Quality control method and application of alhagi sparsifolia effective component for treating ulcerative colitis

Abstract

The invention provides a quality control method of an effective component of alhagi sparsifolia for treating ulcerative colitis and application of the quality control method, and the method comprises the following steps: characterizing the material composition of the effective component of alhagi sparsifolia by using an ultra-high performance liquid chromatography-mass spectrometry technology, and determining key active components by combining network pharmacology prediction and topological analysis; the quality internal control method for the effective components of the alhagi sparsifolia for treating the ulcerative colitis is constructed by adopting a high performance liquid chromatography technology and a thin-layer chromatography technology, the material information of the effective components of the alhagi sparsifolia can be comprehensively reflected, the quality of the effective components of the alhagi sparsifolia in different batches can be effectively monitored, and the method is high in precision, good in reproducibility and suitable for industrial production. The method can be used for controlling the quality of the effective components of the alhagi sparsifolia for treating ulcerative colitis, and provides a basis for solving the problem that the existing standard cannot comprehensively and systematically analyze the material composition and content control of the effective components of the alhagi sparsifolia.

Description

Technical Field

[0001] The present invention relates to the field of quality control of effective components of traditional Chinese medicine, and particularly to a method for quality control of effective components of Alhagi sparsifolia for treating ulcerative colitis and its application. Background Art

[0002] The effective components of Alhagi sparsifolia for treating ulcerative colitis are obtained by removing the fat-soluble small molecule substances from the medicinal residues of Alhagi sparsifolia with ethanol, then extracting by heating under reflux with water, concentrating the extract under reduced pressure, adding ethanol with a volume ratio concentration of 80%, standing for precipitation and impurity removal, and taking the supernatant after centrifugation for concentration and drying. This component has been verified by network pharmacology prediction, acute ulcerative colitis experiments in mice and an inflammation model of rat small intestinal crypt epithelial cells to exert the pharmacological activities of effectively inhibiting the inflammatory expression of acute ulcerative colitis and protecting the intestinal mucosa injury of ulcerative colitis. Due to the complex chemical composition of this effective component, there is currently a lack of scientific quality control methods. Therefore, the present invention uses ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) technology to analyze the component composition of the effective components of Alhagi sparsifolia as a whole, combines network pharmacology prediction to lock key active components, and constructs its quality control method based on the content detection results of multiple batches of effective components of Alhagi sparsifolia, providing a scientific basis for maintaining the stability, effectiveness and quality controllability of different batches of effective components of Alhagi sparsifolia and their pharmaceutical preparations.

[0003] After retrieval:

[0004] 1. Senawar Mangsir. Research on the quality standard of Alhagi sparsifolia medicinal materials and the preparation of chemical reference substances [D], June 2012.

[0005] 2. Senawar Mangsir, Han Kefei, Aimaitijiang Aiyubieke. Research on the quality standard of Alhagi sparsifolia medicinal materials [J]. Herald of Medicine, 2012, 31(8): 1075-1076

[0006] Literature 1.2 jointly describes the determination of the content of isorhamnetin in Alhagi sparsifolia Shap. by HPLC. The sample preparation method is described as follows: "After the Alhagi sparsifolia Shap. medicinal material is ground and passed through a No. 3 sieve, about 2.0 g of the powder is precisely weighed and placed in a stoppered conical flask. Then, 100 ml of 70% methanol is precisely added. After ultrasonic treatment for 30 minutes, cooling, and filtration, the filtrate is concentrated. 25 ml of a mixed solution of methanol - 25% hydrochloric acid solution in a ratio of (4:1) is added. After heating under reflux for 60 minutes and cooling, it is transferred to a 50 ml volumetric flask, then methanol is added to the scale and shaken well, and then filtered through a 0.22 μm filter membrane." In the method, the medicinal material is treated by ultrasonic treatment with 70% methanol, cooling, filtration, and then the filtrate is concentrated again. This process is inconsistent with that in this patent. The more pretreatment steps there are and the more complex the steps are before the determination of the content of the medicinal material, the more loss of the content of the quality marker will occur, which will inevitably lead to inaccurate content of the quality marker. The present invention removes the processes of ultrasonic extraction, filtration, and concentration, and directly uses methanol - 25% hydrochloric acid solution (4:1) for pretreatment, and the measured content of isorhamnetin is more accurate.

[0007] 3. Sainawar Mangsir, Alimujiang Abliz, Zou Guo'an. Study on the HPLC fingerprint of Alhagi sparsifolia Shap. [J]. Arid Zone Research, 2013, 30(05): 873 - 876.

[0008] Literature 3 describes the study on the HPLC fingerprint of Alhagi sparsifolia Shap. Using the HPLC method, isorhamnetin 3 - O - β - D - rutinoside and isorhamnetin - 3 - O - β - D - galactose - (6→1)-α - L - rhamnoside are used as quality markers, which are different from the quality markers in the present invention. The mobile phase of the described chromatographic conditions is acetonitrile (A) - 0.2% formic acid (B), and the gradient elution program is 5% (A) for 0 - 5 min, 5 - 13% (A) for 5 - 15 min, 13 - 30% (A) for 15 - 75 min, 30 - 60% (A) for 75 - 85 min, 60 - 100% (A) for 85 - 95 min, 100% (A) for 95 - 100 min, and the detection wavelength is 280 nm. The above chromatographic conditions are inconsistent with those of the present invention.

[0009] 4. Bao Shuang. Isolation of active components against cervical cancer in Alhagi sparsifolia Shap. and establishment of their content detection methods [D], 2024.5.

[0010] Literature 4 describes the establishment of HPLC content detection methods for two components, alhagiine and narcissin, in Alhagi sparsifolia Shap. The quality markers are alhagiine and narcissin, which are different from the quality markers in the present invention. The biological activities of these two components are against cervical cancer, rather than ulcerative colitis.

[0011] 5. Chen Liang, Xu Xiaoqin, Caesar Sulaiman, et al. Determination of Total Reducing Sugars and Rutin in Alhagi sparsifolia Shap.[J]. China Pharmacy, 2012, 23(47): 4474-4476.

[0012] 6. Chen Liang, Chen Gang, Xu Xiaoqin. Determination of Total Reducing Sugars and Rutin in Different Parts of Alhagi sparsifolia Shap.[J]. Chinese Journal of Experimental Traditional Medical Formulae, 2014, 20(11): 66-69.

[0013] In References 5 and 6, HPLC was used to determine the content of rutin in Alhagi sparsifolia Shap. The quality marker is rutin, not isorhamnetin.

[0014] 7. Xu Xiaoqin, Wei Hongyan, Shi Leiling, et al. Study on the Quality Standard of Uygur Medicine Alhagi sparsifolia Shap.[J]. Research and Practice on Chinese Medicines, 2012, 26(06): 68-71.

[0015] Reference 6 describes the establishment of an HPLC method for determining the content of rutin in Alhagi sparsifolia Shap. The quality marker is rutin, not isorhamnetin.

[0016] 8. Hu Shuchen. Study on the Chemical Constituents of the Petroleum Ether Fraction of Senecio densiserratus Wall. and the Establishment of the Quality Standard of Uygur Medicine Alhagi sparsifolia Shap[D]. Lanzhou University, 2010.

[0017] References 7 and 8 describe the establishment of a method for determining the content of isorhamnetin-3-O-rutinoside by HPLC chromatography. The quality marker is isorhamnetin-3-O-rutinoside, not isorhamnetin. And the detection wavelength in the method is 254 nm. The literature describes the chromatographic conditions as gradient elution with methanol-acetonitrile-0.2% formic acid, which is different from the mobile phase composition and ratio of the present invention.

