinnabar root

[0041] Figure 7 Chromatogram of common peak fingerprint of Sophora tonkinensis

[0042] Figure 8 Specificity test chromatogram

[0043] Figure 9 Precision test chromatogram

[0044] Figure 10 Chromatogram of repeatability experiment

[0045] Figure 11 Chromatogram of stability test

[0046] Figure 12 Chromatogram of recovery experiment

[0047] Figure 13 Chromatograms of ultrasonic and reflux extraction experiments (extraction methods: ultrasonic extraction at the top, reflux extraction at the bottom)

[0048] Figure 14 : Overlay chromatograms of experiments with different solid-liquid ratios (solid-liquid ratios from top to bottom are: 2:20, 1:20, 0.75:20)

[0049] Figure 15 : Overlay chromatograms of experiments with different mobile phases (from top to bottom: methanol-0.2% phosphoric acid aqueous solution, acetonitrile-0.2% phosphoric acid aqueous solution) Detailed Implementation

[0050] Example 1: Method for establishing the fingerprint spectrum of the cinnabar root-sophora root herb pair

[0051] (1) Preparation of reference solutions: Accurately weigh appropriate amounts of gallic acid, matrine, oxymatrine, bergenin, and pterostilbene, and place them separately in volumetric flasks. After dissolving them completely in methanol solution, dilute to volume to obtain concentrations of 2.358, 2.056, 1.020, 1.083, and 1.010 mg·mL, respectively. -1 The reference stock solutions were prepared by adding 10% methanol to obtain mass concentrations of 0.047, 0.123, 0.122, 0.303, and 0.024 mg / mL. -1 The mixed reference solution was stored at 4°C for later use.

[0052] (2) Preparation of the test solution:

[0053] Preparation of freeze-dried powder by decoction: 10g of each of 10 batches of powdered cinnabar root and sophora root were accurately weighed and placed in round-bottom flasks. 8 times the amount of water was added and soaked for 4 hours. The powder was decocted twice, the first time for 2 hours and the second time for 1 hour. The two decoctions were combined, filtered through gauze, concentrated, and freeze-dried to obtain freeze-dried powder of cinnabar root and sophora root.

[0054] Preparation of combined decoction freeze-dried powder: Accurately weigh 10g each of 10 batches of cinnabar root and sophora root powder, mix them, and operate according to the preparation method of single decoction freeze-dried powder to obtain combined decoction freeze-dried powder of cinnabar root and sophora root.

[0055] Take 0.05g of single-decoction lyophilized powder (equivalent to 0.325g of raw medicinal material) and about 0.1g of combined-decoction lyophilized powder (equivalent to 0.75g of raw medicinal material), weigh accurately, place in a stoppered conical flask, accurately add 20mL of 10% methanol solution, stopper tightly, weigh, sonicate (power 500W, frequency 40kHz) for 30min, cool, replenish the lost mass with 10% methanol solution, shake well, take the supernatant and filter through a 0.22μm microporous membrane, take the filtrate, and obtain the test solution;

[0056] (3) Establishment of fingerprint chromatogram: The reference solution and the test solution were detected by high performance liquid chromatography, and the chromatograms were recorded to establish the fingerprint chromatogram of the cinnabar root and sophora root pair; the chromatographic conditions are as follows:

[0057] Chromatographic column: Agilent InfinityLab Poroshell HPH C18 column, 250 nm × 4.6 nm, 4 μm; mobile phase A: acetonitrile, mobile phase B: 0.2% phosphoric acid solution; gradient elution; column temperature: 30℃; flow rate: 0.7 mL / min. -1 The detection wavelength was 210 nm; the column temperature was 30 °C; and the injection volume was 10 μL.

[0058] The gradient elution program is as follows: 0–14 min, 5% A; 14–35 min, 5%–30% A; 35–40 min, 30%–50% A; 40–50 min, 50%–60% A.

[0059] Example 2: Method for establishing the fingerprint spectrum of the cinnabar root-sophora root pair

[0060] (1) Preparation of reference solutions: Accurately weigh appropriate amounts of gallic acid, matrine, oxymatrine, bergenin, and pterostilbene, and place them separately in volumetric flasks. After dissolving them completely in 10% methanol solution, dilute to volume to obtain concentrations of 2.358, 2.056, 1.020, 1.083, and 1.010 mg·mL, respectively. -1 The reference stock solutions were prepared by adding 10% methanol to obtain mass concentrations of 0.047, 0.123, 0.122, 0.303, and 0.024 mg / mL. -1 The mixed reference solution was stored at 4°C for later use.

