Traditional Chinese medicine compound for reducing recurrence of chronic nasosinusitis and preparation method thereof

Abstract

The invention relates to the technical field of traditional Chinese medicine, in particular to a traditional Chinese medicine compound for reducing recurrence of chronic nasosinusitis and a preparation method of the traditional Chinese medicine compound, and the traditional Chinese medicine compound is prepared from, by weight, 14-16 g of radix astragali, 8-10 g of fried radix scutellariae, 8-10 g of radix angelicae, 5-7 g of fructus xanthii, 5-7 g of flos magnoliae, 14-16 g of flos chrysanthemi indici, 11-13 g of fried fructus gardeniae, 5-7 g of radix angelicae sinensis, 5-7 g of flos carthami and 8-10 g of herba menthae. The traditional Chinese medicine composition can significantly improve clinical symptoms of patients with chronic nasosinusitis and reduce the recurrence rate, is good in safety, and does not find abnormal symptoms in clinical use. Meanwhile, the preparation process is simple to operate and suitable for industrial mass production. The raw medicinal materials selected by the traditional Chinese medicine composition are synergistic and complementary in nature and effect, inherit the holism concept of traditional Chinese medicine, emphasize that the traditional Chinese medicine composition emphasizes whole body treatment, plays a role together and inhibits infiltration of inflammatory cells while treating local parts, has small toxic and side effects, fully plays the advantages of treating chronic sinusitis and preventing relapse of the traditional Chinese medicine, relieves the pain of patients, and has a remarkable curative effect on treating chronic sinusitis. The life quality of patients is improved and the recurrence probability is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicines, and in particular to a traditional Chinese medicine compound for reducing the recurrence of chronic sinusitis and a preparation method thereof. Background Art

[0002] Chronic rhinosinusitis (CRS) is a common chronic inflammatory disease in otolaryngology. Its pathological characteristics are mainly manifested as persistent inflammatory response and tissue remodeling of the nasal cavity and sinus mucosa. The clinical diagnostic criteria are symptoms that last for at least 12 weeks or more, mainly including nasal congestion, headache, purulent rhinorrhea, etc. According to Frost & Sullivan's forecast, the number of CRS patients worldwide will increase to 1.2 billion by 2030. These data highlight the far-reaching impact of CRS on global public health and the huge socioeconomic burden it brings.

[0003] Currently, treatment options for CRS primarily encompass two main approaches: surgery and medication. While advancements in medication and surgical techniques have significantly improved disease control in CRS patients, recurrence rates remain high after treatment. Therefore, further improving the overall treatment outcomes for CRS patients and reducing postoperative recurrence rates are critical issues that urgently need to be addressed.

[0004] Inflammatory response is one of the important pathological characteristics of CRS. Multiple studies have suggested that CRS is a chronic, persistent inflammation of the nasal and sinus mucosa caused by a variety of complex infections and inflammatory factors. The interaction between inflammatory factors and inflammatory cells plays a key role in the development of CRS. Under the mediation of cell adhesion molecules (CAMs), various inflammatory cells (neutrophils, lymphocytes, and eosinophils) undergo rolling, tight adhesion, and transendothelial migration, ultimately crossing the endothelial barrier to reach the site of inflammation. At the same time, infiltrating inflammatory cells continuously release large amounts of cytokines and chemokines, further activating vascular endothelial cells and mucosal epithelial cells, and maintaining high expression of CAMs on endothelial cells, causing the inflammatory response to persist, forming a vicious cycle.

[0005] Studies in recent years have shown that ferroptosis plays an important role in the occurrence and development of many chronic diseases, and its mechanism of action varies depending on the type of disease. In chronic inflammatory diseases, the release of inflammatory factors not only recruits the infiltration of inflammatory cells, but also affects iron metabolism and increases the accumulation of iron ions in cells, thereby promoting the occurrence of ferroptosis. Ferroptosis is often accompanied by the release of inflammatory factors (IL-1β and IL-18, etc.), which are significantly correlated with the activation of inflammation-related signaling pathways. Not only that, the oxidative stress and lipid peroxidation caused by ferroptosis can further activate a variety of inflammatory cells and inflammatory factors, forming a vicious circle, leading to continued deterioration of the disease. Summary of the Invention

[0006] The present invention aims to provide a traditional Chinese medicine compound for reducing the recurrence of chronic sinusitis and a preparation method thereof.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a traditional Chinese medicine compound for reducing the recurrence of chronic sinusitis. The traditional Chinese medicine compound is prepared by weight from the following raw materials:

[0009] Astragalus 14-16g, stir-fried Scutellaria baicalensis 8-10g, Angelica dahurica 8-10g, Xanthium sibiricum 5-7g, Magnolia florid 5-7g, Chrysanthemum indicum 14-16g, stir-fried Gardenia jasminoides 11-13g, Angelica sinensis 5-7g, Safflower 5-7g, and Menthol 8-10g.

[0010] Furthermore, the astragalus root 15g, fried scutellaria baicalensis 9g, angelica dahurica 9g, Xanthium sibiricum 6g, magnolia 6g, wild chrysanthemum 15g, fried gardenia 12g, angelica 6g, safflower 6g, and mint 9g.