[0018] 9. Zou Zhizhen, Deng Xiling, Wang Yunlai, et al. Exploring the Mechanism of Action of Alhagi sparsifolia Shap in the Treatment of Sepsis Based on Network Pharmacology and Experimental Verification[J]. World Science and Technology - Modernization of Traditional Chinese Medicine, 2023, 25(09): 3024-3036.

[0019] Reference 9 describes the exploration of the mechanism of action of Alhagi sparsifolia Shap in the treatment of sepsis based on network pharmacology and experimental verification. 30 active ingredients of Alhagi sparsifolia Shap were screened out based on the co-targeting of sepsis. The screened active ingredients are based on the disease of sepsis, not ulcerative colitis. Sepsis is not a commonly used clinical disease. The traditional historical application of Alhagi sparsifolia Shap is for intestinal diseases. The active ingredients screened in Reference 9 are different from those screened in the present invention.

[0020] 10. Xu Xiaoqin; Qing Degang; Chen Liang; Zhang Juan; Sun Yu; Xia Tiguli A buz; Effects of Alhagi pseudalhagi Extract on NLRP3 Inflammasome and Related Cytokines in LPS-Induced IEC-6 Cell Injury Model [J], Journal of Xinjiang Medical University, 2024, 47(5): 740-745.

[0021] 11. Xiaoqin Xu; Juan Zhang; Liang Chen; Yu Sun; Degang Qing; Xuelei Xin; Chunyan Yan; Alhagi pseudalhagi Extract Exerts Protective Effects Against Intestinal Inflammation in Ulcerative Colitis by Affecting TLR4-Dependent NF-κB Signaling Pathways, Frontiers in Pharmacology, 2021, 12(1): 1-12.

[0022] References 10 and 11 describe the preparation method, pharmacological effects and molecular mechanism of the effective components of Alhagi pseudalhagi in the treatment of ulcerative colitis in the present invention.

[0023] Based on the above literature descriptions, the present invention establishes the screening, determination and quality control of the active ingredients in the effective components of Alhagi pseudalhagi for the treatment of ulcerative colitis, providing a quality control method for effectively controlling the effective components of ulcerative colitis. Summary of the Invention

[0024] The object of the present invention is to provide a quality control method and application of the effective components of Alhagi pseudalhagi for the treatment of ulcerative colitis. This method uses ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) technology to characterize the substance composition of the effective components of Alhagi pseudalhagi, combines network pharmacology prediction and topological analysis to determine the key active ingredients, and uses high performance liquid chromatography and thin layer chromatography technologies to construct an internal quality control method for the effective components of Alhagi pseudalhagi. It can comprehensively reflect the substance information of the effective components of Alhagi pseudalhagi, effectively monitor the quality of the effective components of Alhagi pseudalhagi in different batches. This method has high precision and good reproducibility, and is used for the quality control of the effective components of Alhagi pseudalhagi, providing a scientific basis for solving the problem that the existing standards cannot comprehensively and systematically analyze and determine the substance composition and content control of the effective components of Alhagi pseudalhagi.

[0025] The quality control method of the effective components of Alhagi pseudalhagi for the treatment of ulcerative colitis described in the present invention adopts high performance liquid chromatography or thin layer chromatography, and the specific operation is carried out according to the following steps:

[0026] a. Analyze the chemical composition of the effective components of Alhagi sparsifolia by ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS), and 105 chemical components are obtained;

[0027] b. Perform network pharmacology prediction on the chemical components obtained in step a, sort them according to the correlation between the components and diseases, and screen out 9 key active components, namely: epigallocatechin gallate, medicarpin, butein, galangin, quercetin, kaempferol, isorhamnetin, diosmetin, and wogonin;

[0028] High performance liquid chromatography

[0029] c. Preparation of reference solution: Accurately weigh 5.0 mg of isorhamnetin reference substance, add methanol to make a solution of 0.5 mg·mL -1 . Filter through a 0.22 μm filter membrane to obtain the reference solution;

[0030] d. Preparation of test solution: Accurately weigh 1.0 g of the effective components of Alhagi sparsifolia, accurately add 25 mL of methanol-25% hydrochloric acid solution with a volume ratio of 4:1, heat under reflux in a water bath at 70 °C for 1 hour, filter, then add methanol to volume in a 25 mL volumetric flask, shake well, filter through a 0.22 μm filter membrane, and take the filtrate to obtain the test solution;

[0031] e. Chromatographic analysis: Use a chromatographic column filled with octadecylsilane-bonded silica gel, mobile phase: methanol-0.4% phosphoric acid solution with a volume ratio of 55:45, detection wavelength is 360 nm, flow rate is 1.0 mL·min -1 , column temperature is 30 °C, injection volume is 10 μL;

[0032] f. Determination: Accurately pipette 10 μL of the reference solution and the test solution respectively, inject them into the high performance liquid chromatograph, and the obtained target chromatographic peaks include isorhamnetin. The retention time of the isorhamnetin chromatographic peak is 19 - 20 min. Determine and calculate the percentage mass of the effective components of Alhagi sparsifolia;

[0033] Thin layer chromatography 1:

[0034] a. Accurately weigh 20 mg of the effective components of Alhagi sparsifolia, dissolve with 70% methanol and volume to 10 mL in a volumetric flask. Transfer the solution to a 50 mL beaker, concentrate to dryness in a water bath at 60 °C. Add 5 mL of 70% methanol-25% hydrochloric acid mixed solution with a volume ratio of 4:1 to the residue, heat and hydrolyze in a water bath at 60 °C for 1 hour, then immediately cool in pure water at 15 °C, filter the hydrolyzate, transfer the filtrate to a 5 mL volumetric flask, and then volume to the scale with methanol, shake well to obtain the test solution of the effective components of Alhagi sparsifolia;

[0035] b. Accurately weigh the isorhamnetin reference substance, and dissolve it in methanol to prepare a solution with a concentration of 0.4 mg·mL -1 as the reference substance solution;

[0036] c. Accurately weigh 2.5 g of Alhagi sparsifolia powder, place it in a 150 mL stoppered conical flask, accurately add 100 mL of 70% methanol solution, extract with ultrasonic wave at 40 Hz power for 30 min, cool to room temperature, filter by suction, place the filtrate in a water bath at 60 °C until dry, add 25 mL of a 70% methanol - 25% hydrochloric acid mixed solution with a volume ratio of 4:1 to the residue, heat and hydrolyze in a water bath at 60 °C for 1 hour, immediately cool in pure water at 15 °C, filter the hydrolyzed solution, transfer the filtrate to a 25 mL volumetric flask, make up the volume to the mark with methanol, shake well to obtain the Alhagi sparsifolia medicinal material reference solution;

[0037] d. Pipette 10 μL of the Alhagi sparsifolia effective component test solution obtained in step a, 2 μL of the isorhamnetin reference substance solution obtained in step b, and 10 μL of the Alhagi sparsifolia reference medicinal material solution obtained in step c, and spot them on the same silica gel GF 254 thin - layer plate. Use chloroform - methanol - formic acid with a volume ratio of 9:1:0.5 as the developing agent. After saturating for 20 min under closed conditions, develop, air - dry naturally, spray with 1% aluminum trichloride ethanol solution for color development, heat at 60 °C for 5 min until the spots are clearly developed, and examine under a 365 nm ultraviolet lamp. In the test solution chromatogram, fluorescent spots of the same color appear at the corresponding positions of the reference substance chromatogram and the reference medicinal material chromatogram;

[0038] Thin - layer chromatography method 2:

[0039] a. Accurately weigh 40 mg of the Alhagi sparsifolia effective component, add 5 mL of 75% methanol and 0.2 mL of hydrochloric acid, heat and hydrolyze for 1 hour, filter, evaporate the filtrate to dryness, dissolve the residue in 5 mL of water, extract with ethyl acetate twice, 5 mL each time, combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue in 1 mL of methanol to obtain the Alhagi sparsifolia effective component test solution;