[0061] (2) Preparation of the test solution:

[0062] Preparation of freeze-dried powder by decoction: 10g of each of 10 batches of powdered cinnabar root and sophora root were accurately weighed and placed in round-bottom flasks. 6 times the amount of water was added and soaked for 3 hours. The flasks were decocted twice, the first time for 2.5 hours and the second time for 1.5 hours. The two decoctions were combined, filtered through gauze, concentrated, and freeze-dried to obtain freeze-dried powder of cinnabar root and sophora root.

[0063] Preparation of combined decoction freeze-dried powder: Accurately weigh 10g each of 10 batches of cinnabar root and sophora root powder, mix them, and operate according to the preparation method of single decoction freeze-dried powder to obtain combined decoction freeze-dried powder of cinnabar root and sophora root.

[0064] Take 0.05g of single-decoction lyophilized powder (equivalent to 0.325g of raw medicinal material) and about 0.1g of combined-decoction lyophilized powder (equivalent to 0.75g of raw medicinal material), weigh accurately, place in a stoppered conical flask, accurately add 20mL of 10% methanol solution, stopper tightly, weigh, sonicate (power 500W, frequency 40kHz) for 30min, cool, replenish the lost mass with 10% methanol solution, shake well, take the supernatant and filter through a 0.22μm microporous membrane, take the filtrate, and obtain the test solution;

[0065] (3) Establishment of fingerprint chromatogram: The reference solution and the test solution were detected by high performance liquid chromatography, and the chromatograms were recorded to establish the fingerprint chromatogram of the cinnabar root and sophora root pair; the chromatographic conditions are as follows:

[0066] Chromatographic column: Agilent InfinityLab Poroshell HPH C18 column, 250 nm × 4.6 nm, 4 μm; mobile phase A: acetonitrile, mobile phase B: 0.2% phosphoric acid solution; gradient elution; column temperature: 30℃; flow rate: 0.7 mL / min. -1 The detection wavelength was 210 nm; the column temperature was 30 °C; and the injection volume was 10 μL.

[0067] The gradient elution program is as follows: 0–14 min, 5% A; 14–35 min, 5%–30% A; 35–40 min, 30%–50% A; 40–50 min, 50%–60% A.

[0068] Example 3: Method for establishing the fingerprint spectrum of the cinnabar root-sophora root pair

[0069] (1) Preparation of reference solutions: Accurately weigh appropriate amounts of gallic acid, matrine, oxymatrine, bergenin, and pterostilbene, and place them separately in volumetric flasks. After dissolving them completely in 10% methanol solution, dilute to volume to obtain concentrations of 2.358, 2.056, 1.020, 1.083, and 1.010 mg·mL, respectively. -1 The reference stock solutions were prepared by adding 10% methanol to obtain mass concentrations of 0.047, 0.123, 0.122, 0.303, and 0.024 mg / mL. -1 The mixed reference solution was stored at 4°C for later use.

[0070] (2) Preparation of the test solution:

[0071] Preparation of freeze-dried powder by decoction: 10g of each of 10 batches of powdered cinnabar root and sophora root were accurately weighed and placed in round-bottom flasks. 10 times the amount of water was added and soaked for 5 hours. The powder was decocted twice. The first decoction was 3 hours and the second decoction was 2 hours. The two decoction liquids were combined, filtered through gauze, concentrated, and freeze-dried to obtain freeze-dried powder of cinnabar root and sophora root.

[0072] Preparation of combined decoction freeze-dried powder: Accurately weigh 10g each of 10 batches of cinnabar root and sophora root powder, mix them, and operate according to the preparation method of single decoction freeze-dried powder to obtain combined decoction freeze-dried powder of cinnabar root and sophora root.

[0073] Take 0.05g of single-decoction lyophilized powder (equivalent to 0.325g of raw medicinal material) and about 0.1g of combined-decoction lyophilized powder (equivalent to 0.75g of raw medicinal material), weigh accurately, place in a stoppered conical flask, accurately add 20mL of 10% methanol solution, stopper tightly, weigh, sonicate (power 500W, frequency 40kHz) for 30min, cool, replenish the lost mass with 10% methanol solution, shake well, take the supernatant and filter through a 0.22μm microporous membrane, take the filtrate, and obtain the test solution;

[0074] (3) Establishment of fingerprint chromatogram: The reference solution and the test solution were detected by high performance liquid chromatography, and the chromatograms were recorded to establish the fingerprint chromatogram of the cinnabar root and sophora root pair; the chromatographic conditions are as follows:

[0075] Chromatographic column: Agilent InfinityLab Poroshell HPH C18 column, 250 nm × 4.6 nm, 4 μm; mobile phase A: acetonitrile, mobile phase B: 0.2% phosphoric acid solution; gradient elution; column temperature: 30℃; flow rate: 0.7 mL / min. -1 The detection wavelength was 210 nm; the column temperature was 30 °C; and the injection volume was 10 μL.

[0076] The gradient elution program is as follows: 0–14 min, 5% A; 14–35 min, 5%–30% A; 35–40 min, 30%–50% A; 40–50 min, 50%–60% A.

[0077] Example 4: Multi-index content determination method for the cinnabar root-sophora root pair

[0078] (1) Preparation of reference solutions: Accurately weigh appropriate amounts of gallic acid, matrine, oxymatrine, bergenin, and pterostilbene, and place them separately in volumetric flasks. After dissolving them completely in methanol solution, dilute to volume to obtain concentrations of 2.358, 2.056, 1.020, 1.083, and 1.010 mg·mL, respectively. -1 The reference stock solutions were prepared by adding 10% methanol to obtain mass concentrations of 0.047, 0.123, 0.122, 0.303, and 0.024 mg / mL. -1 The mixed reference solution was stored at 4°C for later use.

[0079] (2) Preparation of the test solution:

[0080] Preparation of freeze-dried powder by decoction: Accurately weigh 10g each of powdered cinnabar root and sophora root, place them in round-bottom flasks, add 8 times the amount of water and soak for 4 hours, decoct twice, the first decoction for 2 hours and the second decoction for 1 hour, combine the two decoction liquids, filter with gauze, concentrate, freeze dry to obtain freeze-dried powder of cinnabar root and sophora root.

[0081] Preparation of combined decoction freeze-dried powder: Accurately weigh 10g each of the powdered slices of Cinnamomum cassia and Sophora tonkinensis, mix them, and proceed according to the preparation method of single decoction freeze-dried powder to obtain the combined decoction freeze-dried powder of Cinnamomum cassia and Sophora tonkinensis.

[0082] Take 0.05g of single-decoction lyophilized powder (equivalent to 0.325g of raw medicinal material) and about 0.1g of combined-decoction lyophilized powder (equivalent to 0.75g of raw medicinal material), weigh accurately, place in a stoppered conical flask, accurately add 20mL of 10% methanol solution, stopper tightly, weigh, sonicate (power 500W, frequency 40kHz) for 30min, cool, replenish the lost mass with 10% methanol solution, shake well, take the supernatant and filter through a 0.22μm microporous membrane, take the filtrate, and obtain the test solution;

[0083] (3) Establishment of fingerprint chromatograms: High performance liquid chromatography (HPLC) was used to detect the contents of the reference solution and the test solution, and the chromatographic conditions were as follows:

[0084] Chromatographic column: Agilent InfinityLab Poroshell HPH C18 column, 250 nm × 4.6 nm, 4 μm; mobile phase A: acetonitrile, mobile phase B: 0.2% phosphoric acid solution; gradient elution; column temperature: 30℃; flow rate: 0.7 mL / min. -1 The detection wavelength was 210 nm; the column temperature was 30 °C; and the injection volume was 10 μL.

[0085] The gradient elution program is as follows: 0–14 min, 5% A; 14–35 min, 5%–30% A; 35–40 min, 30%–50% A; 40–50 min, 50%–60% A.

[0086] To further verify the feasibility of the present invention, the inventors conducted a series of experiments, as follows:

[0087] 1.1 Instruments

[0088] Agilent 1260 high-performance liquid chromatograph (Agilent Technologies, USA, equipped with G1315B DAD detector and Agilent Chemical Chromatography workstation); AG135 electronic balance (Mettler-Toledo, Switzerland); PTHW general-purpose constant temperature electric heating mantle (Gongyi Yuhua Instrument Co., Ltd.); KQ-500DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); YXQ-S0SII vertical pressure steam sterilizer (Shanghai Dongya Pressure Vessel Manufacturing Co., Ltd.).

[0089] 1.2 Materials

[0090] Gallic acid reference standard (batch number PS000688, purity ≥98%), matrine (batch number PS011495, purity ≥98%), oxymatrine (batch number PS011495, purity ≥98%), bergenin reference standard (batch number PS000991, purity ≥98%), and pterostilbene reference standard (batch number PS012410, purity ≥98%) were all purchased from Chengdu Pusi Biotechnology Co., Ltd.; acetonitrile (chromatographic grade, Tiandi Inc., USA); methanol (Sinopharm Chemical Reagent Co., Ltd.); phosphoric acid (chromatographic grade, Tianjin Kemeio Chemical Reagent Co., Ltd.); water was ultrapure water, and all other reagents were of analytical grade.