[0040] b. Accurately weigh the kaempferol and quercetin reference substances, and dissolve them in methanol respectively to prepare solutions with a concentration of 0.5 mg·mL -1 as the reference substance solutions;

[0041] c. Accurately weigh 2.0 g of Alhagi sparsifolia medicinal material, add 25 mL of 75% methanol and 1 mL of hydrochloric acid, heat and hydrolyze for 1 hour, filter, evaporate the filtrate in a water bath at 60 °C to dryness, dissolve the residue in 10 mL of water, extract with ethyl acetate twice, 10 mL each time, combine the ethyl acetate extracts, evaporate to dryness at 60 °C, dissolve the residue in 2 mL of methanol to obtain the Alhagi sparsifolia reference medicinal material solution;

[0042] d. Pipette 10 μL of the test solution of the effective components of Alhagi sparsifolia obtained in step a, 5 μL each of the control solutions of kaempferol and quercetin obtained in step b, and 10 μL of the solution of the control crude drug of Alhagi sparsifolia obtained in step c onto the same silica gel G thin-layer plate. Using xylene - ethyl acetate - formic acid with a volume ratio of 10:8:1 as the developing solvent, saturate for 20 min under closed conditions, then develop, take out, air-dry, spray with 3% aluminum trichloride ethanol solution, heat at 105 °C for 5 min, and examine under a 365 nm ultraviolet lamp. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as those in the chromatograms of the control substances and the control crude drug.

[0043] Use of the method for quality control of the effective components of Alhagi sparsifolia for treating ulcerative colitis in detecting or identifying chemical components in Alhagi sparsifolia including isorhamnetin, kaempferol, and quercetin.

[0044] The method for quality control of the effective components of Alhagi sparsifolia for treating ulcerative colitis according to the present invention, and the preparation method of the effective components of Alhagi sparsifolia involved in this method is prepared according to the following steps:

[0045] Weigh 10 kg of the crude powder of Alhagi sparsifolia, extract with 10 times the amount of 95% ethanol for 1 hour, discard the small molecule alcohol-soluble substances, dry the drug residue at 60 °C, then add distilled water according to a solid - liquid ratio of 1:18, reflux and extract for 1.0 hour, extract 3 times, combine the extracts, filter, concentrate the filtrate under reduced pressure at 60 °C and 0.08 Mpa, with a concentration volume ratio of 5:1. Add ethanol to the concentrated solution to make the alcohol precipitation volume concentration 80%, refrigerate and stand for 6 hours, then centrifuge at 3500 r / min at room temperature for 5 min, collect the upper layer of the medicinal liquid, concentrate under reduced pressure at 60 °C, and dry to obtain the Alhagi sparsifolia component (APE2).

[0046] Compared with the prior art, the beneficial effects of the present invention are:

[0047] The present invention uses ultra-high performance liquid chromatography - mass spectrometry (UPLC-QE-MS) technology to characterize the substance composition of the effective components of Alhagi sparsifolia, combines network pharmacology prediction and topological analysis to determine the key active components, and uses high performance liquid chromatography and thin layer chromatography to construct a set of internal quality control methods for the effective components of Alhagi sparsifolia. As a whole, it can comprehensively reflect the substance information of the effective components of Alhagi sparsifolia, monitor the quality of the effective components in different batches. This method has high precision and good reproducibility, and can be used for the quality control of the effective components of Alhagi sparsifolia for treating ulcerative colitis.

[0048] The method is simple, fast and easy to operate. The method for determining the composition and content of the effective components of Alhagi sparsifolia is more comprehensive and systematic than the traditional method, and has a high correlation with ulcerative colitis. It can comprehensively reflect the overall material characteristics of the effective components of Alhagi sparsifolia in the treatment of ulcerative colitis, scientifically evaluate its quality, and improve the characteristics of uniformity, stability and effectiveness of the effective components. Description of the Drawings

[0049] Figure 1 This is the TIC diagram of the effective components of Alhagi sparsifolia under the positive and negative ion modes of the present invention, where A: total ion current diagram under positive ion mode; B: total ion current diagram under negative ion mode;

[0050] Figure 2 This is the Venn diagram of the mapping between different gene groups of the present invention, where a. the mutual mapping diagram of disease genes and marketed drug targets; b. the mutual mapping diagram of disease genes, marketed drug targets and potential targets of traditional Chinese medicine components of Alhagi sparsifolia; A disease genes (Genecards database), B differentially expressed genes (GEO databases GSE3365, GSE11223), C marketed drug targets (Drugbank database), D marketed drug targets (KEGG database), E disease gene set (A&B), F marketed drug targets (C&D), G potential targets of Alhagi sparsifolia (TCMSP, ETCM, Pubchem databases);

[0051] Figure 3 This is the PPI network analysis diagram of the components-targets of Alhagi sparsifolia of the present invention, where the orange diamond nodes represent the components of Alhagi sparsifolia; the blue circular nodes represent the targets acted on by the components of Alhagi sparsifolia, and the pink circular nodes represent the common targets of the potential targets of Alhagi sparsifolia and the disease targets of ulcerative colitis. The links between the nodes represent their relationships;

[0052] Figure 4 This is the HPLC content determination chromatogram of the effective components of Alhagi sparsifolia of the present invention, where 1. isorhamnetin reference substance; 2. effective components of Alhagi sparsifolia; 3. Alhagi sparsifolia medicinal materials; 4. negative reference substance;

[0053] Figure 5 This is the TLC chromatogram for the qualitative analysis of isorhamnetin in the effective components of Alhagi sparsifolia of the present invention, where 1-3 batches of effective components of Alhagi sparsifolia; 4. Alhagi sparsifolia medicinal materials; 5. isorhamnetin reference substance;

[0054] Figure 6 This is the TLC chromatogram for the qualitative analysis of kaempferol and quercetin in the effective components of Alhagi sparsifolia of the present invention, where A. developed with 3% aluminum trichloride ethanol solution and inspected under natural light; B. developed with 3% aluminum trichloride ethanol solution and inspected under a 365 nm ultraviolet lamp; 1-3 batches of effective components of Alhagi sparsifolia; 4. Alhagi sparsifolia medicinal materials; 5. kaempferol reference substance; 6. quercetin reference substance. Detailed implementation mode

[0055] Example 1

[0056] a. Analyze the chemical composition of the active components of Alhagi sparsifolia by ultra-high performance liquid chromatography-mass spectrometry (UPLC-QE-MS) to obtain 105 chemical components;

[0057] b. Combine the chemical components obtained in step a with the reported active components of Alhagi sparsifolia for network pharmacology prediction, sort according to the relevance between the components and diseases, and screen out 9 key active components, namely: epigallocatechin gallate, medicarpin, brazilin, galangin, quercetin, kaempferol, isorhamnetin, diosmetin and wogonin;

[0058] High performance liquid chromatography

[0059] c. Preparation of reference substance solution: Weigh accurately 5.0 mg of isorhamnetin reference substance, add methanol to make a solution of 0.5074 mg·mL -1 , filter through a 0.22 μm filter membrane to obtain the reference substance solution;

[0060] d. Preparation of test solution: Weigh accurately 1.0 g of the active components of Alhagi sparsifolia, accurately add 25 mL of methanol-25% hydrochloric acid solution with a volume ratio of 4:1, heat under reflux in a water bath at 70 °C for 1 hour, filter, and then add methanol to a constant volume in a 25 mL volumetric flask, shake well, filter through a 0.22 μm filter membrane, and take the filtrate to obtain the test solution;

[0061] e. Chromatographic analysis: A chromatographic column filled with octadecylsilane-bonded silica gel, mobile phase: methanol-0.4% phosphoric acid solution with a volume ratio of 55:45, detection wavelength is 360 nm, flow rate is 1.0 mL·min -1 , column temperature is 30 °C, injection volume is 10 μL;