[0091] Ten batches of *Ardisia crenata* and *Sophora tonkinensis* used in the experiment were purchased from Guizhou Sanli Pharmaceutical Co., Ltd. and the market. Source information is shown in Table 1. Professor Wei Shenghua of the School of Pharmacy, Guizhou University of Traditional Chinese Medicine, identified *Ardisia crenata* as the dried root of *Ardisia crenata* Sims (family Myrsinaceae) and *Sophora tonkinensis* Gagnep. (family Fabaceae) as the dried root and rhizome of *Sophora tonkinensis* Gagnep. (family Fabaceae). The following *Ardisia crenata* (Z1-Z10) and *Sophora tonkinensis* (S1-S10) were paired sequentially to obtain *Ardisia crenata*-*Sophora tonkinensis* herbal pairs, as shown in Table 1.

[0092] Table 1 Source information of Cinnamomum cassia and Sophora tonkinensis

[0093]

[0094] 2.1 Chromatographic conditions

[0095] The chromatographic conditions were as follows: an octadecylsilane-bonded silica column with a size of 250 nm × 4.6 nm and a diameter of 4 μm was used; mobile phase A was acetonitrile, and mobile phase B was an aqueous phosphoric acid solution; gradient elution was used: 0–14 min, 5% A; 14–35 min, 5%–30% A; 35–40 min, 30%–50% A; 40–50 min, 50%–60% A; column temperature: 30 °C; flow rate: 0.7 mL·min⁻¹; detection wavelength: 210 nm; column temperature: 30 °C; injection volume: 10 μL.

[0096] 2.2 Solution Preparation

[0097] 2.2.1 Preparation of Reference Solutions: Accurately weigh appropriate amounts of gallic acid, matrine, oxymatrine, bergenin, and pterostilbene, and add 10% methanol to prepare solutions with mass concentrations of 2.358, 2.056, 1.020, 1.083, and 1.010 mg·mL, respectively. -1 The reference stock solution.

[0098] 2.2.2 Preparation of Mixed Reference Solutions Take appropriate amounts of the stock solutions of gallic acid, matrine, oxymatrine, bergenin, and pterostilbene reference standards from section “2.2.1”, and add 10% methanol to prepare solutions with mass concentrations of 0.047, 0.123, 0.122, 0.303, and 0.024 mg·mL, respectively. -1 The mixed reference solution was stored at 4°C for later use.

[0099] 2.2.3 Preparation of the test solution

[0100] Preparation of freeze-dried powders for single and combined decoction

[0101] Preparation of freeze-dried powder by decoction: 10g of each of 10 batches of powdered cinnabar root and sophora root (passed through a No. 2 sieve) were accurately weighed and placed in round-bottom flasks. 8 times the amount of water was added and soaked for 4 hours. The flasks were decocted twice, the first time for 2 hours and the second time for 1 hour. The two decoctions were combined, filtered through gauze (8 layers), concentrated, and freeze-dried to obtain freeze-dried cinnabar root and sophora root powders, numbered Z1-Z10 and S1-S10, respectively.

[0102] Preparation of freeze-dried powder by decoction: Accurately weigh 10g each of 10 batches of powdered cinnabar root and sophora root (passed through a No. 2 sieve), mix them, and operate according to the preparation method of freeze-dried powder by decoction in "2.2.3" to obtain freeze-dried powder of cinnabar root and sophora root by decoction, numbered ZS1-ZS10.

[0103] Weigh 0.05g of the single-decoction lyophilized powder (containing 0.375g of raw medicinal material) and 0.1g of the combined-decoction lyophilized powder (containing 0.75g of raw medicinal material) from different batches of the above-mentioned samples. Accurately weigh the samples and place them in a stoppered conical flask. Accurately add 20mL of 10% methanol, seal the flask tightly, weigh the samples, and sonicate them (500W power, 40kHz frequency) for 30min. Cool the flask, replenish the lost mass with 10% methanol, shake well, and filter the supernatant through a 0.22μm microporous membrane. Collect the filtrate to obtain the test solution, which is numbered Z1-Z10, S1-S10, and ZS1-ZS10, respectively.

[0104] 2.2.4 Preparation of negative sample solution

[0105] Prepare two negative samples, one lacking Cinnamomum cassia root and the other lacking Sophora tonkinensis root, and prepare them into negative sample solutions according to the preparation method of the test sample solution in step (3).

[0106] 2.3 Establishment and Method Validation of HPLC Fingerprint

[0107] 2.3.1 Precision Test

[0108] Take the lyophilized powder of the test sample (ZS8), prepare the test solution according to the method in section "2.2.3", and inject it continuously 6 times under the chromatographic conditions in section "2.1". Using the chromatographic peak of Bergenin No. 8 as the reference peak (S), the RSD of the relative retention time of each common peak was calculated to be 0-1.02%, and the RSD of the relative peak area was 0-1.75%, indicating that the instrument is precise and in good condition. See Tables 2-3.

[0109] Table 2 Results of the test on the relative retention time of the common peak precision

[0110]

[0111]

[0112] Table 3. Results of the test on the relative peak area and precision of the common peak.

[0113]

[0114] 2.3.2 Repeatability Test

[0115] Take 6 portions of the lyophilized powder (ZS8) of the test sample and prepare 6 test sample solutions in parallel according to the method in section "2.2.3". Determine the solution under the chromatographic conditions in section "2.1" and use the chromatographic peak of Bergenin No. 8 as the reference peak (S). The RSD of the relative retention time of each common peak is calculated to be 0-0.33% and the RSD of the relative peak area is 0-2.90%, indicating that the method has good repeatability. See Tables 4-5.

[0116] Table 4 Results of the relative retention time test for common peak repeatability

[0117]

[0118] Table 5 Results of the relative peak area test for common peak repeatability

[0119]

[0120]

[0121] 2.3.3 Stability Assessment

[0122] The test sample lyophilized powder (ZS8) was prepared into a test solution according to the method in section "2.2.3". Chromatographic determination was performed at 0, 3, 6, 12, 24 and 48 h according to the chromatographic conditions in section "2.1". The chromatographic peak of bergenin No. 6 was used as the reference peak (S). The RSD of the relative retention time of each common peak was calculated to be 0 to 0.63%, and the RSD of the relative retention peak area was 0 to 2.77%, indicating that the test sample solution had good stability within 48 hours. See Tables 6-7.

[0123] Table 6. Test results of relative retention time of common peak stability

[0124]

[0125] Table 7. Test results of relative peak area and stability of common peak.

[0126]

[0127] 2.3.4 Establishment and Evaluation of Fingerprint Patterns

[0128] Ten batches of single and mixed decoctions of *Ardisia crenata* and *Sophora tonkinensis* were tested and injected separately. According to the obtained chromatograms, five common peaks were identified in the 10 batches of single decoction of *Ardisia crenata*, among which peak No. 4 was the chromatographic peak of bergenin and was selected as the reference peak (S); eleven common peaks were identified in the 10 batches of single decoction of *Sophora tonkinensis*, among which peak No. 3 was the chromatographic peak of oxymatrine and was selected as the reference peak (S); and fourteen common peaks were identified in the 10 batches of combined decoction of *Ardisia crenata* and *Sophora tonkinensis*, among which peak No. 8 was the chromatographic peak of bergenin and was selected as the reference peak (S). The common peaks in all three batches accounted for more than 90% of the total peak area. Using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (version 2012.130723), after peak selection and matching mode settings, chromatographic peaks were automatically matched. The chromatograms of 10 batches of single-origin decoctions of *Ardisia crenata*, single-origin decoctions of *Sophora tonkinensis*, and combined decoctions were matched, and the control fingerprint chromatograms generated from these 10 batches were used as a reference. The differences in chromatographic peaks and overall similarity were evaluated, and the similarity of each chromatogram was calculated using the median method. Figure 1-3 The chromatograms of the tested samples of single decoction of *Aralia elata* root, single decoction of *Sophora tonkinensis* root, and combined decoction showed a similarity of more than 0.90 with their respective reference fingerprint chromatograms, indicating that the drug is stable in quality for each batch of medicinal materials and freeze-dried powder, and the method is reasonable and stable (see Tables 8-10).

[0129] Table 8. Similarity of HPLC fingerprint chromatograms of 10 batches of single-ingredient decoction of *Ardisia crenata* root

[0130]

[0131] Table 9. Similarity of HPLC fingerprint chromatograms of 10 batches of Sophora tonkinensis decoction solutions

[0132]

[0133] Table 10. Similarity of HPLC fingerprint chromatograms of decoctions of *Ardisia crenata* and *Sophora tonkinensis* from 10 batches.

[0134]

[0135] 2.3.5 Study on the assignment of chromatographic peaks

[0136] By comparing the spectra of the single decoction, combined decoction, and mixed reference standard of the herbal pair, the common peaks in the combined decoction of *Ardisia crenata* and *Sophora tonkinensis* were preliminarily assigned. Peaks 1–3, 8, and 12 were basically determined to originate from *Ardisia crenata*, and peaks 4–7 and 9–14 from *Sophora tonkinensis*. Through retention time and UV spectral comparison, peak 3 was identified as gallic acid, peak 4 as matrine, peak 6 as oxymatrine, peak 8 as bergenin, and peak 14 as pterostilbene glycoside.