[0062] f. Determination: Accurately pipette 10 μL of the reference substance solution and the test solution respectively, inject into the high performance liquid chromatograph, the obtained target chromatographic peaks include isorhamnetin, the retention time of the isorhamnetin chromatographic peak is 19 - 20 min, measure and calculate the percentage mass of the active components of Alhagi sparsifolia;

[0063] Thin layer chromatography 1:

[0064] a. Accurately weigh three portions of the effective components of Alhagi sparsifolia, namely 20.43 mg, 20.35 mg, and 20.28 mg. Dissolve them separately in 70% methanol and dilute to a volume of 10 mL in a volumetric flask. Transfer the solution to a 50 mL beaker, and concentrate it to dryness in a water bath at 60 °C. Add 5 mL of a mixed solution of 70% methanol - 25% hydrochloric acid with a volume ratio of 4:1 to the residue, heat and hydrolyze it in a water bath at 60 °C for 1 hour, then immediately cool it in pure water at 15 °C. Filter the hydrolyzate, transfer the filtrate to a 5 mL volumetric flask, and dilute it to the mark with methanol. Shake well to obtain the test solution of the effective components of Alhagi sparsifolia;

[0065] b. Accurately weigh 4.60 mg of isorhamnetin reference substance, dissolve it in methanol and dilute to a volume of 10 mL in a volumetric flask to prepare a solution with a concentration of 0.4 mg·mL -1 as the reference solution;

[0066] c. Accurately weigh 2.50558 g of Alhagi sparsifolia powder, place it in a 150 mL stoppered conical flask, accurately add 100 mL of 70% methanol solution, extract it by ultrasonic wave at 40 Hz for 30 min, then cool it to room temperature, filter by suction. Place the filtrate in a water bath at 60 °C until dry. Add 25 mL of a mixed solution of 70% methanol - 25% hydrochloric acid with a volume ratio of 4:1 to the residue, heat and hydrolyze it in a water bath at 60 °C for 1 hour, then immediately cool it in pure water at 15 °C. Filter the hydrolyzate, transfer the filtrate to a 25 mL volumetric flask, and dilute it to the mark with methanol. Shake well to obtain the control solution of Alhagi sparsifolia medicinal material;

[0067] d. Pipette 10 μL of the test solution of the effective components of Alhagi sparsifolia obtained in step a, 2 μL of the isorhamnetin reference solution obtained in step b, and 10 μL of the control solution of Alhagi sparsifolia medicinal material obtained in step c, and spot them on the same silica gel GF 254 thin-layer plate. Use a developing agent of chloroform - methanol - formic acid with a volume ratio of 9:1:0.5. Saturate it for 20 min under closed conditions, then develop. After air-drying naturally, spray it with 1% aluminum trichloride ethanol solution for color development, heat it at 60 °C for 5 min until the spots are clearly developed, and examine it under a 365 nm ultraviolet lamp. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as those in the chromatograms of the reference substance and the control medicinal material;

[0068] Thin-layer chromatography method 2:

[0069] a. Accurately weigh 40.05 mg, 40.06 mg, and 40.08 mg of the effective components of Alhagi sparsifolia, add 5 mL of 75% methanol and 0.2 mL of hydrochloric acid respectively, heat and hydrolyze for 1 hour, filter, evaporate the filtrate to dryness, dissolve the residue in 5 mL of water, extract it twice with 5 mL of ethyl acetate each time, combine the ethyl acetate solutions, evaporate to dryness, and dissolve the residue in 1 mL of methanol to obtain the test solution of the effective components of Alhagi sparsifolia;

[0070] b. Accurately weigh 2.57 mg of kaempferol reference substance and 2.52 mg of quercetin reference substance, and separately dissolve them in methanol to prepare solutions with a concentration of 0.5 mg·mL -1 for use as reference substance solutions;

[0071] c. Accurately weigh 2.00567 g of Alhagi sparsifolia Shap. medicinal material, add 25 mL of 75% methanol and 1 mL of hydrochloric acid, heat for hydrolysis for 1 hour, filter, evaporate the filtrate to dryness in a water bath at 60°C, dissolve the residue in 10 mL of water, extract with ethyl acetate twice, 10 mL each time, combine the ethyl acetate extracts, evaporate to dryness at 60°C, dissolve the residue in 2 mL of methanol for use as the reference medicinal material solution of Alhagi sparsifolia Shap.;

[0072] d. Pipette 10 μL of the test solution of the active components of Alhagi sparsifolia Shap. obtained in step a, 5 μL each of the kaempferol and quercetin reference substance solutions obtained in step b, and 10 μL of the reference medicinal material solution of Alhagi sparsifolia Shap. obtained in step c, and spot them on the same silica gel G thin-layer plate respectively. Use xylene - ethyl acetate - formic acid with a volume ratio of 10:8:1 as the developing agent. After saturation for 20 min under closed conditions, develop, take out, air-dry, spray with 3% aluminum trichloride ethanol solution, heat at 105°C for 5 min, and examine under a 365 nm ultraviolet lamp. In the test sample chromatogram, fluorescent spots of the same color appear at the corresponding positions of the reference substance chromatogram and the reference medicinal material chromatogram.

[0073] Example 2

[0074] 1. Analysis of the chemical components of the active components (APE2) of Alhagi sparsifolia Shap.;

[0075] 1.1 Establishment of an ultra-high performance liquid chromatography - mass spectrometry (UPLC-QE-MS) analysis method;

[0076] (1) Detection conditions:

[0077] Ultra-high performance liquid chromatography detection conditions: The chromatographic column is Waters UPLC BEH Amide C 18 (2.1 mm × 100 mm, 1.7 μm), the column temperature is 30°C, and the flow rate is 0.4 mL·min -1, the injection volume was 5 μL; the mobile phase was 0.1% formic acid aqueous solution (A) and 0.1% formic acid acetonitrile aqueous solution (B); the binary linear gradient elution program was 0 - 3.5 min, 95 - 85% A; 3.5 - 6 min, 85 - 70% A; 6 - 6.5 min, 70 - 70% A; 6.5 - 12 min, 70 - 30% A; 12 - 12.5 min, 30 - 30% A; 12.5 - 18 min, 30 - 0% A; 18 - 25 min 0 - 0% A; 25 - 26 min, 0 - 95% A; 26 - 30 min 95 - 95% A;

[0078] Mass spectrometry detection conditions: The ion source was an electrospray ionization source (ESI), detected in positive and negative ion modes, collected based on the information-dependent acquisition mode (IDA), using a Q Exactive Focus mass spectrometer combined with data acquisition and analysis software (Xcalibur 4.0, Thermo Fisher Scientific Inc., USA), and performing primary and secondary mass spectrometry data acquisition based on the FullScan-ddMS2 function; in each acquisition cycle, the mass spectrometry acquisition range (m / z) was between 100 - 1500, screening the top 3 mass spectra in each cycle, and further obtaining the corresponding secondary mass spectrometry data;

[0079] The mass spectrometry parameters were as follows: The sheath gas was nitrogen, flow rate: 45 arb, the auxiliary gas was nitrogen, flow rate: 15 arb, the ion source temperature: 400 °C, the full-scan MS resolution: 70000 FWHM (m / z 200), the secondary resolution FWHM (m / z 200): 17500, the collision energy multi-stage NCE mode: 15, 30, 45 eV, the spray voltage: 4.0 kV positive ion mode or -3.6 kV negative ion mode;

[0080] 1.2 Preparation of the sample solution:

[0081] Weigh 100 mg of the effective component of Alhagi sparsifolia (APE2), add 1000 μL of the extraction solution (methanol:water = 4:1, L-2-chlorophenylalanine = 1000:10), grind at 45 Hz / 240 s, ultrasonicate in an ice-water bath for 1 h, let stand at -20 °C for 1 h, then centrifuge the sample at 4 °C, 12000 rpm centrifugal force 13800 (×g), radius 8.6 cm for 15 min, take the supernatant, filter it through a 0.22 μm filter membrane, and place it in an injection vial for on-machine detection;

[0082] 1.3 UPLC-QE-MS analysis results of APE2:

[0083] First, the raw mass spectrometry data was imported using XCMS software, and processes such as retention time correction, peak identification, peak extraction, peak integration, and peak alignment were carried out to obtain the positive and negative ion first-order and second-order mass spectrometry diagrams under the electrospray ionization (ESI) condition (see Figure 1 ), taking the quasi-molecular ions and adduct ions [M+H] + , [M+Na] + , M + , M - , [M-H] - and [M+HCOO] - of the compound as references, the exact relative molecular mass of the first-order mass spectrometry was speculated, and compounds within the range of error within 10 ppm (1 ppm = 1×10 -6 ) (δ<10 ppm) were selected. The corresponding molecular formulas were calculated by fitting with software Xcalibur4.0 to complete the preliminary speculation of the molecular formula. Then, based on the first-order mass spectrometry data, second-order mass spectrometry data information, combined with the reference substance information, using the BIOTREE TCM second-order mass spectrometry database and Scifinder, TCMID, TCMSP, CNKI, ChemSpider, PubChem, ChemicalBook databases for retrieval, and the MassBank, Pubchem, HMDB, ChemSpider databases for comparative analysis of the compound composition, see Table 1;

[0084] Table 1 UPLC-QE-MS identification results of APE2

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091] 105 compounds were identified from the effective components (APE2) of Alhagi sparsifolia, including 56 flavonoids, 21 phenylpropanoids, 9 phenolic acids, 10 terpenoids, 6 alkaloids, and 3 other types of compounds.

[0092] Example 3

[0093] Network pharmacology predicts the key active components of the effective components (APE2) of Alhagi sparsifolia:

[0094] Query and collection of Alhagi sparsifolia compound information:

[0095] Search for reported Alhagi sparsifolia compounds through CNKI, VIP, and Wanfang databases, merge the compounds analyzed by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QE-MS) data acquisition, construct a compound database, query the chemical composition information of the above compounds from the Pubchem database, Huayuanwang, and Aihuaxue website, use the FAFDrugs4 database with the Drug-Like soft index combination (physchem Filters), oral bioavailability (OB) ≥ 30%, and drug-likeness (DL) ≥ 0.18 as the screening criteria for active ingredients, and then combine relevant literature to include compounds with reported biological activities and pharmacological effects that do not meet the screening criteria into candidate ingredients;

[0096] Collection of targets of Alhagi sparsifolia, ulcerative colitis (UC), and marketed drugs:

[0097] (1) Obtaining potential targets of Alhagi sparsifolia compounds:

[0098] Use the System Pharmacology Database and Analysis Platform to calculate the action targets of Alhagi sparsifolia components in the TCMSP, ETCM databases, and Pubchem databases; predict targets through MedChem Studio in ETCM, select targets with a confidence index ≥ 0.8, and query compound-protein interaction information from three parts of the Pubchem database: chemical-gene co-occurrence in the literature, three-dimensional structure of protein binding, and bioassay results, and collect potential targets; use the UniprotKB and Retrieve / ID mapping retrieval functions in the Uniprot database and the SEARCH function in the STRING database, limit the species to Homo sapiens, correct the target names, and eliminate active ingredients without targets, so as to obtain information on active ingredients and related target genes and construct a dataset of potential action target information of Alhagi sparsifolia compounds;

[0099] (2) Obtaining targets of ulcerative colitis (UC) disease:

[0100] Search for key disease target information of ulcerative colitis through the GeneCards database, GEO database, and Disgenet database using the keywords UlcerativeColitis, UC, integrate and remove duplicates to construct a dataset of ulcerative colitis disease target information, and then standardize and normalize the potential targets using the STRING and Uniprot databases;

[0101] (3) Obtaining targets of marketed drugs:

[0102] Retrieve the target gene information of marketed drugs for ulcerative colitis (UC) by combining the TTD database, Drugbank database, and KEGG database with the keywords "Ulcerative Colitis, UC", integrate and deduplicate them to construct a target dataset of marketed drugs, and then standardize and normalize the potential targets using the STRING and Uniprot databases;

[0103] (4) Target mapping and confirmation of important targets:

[0104] Mutually map the disease gene GeneCards dataset, GEO dataset with the KEGG dataset and Drugbank dataset of marketed drugs to obtain intersection genes and key targets of marketed drugs; then deduplicate and merge the diseases and differentially expressed genes in the GeneCards and GEO datasets into the E dataset, and deduplicate and merge the Drugbank and KEGG marketed drug targets into the F dataset. Map and analyze the E and F data with the potential target data of Alhagi sparsifolia, and use an online tool (http: / / bioinformatics.psb.ugent.be / webtools / venn / , Firefox browser) to draw a Venn diagram to obtain important target information for Alhagi sparsifolia against UC disease;

[0105] Construction and visualization analysis of the PPI network:

[0106] (1) Construction and visualization analysis of the compound-target-disease PPI network of Alhagi sparsifolia:

[0107] Import the compound, target, and disease target data of Alhagi sparsifolia into the Cytoscape 3.6.0 software to construct a visual regulatory network diagram of the compound-disease targets of Alhagi sparsifolia, and use the CytoNCA and Network Analyzer functions for network topology analysis to calculate the degree centrality (DC), betweenness centrality (BC), and closeness centrality (CC). Screen effective active ingredients with DC value > 2 times the median, BC value > 0.01, and CC value > 0.30;

[0108] 2.4 Results of network pharmacology analysis:

[0109] (1) Screening results of potential targets of Alhagi sparsifolia compounds:

[0110] 178 active compounds of Alhagi sparsifolia were retrieved and collected from CNKI, VIP, and Wanfang databases. 105 compounds were identified by UPLC-TOF-MS. 30 active compounds of Alhagi sparsifolia were screened using the Drug-Like soft index combination of the FAFDrugs4 database, with OB≥30% and DL≥0.18 as the criteria, and 276 potential action targets were collected;

[0111] (2) Screening results of disease-related targets for ulcerative colitis (UC):

[0112] 4844 disease gene information was obtained through the GeneCards database. 545 were screened and confirmed with Score≥5.0. Gene data related to disease research were analyzed by querying the GEO database (differentially expressed genes were screened with P-value<0.05, |logFC, Fold change|≥1.0). 252 significantly expressed genes were obtained from Reference Series GSE3365, and 35 differentially expressed genes were obtained from GSE11223. After removing duplicates and merging, there were 284 in total;

[0113] (3) Screening results of target drugs for ulcerative colitis (UC) on the market:

[0114] Anti-inflammatory drugs and immunosuppressive agents are the main categories of drugs on the market for ulcerative colitis (UC). 14 drugs and 60 related targets were obtained through querying Drugbank, and 8 drugs on the market and 18 targets were obtained through KEGG query;

[0115] (4) Results of target mapping and confirmation of important targets:

[0116] The GeneCards dataset and the GEO dataset were mapped to each other. The results showed that 27 out of 545 disease genes were involved in the differential expression of experimental ulcerative colitis (UC) in the GEO dataset, accounting for 4.95% of all disease genes. There were 3 common genes between the Drugbank and KEGG datasets of drugs on the market, accounting for 16.7% of the target genes in the KEGG database. By checking the drugs and targets in the two databases, the results showed that the KEGG database only collected specific therapeutic targets of drugs, while Drugbank included more proteins that interact with drugs (see Figure 2 -a);