[0137] 3. Determination of sample content

[0138] 3.1 Specificity Test: Accurately pipette the mixed reference solution, test solution, and 10% methanol blank solution from section "2.2.2". Prepare negative solutions of *Sophora tonkinensis* and *Ardisia crenata* according to section "2.2.3". Take 10 μL of each solution and inject them under the above chromatographic conditions to obtain the corresponding chromatograms. In the chromatogram of the test solution, chromatographic peaks with the same retention times at the corresponding chromatographic peak positions of gallic acid, matrine, oxymatrine, bergenin, and pterostilbene in the mixed reference solution chromatogram were observed. The results showed no interference from the blank solution, indicating good method specificity. (See [reference]). Figure 8 .

[0139] 3.2 Linearity Examination: Accurately pipette appropriate amounts of the stock reference solutions of gallic acid, matrine, oxymatrine, bergenin, and pterostilbene from section "2.2.1" and place them in the same volumetric flask. Dilute with 10% methanol to obtain a series of mixed reference solutions. Determine the peak area according to the chromatographic conditions in section "2.1". Plot a standard curve with peak area as the ordinate (Y) and reference concentration as the abscissa (X). Calculate the regression equation and correlation coefficient (r). 2 The results are shown in Table 11.

[0140] Table 11 Regression equations, correlation coefficients, and linear ranges for the five components.

[0141]

[0142] 3.3 Precision Test: The lyophilized sample powder (ZS8) was injected six times consecutively under the chromatographic conditions described in section "2.1". The peak area values ​​of five components—gallic acid, matrine, oxymatrine, bergenin, and pterostilbene—were recorded. The RSD values ​​of the peak areas of the five components were calculated to be 0.19%, 0.37%, 1.08%, 0.18%, and 0.75%, respectively, indicating good instrument precision (see Table 12). Figure 9 .

[0143] Table 12 Results of Precision Peak Area Test for Each Component

[0144]

[0145] 3.4 Repeatability Test: Six lyophilized powders (ZS8) were taken, and six parallel test solutions were prepared according to the method described in section "2.2.3". The solutions were then analyzed under the chromatographic conditions described in section "2.1". The peak area values ​​of the five components—gallic acid, matrine, oxymatrine, bergenin, and pterostilbene—were recorded. The calculated RSDs for the five components were 2.10%, 2.36%, 2.90%, 2.80%, and 2.24%, respectively, indicating good repeatability of the method (see Table 13). Figure 10 .

[0146] Table 13 Results of repeatability peak area tests for each component

[0147]

[0148] 3.5 Stability Test: The lyophilized powder of the test sample (ZS8) was injected and analyzed at 0, 3, 6, 12, 24, and 48 h according to the chromatographic conditions described in section "2.1". The peak area values ​​of five components—gallic acid, matrine, oxymatrine, bergenin, and pterostilbene—were recorded. The calculated RSD values ​​of the peak areas of the five components were 0.13%, 0.18%, 2.10%, 0.18%, and 2.33%, respectively. The results indicate that the test sample solution exhibits good stability within 48 h (see Table 14). Figure 11 .

[0149] Table 14 Results of peak area tests for stability of each component

[0150]

[0151] 3.6 Recovery Rate Six portions of the lyophilized sample (ZS8) were accurately weighed, each containing 0.375 g of crude drug. The mixed reference solution was accurately added at a concentration level (1:1). Six test solutions were prepared for each portion according to the method described in section "2.2.3". The solutions were injected, determined, and the recoveries of gallic acid, matrine, oxymatrine, bergenin, and pterostilbene were calculated to be 102.5%, 101.7%, 99.6%, 99.7%, and 100.7%, respectively, with RSDs of 2.21%, 2.42%, 1.97%, 2.46%, and 2.78%, respectively. The results indicate that this method meets the requirements of analytical methodology. (See...) Figure 12 .

[0152] 3.7 Determination of the content of single-ingredient and combined-ingredient samples: Take the test solution under section “2.2.3” and perform the determination according to the chromatographic conditions under section “2.1”. Record the peak area values ​​of gallic acid, matrine, oxymatrine, bergenin, and pterostilbene in the chromatograms of different batches of single-ingredient and combined-ingredient solutions. Calculate the content of the five components in the samples. The results are shown in Table 15.