[0117] The diseases and differentially expressed genes in the GeneCards and GEO datasets were removed duplicates and merged into dataset E (800 genes), and the target drugs on the market in Drugbank and KEGG were removed duplicates and merged into dataset F (75 genes). Dataset E and F were mapped and analyzed with the potential target data of Alhagi sparsifolia (276 genes), see Figure 2-b, the results were analyzed for three datasets and their overlapping situations. Among the 276 potential targets of Alhagi sparsifolia, 67 are disease targets, 9 are targets of marketed drugs, and 10 are common targets, namely: Arachidonate 5-lipoxygenase (ALOX5), Peroxisome proliferator-activated receptor gamma (PPARG), Tumor necrosis factor-α (TNF), Prostaglandin G / H synthase 2 (PTGS2), Prostaglandin G / H synthase 1 (PTGS1), Nitric oxide synthase 2 (NOS2), Nuclear receptor subfamily 3 group C member 1 (NR3C1), Nuclear receptor subfamily 1 group I member 2 (NR1I2), Xanthine dehydrogenase (XDH), Cytochrome P450 family 1 subfamily B member 1 (CYP1B1). The above results predict that these 10 targets are potential action targets for Alhagi sparsifolia in the prevention and treatment of ulcerative colitis. Based on the cross-analysis results, it is speculated that the above genes are targeted by components such as kaempferol, rutin, isorhamnetin, and dihydroquercetin in Alhagi sparsifolia medicinal materials;

[0118] (5) PPI network construction and visualization analysis:

[0119] The data of Alhagi sparsifolia compounds, targets, and disease-related targets were imported into Cytoscape 3.6.0 software to draw a PPI network diagram, as shown in Figure 3 , through network topology structure analysis, the Degree values were sorted from high to low, and 9 key active components were determined by screening with Degree > 28 (median) and Betweenness Centrality > 0.02. The results are shown in Table 2:

[0120] Table 2 Core compound information of effective components of Alhagi sparsifolia

[0121]

[0122] It can be seen from Table 2 that the Degree values of 9 components, namely epigallocatechin gallate, medicarpin, butein, galangin, quercetin, kaempferol, isorhamnetin, diosmetin and wogonin, rank among the top, and they are the key active components in the effective components of Alhagi sparsifolia Shap.

[0123] Example 4

[0124] Construction of the quality control method for the effective components (APE2) of Alhagi sparsifolia Shap:

[0125] Chromatographic conditions and system suitability: Chromatographic column: Waters XTERRA RP 18 column (4.6×250 mm, 5 μm); Mobile phase: methanol - 0.4% phosphoric acid solution with a volume ratio of 55:45, detection wavelength is 360 nm, flow rate is 1.0 mL·min -1 , column temperature is 30 °C, injection volume is 10 μL;

[0126] Preparation of solutions:

[0127] Preparation of reference substance solution: Accurately weigh 5.12 mg of isorhamnetin reference substance, add methanol to make a solution of 0.5074 mg·mL -1 , filter through a 0.22 μm filter membrane to obtain the reference substance solution;

[0128] Preparation of negative control solution: Accurately measure 25 mL of methanol - 25% hydrochloric acid solution with a volume ratio of 4:1, heat and reflux for extraction in a water bath at 70 °C for 1 hour, transfer to a 25 mL volumetric flask, make up the volume with methanol, shake well, filter through a 0.22 μm filter membrane, and take the filtrate to obtain the negative control solution;

[0129] Preparation of test solution: Accurately weigh 1.0 g of the effective components of Alhagi sparsifolia Shap, place it in a stoppered conical flask, accurately add a total of 25 mL of methanol - 25% hydrochloric acid solution with a volume ratio of 4:1, heat and reflux for extraction in a water bath at 70 °C for 1 hour, take out and filter, make up the volume with methanol to 25 mL in a volumetric flask, shake well, filter through a 0.22 μm filter membrane, and take the filtrate to obtain the test solution;

[0130] Determination method:

[0131] Accurately pipette 10 μL each of the reference substance solution and the test solution, inject them into the high - performance liquid chromatograph, measure the peak area values, calculate the percentage content, and the calculation formula:

[0132] Percentage content of isorhamnetin (%) = (c×V×D) / w×100%

[0133] Note: c: Concentration of isorhamnetin in the solution (mg·mL -1 ), V: Fixed volume of the solution (mL), D: Dilution factor, m:

[0134] Sample mass (mg);

[0135] Methodology verification:

[0136] Specificity test:

[0137] Use an HPLC chromatograph to automatically inject the reference solution, negative control solution, and test solution. The injection volume is 10 μL. Examine whether there are any interfering peaks in the negative control solution, and record the elution time of the target peak, chromatographic information such as resolution, and number of theoretical plates;

[0138] Linearity range investigation:

[0139] Precisely pipette 0.2, 0.5, 1, 3, 6, 9, 12, and 15 μL of the reference stock solution and inject them into the liquid chromatograph. Record the peak areas. Perform linear regression with the peak areas as the abscissa and the concentrations as the ordinate to obtain the standard curve equation and the linear range;

[0140] Accuracy test:

[0141] Adopt the standard addition method. Precisely weigh 0.3 g of the test sample of the effective components of Alhagi sparsifolia with known content, a total of 6 portions. Place them in 25-mL stoppered conical flasks respectively. Add the rhamnetin reference substance according to the mass ratio of rhamnetin in the test sample of 1:1. Prepare according to the test sample preparation method and inject for determination under the chromatographic conditions for content determination. Calculate the recovery rate and its RSD value;

[0142] Precision test:

[0143] Intra-day precision: For the same batch of test solutions, place them at room temperature and inject for analysis at 0, 2, 4, 6, 8, and 10 hours respectively. Record the peak area values and calculate the RSD value;

[0144] Inter-day precision: For the same batch of test solutions, repeat the determination continuously for 5 days respectively. Record the peak area values and calculate the RSD value.

[0145] Repeatability test: Precisely weigh 6 portions of the same batch of samples, prepare the solutions according to the method under the preparation of the test solution item, inject for determination under the above chromatographic conditions, record the peak area values, and calculate the content of rhamnetin in the samples and the RSD value;

[0146] Detection limit determination:

[0147] Dilute the reference solution by 1-fold, 10-fold, 50-fold, 100-fold, 250-fold, 500-fold, and 2250-fold respectively, inject 10 μL for determination in sequence, record the signal-to-noise ratio (S / N), and the corresponding concentration when the signal-to-noise ratio (S / N) is 3:1;

[0148] HPLC content determination results of rhamnetin:

[0149] Specificity test:

[0150] Prepare the test solution, reference solution and negative blank solution according to the solution preparation method. Under the above chromatographic conditions, automatically inject 10 μL of the reference solution, negative control solution and test solution for testing to investigate the specificity of this method; see the chromatographic separation effect in Figure 4 , compared with the reference chromatogram, there are no interfering impurity peaks in the negative control solution, and the separation is good. The retention time of isorhamnetin is 19.2 min, the theoretical plate number is greater than 6000, the resolution is greater than 1.5, and the tailing factor is 1.06. The chromatographic peak of isorhamnetin is chromatographically separated, indicating that this method has good specificity;

[0151] Linearity range investigation:

[0152] Precisely pipette 0.2, 0.5, 1, 3, 6, 9, 12, and 15 μL of the reference stock solution at 0.5074 mg·mL -1 into the liquid chromatograph and record the peak areas; use the peak areas as the abscissa and the concentrations (mg·mL -1 ) as the ordinate for regression to obtain the standard curve equation: Y = 304.78X - 2.0137, r 2 = 0.9999;

[0153] The above results show that there is a good linear relationship between the concentration of isorhamnetin and the peak area in the range of 0.01 - 0.76 mg·mL -1 , as shown in Table 3;

[0154] Table 3 Results of linear relationship test

[0155]

[0156] Accuracy test:

[0157] Precisely weigh 0.3 g of the effective component sample of Alhagi sparsifolia, a total of 6 portions, place them in 25-mL stoppered conical flasks respectively, add 4.06 mg of isorhamnetin reference substance to each, after treating according to the test solution preparation method, inject 10 μL under the chromatographic conditions for content determination, and calculate the recovery rate. The results show that the average recovery rate of the sample is 95.91% (n = 6), and the RSD is 2.87% (see Table 4), indicating that the method is accurate;

[0158] Table 4 Results of recovery rate determination

[0159]

[0160] Precision test:

[0161] Intraday precision: For the same batch of test sample solutions, they were placed at room temperature and injected for analysis at 0, 2, 4, 6, 8, and 10 hours respectively. The peak area values were recorded. The results showed that the RSD value of the determination results at different times on the same day was 0.22% (see Table 5), indicating that the test sample solution was stable within 10 hours;

[0162] Table 5 Results of stability (intraday difference) test

[0163]

[0164] Interday precision: For the same batch of test sample solutions, they were repeatedly determined continuously for 5 days, and the peak area values were recorded. The results showed that the RSD value of the test sample within 5 days was 0.22% (see Table 6), indicating that the content of the test sample solution was stable within 5 days at room temperature;

[0165] Table 6 Results of stability (interday difference) test

[0166]

[0167] Repeatability test:

[0168] Six test sample solutions were repeatedly prepared, and the determination results showed that the average content of isorhamnetin in the test sample was 1.1598%, and the RSD value was 0.30% (see Table 7), indicating that this method had good reproducibility;

[0169] Table 7 Results of repeatability test

[0170]

[0171] Determination of minimum detection limit:

[0172] When the signal-to-noise ratio (S / N) was 3:1, the detection limit of the sample was 0.23 ng·mL -1 (see Table 8).

[0173] Table 8 Results of minimum detection limit determination

[0174]

[0175] Results of sample content determination:

[0176] The average content of isorhamnetin in the effective components (APE2) of three batches of Alhagi sparsifolia was 1.1690%, and the RSD value was 0.35% (see Table 9);

[0177] Table 9 Results of content determination of isorhamnetin in APE2

[0178]

[0179] Example 5

[0180] Thin-layer identification of isorhamnetin, kaempferol, and quercetin in the effective components (APE2) of Alhagi sparsifolia Shap.:

[0181] (1) Thin-layer identification of isorhamnetin:

[0182] Preparation of solutions:

[0183] Preparation of the reference substance solution: Precisely weigh 4.60 mg of isorhamnetin reference substance, dissolve it in methanol and make up the volume to 10 mL in a volumetric flask to prepare a solution with a concentration of 0.4 mg·mL -1 . That is how to obtain it;

[0184] Preparation of the Alhagi sparsifolia Shap. medicinal material solution: Precisely weigh 2.50558 g of Alhagi sparsifolia Shap. powder (passed through No. 3 sieve), place it in a 150 mL stoppered conical flask, precisely add 100 mL of methanol solution with a volume ratio of 70%, ultrasonically extract at 40 Hz power for 30 min, cool to room temperature, filter by suction, place the filtrate in a water bath at 60 °C until dry, add a total of 25 mL of a 70% methanol - 25% hydrochloric acid mixed solution with a volume ratio of 4:1 to the residue, heat and hydrolyze in a water bath at 60 °C for 1 hour, immediately cool in pure water at 15 °C, filter the hydrolyzed solution, transfer the filtrate to a 25 mL volumetric flask, make up the volume to the mark with methanol, and shake well to obtain the medicinal material reference solution;

[0185] Preparation of the test solution: Precisely weigh 20.43 mg, 20.35 mg, and 20.28 mg of the effective components of Alhagi sparsifolia Shap., dissolve them in methanol with a concentration of 70% respectively and make up the volume to 10 mL in a volumetric flask, transfer the solution to a 50 mL beaker, concentrate it to dryness in a water bath at 60 °C, add a total of 5 mL of a 70% methanol - 25% hydrochloric acid mixed solution with a volume ratio of 4:1 to the residue, heat and hydrolyze in a water bath at 60 °C for 1 hour, immediately cool in pure water at 15 °C, filter the hydrolyzed solution, transfer the filtrate to a 5 mL volumetric flask, make up the volume to the mark with methanol, and shake well to obtain the test solution of the effective components APE2 of Alhagi sparsifolia Shap.;

[0186] Thin-layer identification method

[0187] According to the thin-layer chromatography method (Appendix Ⅵ B) in the Chinese Pharmacopoeia 2020 edition, absorb 10 μL of the test solution of the effective components of Alhagi sparsifolia Shap. and the reference medicinal material solution of Alhagi sparsifolia Shap., and 2 μL of the isorhamnetin reference substance solution, and spot them on the same silica gel GF 254 thin-layer plate respectively. Use chloroform - methanol - formic acid with a volume ratio of 9:1:0.5 as the developing agent. After saturating for 20 min under closed conditions, develop, air-dry naturally, then spray with 1% aluminum trichloride ethanol solution for color development, heat the plate at 60 °C for 5 min until the spots on the thin-layer plate are clearly developed, and examine under a 365 nm ultraviolet lamp. Observe the spots and the color development situation at the corresponding positions of the test solution chromatogram and the reference substance chromatogram.

[0188] (2) Thin-layer identification of kaempferol and quercetin:

[0189] Preparation of solutions:

[0190] Preparation of reference substance solution: Weigh accurately 2.57 mg of kaempferol reference substance and 2.52 mg of quercetin reference substance, and dissolve them separately in methanol to make solutions with a concentration of 0.5 mg·mL -1 as the reference substance solution;

[0191] Preparation of the solution of Alhagi sparsifolia Shap. medicinal material: Weigh accurately 2.00567 g of Alhagi sparsifolia Shap. medicinal material, add 25 mL of 75% methanol and 1 mL of hydrochloric acid, heat and hydrolyze for 1 hour, filter, evaporate the filtrate to dryness in a water bath at 60 °C, dissolve the residue in 10 mL of water, extract with ethyl acetate twice, 10 mL each time, combine the ethyl acetate extracts, evaporate to dryness at 60 °C, and dissolve the residue in 2 mL of methanol to make the reference medicinal material solution;

[0192] Preparation of the test solution: Weigh accurately three portions of 40.05 mg, 40.06 mg, and 40.08 mg of the active components of Alhagi sparsifolia Shap., add 5 mL of 75% methanol and 0.2 mL of hydrochloric acid respectively, heat and hydrolyze for 1 hour, filter, evaporate the filtrate to dryness, dissolve the residue in 5 mL of water, extract with ethyl acetate twice, 5 mL each time, combine the ethyl acetate solutions, evaporate to dryness, and dissolve the residue in 1 mL of methanol to make the test solution;

[0193] Thin-layer identification method

[0194] According to the thin-layer chromatography method (Appendix Ⅵ B) in the Chinese Pharmacopoeia 2020 Edition, absorb 10 μL of the test solution of the active components of Alhagi sparsifolia Shap., 10 μL of the reference medicinal material solution of Alhagi sparsifolia Shap., and 5 μL of the reference substance solutions of kaempferol and quercetin, and spot them on the same silica gel G thin-layer plate respectively. Use xylene-ethyl acetate-formic acid with a volume ratio of 10:8:1 as the developing agent. After saturating for 20 min under closed conditions, develop, take out, dry, spray with 3% aluminum trichloride ethanol solution, heat at 105 °C for 5 min, and examine under a 365 nm ultraviolet lamp. Observe the spots and the color development at the corresponding positions of the test solution chromatogram and the reference substance chromatogram.