[0153] Table 15. Results of determination of five active ingredients in 10 batches of samples of *Ardisia crenata*, *Sophora tonkinensis*, and *Ardisia crenata-Sophora tonkinensis*.

[0154]

[0155] 4. Summary of Detection Methods

[0156] 4.1 Selection of chromatographic conditions

[0157] This study optimized the mobile phase type (acetonitrile-0.2% phosphoric acid aqueous solution, methanol-0.2% phosphoric acid aqueous solution), extraction method (ultrasonic extraction, reflux extraction), and solid-liquid ratio (0.75:20, 1:20, 2:20). The maximum absorption wavelength obtained from full-wavelength scanning was used as the chromatographic detection wavelength. Taking into account peak shape, resolution, and analysis time, the optimal chromatographic conditions were finally selected. See Figures 13 to 15 .

[0158] 4.2 Sample Batch Selection

[0159] In this study, fingerprint profiles of different batches of Cinnamomum cassia and Sophora tonkinensis were constructed for testing when they were combined. Different batches of samples meant that the origin, processing method, drying method, and storage method of the medicinal slices were different, so the content of different batches of the combination was different.

[0160] 4.3 Selection of Indicator Components

[0161] By comparing the chromatograms of the single-decocted, combined-decocted, and mixed reference solutions, 10 batches of *Ardisia crenata*, *Sophora tonkinensis*, and *Ardisia crenata*-Sophora tonkinensis* herbal pairs were identified, with common peaks of 5, 11, and 14, respectively. Chromatin, matrine, oxymatrine, bergenin, and trifolin stigmacin were also identified as chromatographic peaks. Based on literature review and pharmacological studies of active components of traditional Chinese medicine, alkaloids are the main components and active ingredients of *Sophora tonkinensis*. Matrine and oxymatrine are listed in the 2020 edition of the *Chinese Pharmacopoeia*. The target components, which are controlled in terms of quantity, possess pharmacological effects such as antiviral, anti-inflammatory, immunomodulatory, and antitumor activity. Besides alkaloids, flavonoids are also among the active components of *Sophora tonkinensis*. Pterostilbene trifoliata has pharmacological effects including antitumor, anti-inflammatory, antioxidant, antibacterial, antiviral, and anti-cardiovascular disease activity. Bergenin and gallic acid in *Ardisia crenata* have anti-inflammatory, antibacterial, and antiviral pharmacological effects. Based on the consistency between the pharmacological activities of these components and the therapeutic indications of the *Ardisia crenata*-*Sophora tonkinensis* herbal pair, this study conducted a content determination study on the above five components. The content determination results showed that the content of the target components varied among different batches of samples. The total content of matrine and oxymatrine in the single-decocted herbs was significantly lower than that in the combined-decocted herbs. Studies have shown a positive correlation between the degree of toxicity and the content of toxic components. Therefore, this component was selected as the quality control standard to provide a foundation for the quality control of the *Ardisia crenata*-*Sophora tonkinensis* herbal pair and its preparations, and to provide a reference for quality standards and subsequent related research.

[0162] 4.4 Changes in HPLC fingerprints and main chemical components before and after compatibility and their significance

[0163] This study found significant changes in the fingerprint spectrum after the combination of medicinal pairs, with most chemical components showing an increase in content and a few components showing a decrease. Specifically, oxymatrine underwent conversion and was partially reduced after decoction, indicating that the dissolution of the main active components of *Dioscorea opposita* (Zhu Shan Yao) pairs affects each other. However, this change should be understood dialectically and comprehensively; the increase or decrease in content does not necessarily mean an increase or decrease in efficacy after combination. This aligns with the traditional Chinese medicine theory of "restraining the reduction of toxicity" in medicinal pairings and has practical value in clinical application.