[0195] Results of thin-layer identification:

[0196] (1) Results of thin-layer identification of isorhamnetin:

[0197] In the chromatogram of the test solution APE2 of the active components of Alhagi sparsifolia Shap., at the corresponding horizontal positions of the isorhamnetin reference substance chromatogram and the Alhagi sparsifolia Shap. reference medicinal material chromatogram, fluorescent spots of the same color appear. The migration distance ratio value Rf of isorhamnetin is 4.5 / 7.5 = 0.6, and the results are shown in Figure 5 ;

[0198] (2) TLC identification results of kaempferol and quercetin:

[0199] In the test sample chromatogram of the effective component APE2 of Alhagi sparsifolia, fluorescent spots of the same color appeared at the corresponding positions as those in the reference substance chromatograms of kaempferol and quercetin and the reference medicinal material chromatogram of Alhagi sparsifolia. The migration distance ratio value Rf of kaempferol was 5.2 / 7.5 = 0.69, and that of quercetin was Rf = 4.5 / 7.5 = 0.6. The results are shown in Figure 6 .

[0200] Through the above examples, a quality control method for the effective components of Alhagi sparsifolia for the treatment of ulcerative colitis was obtained. This method has high precision and good reproducibility and can be used for the quality control of the effective components of Alhagi sparsifolia and its products.

Claims

1. A method for quality control of the effective components of Alhagi sparsifolia for treating ulcerative colitis, characterized in that, The high performance liquid chromatography (HPLC) method or thin layer chromatography (TLC) method is adopted, and the specific operation is carried out according to the following steps: a. Analyze the chemical composition of the effective components of Alhagi sparsifolia by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) to obtain 105 chemical components; b. Conduct network pharmacology prediction on the chemical components obtained in step a, sort them according to the correlation between the components and diseases, and screen out 9 key active components, namely: epigallocatechin gallate, medicarpin, butein, galangin, quercetin, kaempferol, isorhamnetin, diosmetin, and wogonin; High performance liquid chromatography c. Preparation of reference solution: Weigh accurately 5.0 mg of isorhamnetin reference substance, dissolve it in methanol to make a solution of 0.5 mg·mL -1 . Filter it through a 0.22 μm membrane filter to obtain the reference solution; d. Preparation of the test solution: Accurately weigh 1.0 g of the effective components of Alhagi sparsifolia, accurately add 25 mL of a methanol-25% hydrochloric acid solution with a volume ratio of 4:1, heat under reflux in a water bath at 70 °C for 1 hour, filter, then add methanol to make up the volume to 25 mL in a volumetric flask, shake well, filter through a 0.22 μm filter membrane, and take the filtrate to obtain the test solution; e. Chromatographic analysis: A chromatographic column packed with octadecylsilane chemically bonded silica gel, mobile phase: methanol - 0.4% phosphoric acid solution with a volume ratio of 55:45, detection wavelength at 360 nm, flow rate at 1.0 mL·min -1 , column temperature at 30 °C, injection volume at 10 μL; f. Determination: Accurately pipette 10 μL of the reference solution and the test solution respectively and inject them into the high performance liquid chromatograph. The target chromatographic peaks obtained include isorhamnetin. The retention time of the isorhamnetin chromatographic peak is 19 - 20 min. Determine and calculate the percentage mass of the effective components of Alhagi sparsifolia; Thin layer chromatography 1: a. Accurately weigh 20 mg of the effective components of Alhagi sparsifolia, dissolve with 70% methanol and make up the volume to 10 mL in a volumetric flask. Transfer the solution to a 50 mL beaker, concentrate it to dryness in a water bath at 60 °C. Add 5 mL of a 70% methanol-25% hydrochloric acid mixed solution with a volume ratio of 4:1 to the residue, heat and hydrolyze in a water bath at 60 °C for 1 hour, then immediately cool in pure water at 15 °C, filter the hydrolyzate, transfer the filtrate to a 5 mL volumetric flask, and then make up the volume to the mark with methanol, shake well to obtain the test solution of the effective components of Alhagi sparsifolia; b. Accurately weigh the isorhamnetin reference substance and add methanol to make the concentration 0.4 mg·mL -1 The solution was used as the reference solution; c. Accurately weigh 2.5 g of Alhagi sparsifolia powder, place it in a 150 mL stoppered conical flask, accurately add 100 mL of 70% methanol solution, ultrasonically extract at a power of 40 Hz for 30 min, cool to room temperature, filter by suction. Place the filtrate in a water bath at 60 °C until dry. Add 25 mL of a 70% methanol-25% hydrochloric acid mixed solution with a volume ratio of 4:1 to the residue, heat and hydrolyze in a water bath at 60 °C for 1 hour, then immediately cool in pure water at 15 °C, filter the hydrolyzate, transfer the filtrate to a 25 mL volumetric flask, make up the volume to the mark with methanol, shake well to obtain the reference solution of Alhagi sparsifolia medicinal material; d. Take 10 μL of the camel thorn active ingredient test solution obtained in step a, 2 μL of the isorhamnetin reference solution obtained in step b, and 10 μL of the camel thorn reference medicinal material solution obtained in step c, and apply them to the same silica gel GF. 254 On the thin layer plate, chloroform-methanol-formic acid with a volume ratio of 9:1:0.5 was used as the developing solvent. After saturation for 20 minutes under closed conditions, it was developed and naturally dried. Then, 1% aluminum chloride ethanol solution was sprayed for color development. The temperature was heated at 60°C for 5 minutes until the spots were clearly colored. The plates were placed under a 365nm ultraviolet lamp for inspection. In the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions of the chromatograms of the reference sample and the reference medicinal material. Thin layer chromatography 2: a. Accurately weigh 40 mg of the effective components of Alhagi sparsifolia, add 5 mL of 75% methanol and 0.2 mL of hydrochloric acid, heat and hydrolyze for 1 hour, filter, evaporate the filtrate to dryness. Dissolve the residue in 5 mL of water, extract with ethyl acetate twice, 5 mL each time. Combine the ethyl acetate layers, evaporate to dryness, dissolve the residue in 1 mL of methanol to obtain the test solution; b. Weigh accurately kaempferol and quercetin reference substances, and separately dissolve them in methanol to prepare solutions with a concentration of 0.5 mg·mL -1 . These are used as reference substance solutions; c. Weigh accurately 2.0 g of the Alhagi sparsifolia Shap. medicinal material, add 25 mL of 75% methanol and 1 mL of hydrochloric acid, heat for hydrolysis for 1 hour, filter, evaporate the filtrate to dryness in a water bath at 60°C, dissolve the residue in 10 mL of water, extract with ethyl acetate twice, 10 mL each time, combine the ethyl acetate extracts, evaporate to dryness at 60°C, dissolve the residue in 2 mL of methanol to obtain the control medicinal material solution of Alhagi sparsifolia Shap.; d. Pipette 10 μL of the test solution of the active components of Alhagi sparsifolia Shap. obtained in step a, 5 μL each of the control solutions of kaempferol and quercetin obtained in step b, and 10 μL of the control medicinal material solution of Alhagi sparsifolia Shap. obtained in step c, spot them on the same silica gel G thin-layer plate respectively, use xylene-ethyl acetate-formic acid with a volume ratio of 10:8:1 as the developing agent, saturate for 20 min under closed conditions, develop, take out, air-dry, spray with 3% aluminum trichloride ethanol solution, heat at 105°C for 5 min, and examine under a 365 nm ultraviolet lamp. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions of the chromatograms of the control samples and the control medicinal material.

2. According to the method for quality control of the active components of Alhagi sparsifolia Shap. for treating ulcerative colitis as described in claim 1, the chemical components detected or identified in the active components of Alhagi sparsifolia Shap. include isorhamnetin, kaempferol, and quercetin.

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