[0164] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for establishing the fingerprint spectrum of a medicinal pair of *Ardisia crenata* and *Sophora tonkinensis*, characterized in that, The method employs HPLC-DAD fingerprinting detection and includes the following steps: (1) Preparation of reference solutions: Accurately weigh appropriate amounts of gallic acid, matrine, oxymatrine, bergenin, and pterostilbene, place them in volumetric flasks, add 10% methanol solution to dissolve them completely, and then make up to volume to obtain the stock solutions of each reference standard; accurately measure appropriate amounts of each stock solution of the reference standard and dilute with 10% methanol solution to prepare a mixed reference solution. (2) Preparation of the test solution: Preparation of freeze-dried powder by decoction: 10g of each of the powders obtained after pulverizing 10 batches of cinnabar root and sophora root slices through a No. 2 sieve were accurately weighed and placed in round-bottom flasks. 8 times the amount of water was added and soaked for 4 hours. The flasks were decocted twice, the first time for 2 hours and the second time for 1 hour. The two decoctions were combined and filtered through 8 layers of gauze. The filtrate was concentrated to near dryness in a 70℃ water bath to obtain a concentrated solution. The concentrated solution was pre-frozen at -80℃ for 8 hours and then freeze-dried to obtain freeze-dried powder of cinnabar root and sophora root. Preparation of freeze-dried powder by decoction: 10g of each of the powders obtained after pulverizing 10 batches of cinnabar root and sophora root slices through a No. 2 sieve are accurately weighed, mixed, and prepared according to the method for preparing freeze-dried powder by decoction. The freeze-dried powder of cinnabar root and sophora root by decoction is obtained. Accurately weigh 0.05 g of single-decoction lyophilized powder and 0.1 g of combined-decoction lyophilized powder, place them in a stoppered conical flask, accurately add 20 mL of 10% methanol solution, seal tightly, weigh, and sonicate for 30 min at a power of 500 W and a frequency of 40 kHz. Cool, replenish the lost mass with 10% methanol solution, shake well, and filter the supernatant through a 0.22 μm microporous membrane. Collect the filtrate to obtain the test solution. (3) Establishment of fingerprint chromatogram: The reference solution and the test solution were detected by high performance liquid chromatography, and the chromatograms were recorded to establish the fingerprint chromatogram of the cinnabar root and sophora root pair; the chromatographic conditions are as follows: Chromatographic column: Agilent InfinityLab Poroshell HPH C18 column, 250 mm × 4.6 mm, 4 μm; mobile phase A: acetonitrile, mobile phase B: 0.2% phosphoric acid solution; gradient elution; flow rate: 0.7 mL / min. -1 The detection wavelength was 210 nm; the column temperature was 30℃; and the injection volume was 10 μL. The gradient elution program is as follows: 0~14 min, 5% A; 14~35 min, 5%~30% A; 35~40 min, 30%~50% A; 40~50 min, 50%~60% A.

2. A method for determining the content of multiple indicators in a medicinal pair of *Ardisia crenata* and *Sophora tonkinensis*, characterized in that, The method includes the following steps: (1) Preparation of reference solution: Accurately weigh appropriate amounts of gallic acid, matrine, oxymatrine, bergenin, and pterostilbene, place them in volumetric flasks respectively, add solvent to dissolve them completely, and then dilute to volume to prepare a solution with a mass concentration of 1-2 mg / mL. -1 Prepare stock solutions of each reference standard; accurately measure an appropriate amount of each stock solution of the reference standard and dilute it with a solvent to prepare a mixed reference standard solution; (2) Preparation of the test solution: Preparation of freeze-dried powder by decoction: Accurately weigh 10g each of powdered cinnabar root and sophora root, place them in round-bottom flasks, add 8 times the amount of water and soak for 4 hours, decoct twice, the first decoction for 2 hours and the second decoction for 1 hour, combine the two decoction liquids, filter with gauze, concentrate, freeze dry to obtain freeze-dried powder of cinnabar root and sophora root. Preparation of combined decoction freeze-dried powder: Accurately weigh 10g each of the powdered slices of Cinnamomum cassia and Sophora tonkinensis, mix them, and proceed according to the preparation method of single decoction freeze-dried powder to obtain the combined decoction freeze-dried powder of Cinnamomum cassia and Sophora tonkinensis. Take the single-decoction lyophilized powder and the combined-decoction lyophilized powder, weigh them accurately, place them in a stoppered conical flask, add 10% methanol solution to dissolve them accurately, stopper tightly, weigh them, sonicate for 30 min, cool them, make up the lost mass with 10% methanol solution, shake well, take the supernatant and filter it through a 0.22 μm microporous membrane, take the filtrate, and you will get the test solution. (3) Content determination: The content of the reference solution and the test solution was determined by high performance liquid chromatography (HPLC); the chromatographic conditions are as follows: Chromatographic column: Agilent InfinityLab Poroshell HPH C18 column, 250 mm × 4.6 mm, 4 μm; mobile phase A: acetonitrile, mobile phase B: 0.2% phosphoric acid solution; gradient elution; column temperature: 30℃; flow rate: 0.7 mL / min. -1 The detection wavelength was 210 nm; the column temperature was 30℃; and the injection volume was 10 μL. The gradient elution procedure is as follows: 0~14 min, 5% A; 14~35 min, 5%~30% A; 35~40 min, 30%~50% A; 40~50 min, 50%~60% A.

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