[0011] The present invention also includes a preparation method of the above-mentioned traditional Chinese medicine compound, comprising the following steps: weighing each raw material, crushing and mixing them respectively to obtain a medicine mixture; adding cold water equivalent to 3-5 times the total weight of the medicine mixture, soaking for 30 minutes, boiling over high heat and then decocting over low heat for 30-60 minutes, filtering, collecting a first filtrate, adding water to the filter residue and decocting again as above, filtering, and collecting a second filtrate; and mixing the two collected filtrates to obtain a traditional Chinese medicine compound decoction.

[0012] Furthermore, the traditional Chinese medicine compound decoction is vacuum freeze-dried into powder, pharmaceutically acceptable excipients are added to the powder, and a drug preparation other than the decoction is prepared according to conventional processes.

[0013] Furthermore, the administration preparation is a tablet, pill, powder, granule, oral liquid or capsule.

[0014] Compared with the prior art, the present invention can significantly improve the clinical symptoms of patients with chronic sinusitis, reduce the recurrence rate, and has good safety. No abnormal symptoms have been found in clinical use. At the same time, the preparation process of the present invention is simple to operate and is suitable for large-scale industrial production. The Chinese medicine compound of the present invention has 101 active ingredients, of which 60 ingredients inhibit the adhesion and invasion of inflammatory cells in nasal mucosal tissue by targeting the NFKB signaling pathway, and 57 ingredients inhibit the inflammatory response induced by ferroptosis by targeting the ferroptosis signaling pathway. The raw medicinal materials selected by the present invention are both synergistic and complementary in nature and efficacy. Adhering to the holistic concept of traditional Chinese medicine, it emphasizes that while treating local areas, it also attaches importance to systemic treatment, works together to inhibit the infiltration of inflammatory cells, and has little toxic and side effects, giving full play to the advantages of traditional Chinese medicine in treating chronic sinusitis and preventing recurrence, alleviating patients' pain, improving patients' quality of life, and reducing the chance of recurrence. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The figure shows the effects of different concentrations of the Chinese herbal compound of the present invention on the cell cycle distribution of HNEpC cells.

[0016] Figure 2 The figure shows the effects of different concentrations of the Chinese herbal compound of the present invention on HNEpC cell apoptosis.

[0017] Figure 3 The figure shows the effects of different concentrations of the Chinese herbal compound of the present invention on the invasion ability of U937 cells.

[0018] Figure 4 The figure shows the effects of different concentrations of the Chinese herbal compound of the present invention on the adhesion ability of U937 cells.

[0019] Figure 5 The figure shows the effects of different concentrations of the Chinese herbal compound of the present invention on the expression of adhesion proteins ICAM-1, VCAM-1 and E-selectin on the surface of endothelial cells.

[0020] Figure 6 The figure shows the effects of different concentrations of the Chinese herbal compound of the present invention on the expression of proteins related to the NF-κB signaling pathway in HUVECs cells.

[0021] Figure 7 This is the effect of the Chinese medicinal compound of the present invention on the active oxygen of HNEpC cells.

[0022] Figure 8 This is the effect of the Chinese herbal compound of the present invention on lipid peroxidation in HNEpC.

[0023] Figure 9 The Chinese medicinal compound of the present invention inhibits mitochondrial deformation of ferroptotic cells.

[0024] Figure 10 This is the effect of the Chinese herbal compound of the present invention on the levels of ferroptosis-related proteins in HNEpC cells.

[0025] Figure 11 This is the effect of the Chinese medicinal compound of the present invention on inflammatory cells.

[0026] Figure 12 This is the effect of the Chinese medicinal compound of the present invention on the expression levels of TNF-α and IFN-γ.

[0027] Figure 13 The recovery of sinus mucosa in the two groups was shown before surgery and within 6 months after surgery.

[0028] Figure 14 This is the positive ion current diagram of the Chinese medicine compound powder of the present invention.

[0029] Figure 15 This is the negative ion flow diagram of the Chinese medicine compound powder of the present invention.

[0030] Figure 16 This is a "component-intersection target" network diagram of the NF-κB signaling pathway of the traditional Chinese medicine compound of the present invention.

[0031] Figure 17 This is a "component-intersection target" network diagram of the ferroptosis signaling pathway of the traditional Chinese medicine compound of the present invention. DETAILED DESCRIPTION

[0032] The technical solutions of the present invention are further described below in conjunction with specific examples. It should be understood that the following examples are merely exemplary illustrations and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope that the present invention is intended to protect.

[0033] A traditional Chinese medicine compound for reducing the recurrence of chronic sinusitis, the traditional Chinese medicine compound being prepared by weight from the following raw materials:

[0034] Astragalus 14-16g, stir-fried Scutellaria baicalensis 8-10g, Angelica dahurica 8-10g, Xanthium sibiricum 5-7g, Magnolia florid 5-7g, Chrysanthemum indicum 14-16g, stir-fried Gardenia jasminoides 11-13g, Angelica sinensis 5-7g, Safflower 5-7g, and Menthol 8-10g.

[0035] The preparation method of the above-mentioned traditional Chinese medicine compound comprises the following steps: weighing each raw material, crushing and mixing them separately to obtain a drug mixture; adding cold water equivalent to 3-5 times the total weight of the drug mixture, soaking for 30 minutes, boiling over high heat and then decocting over low heat for 30-60 minutes, filtering, collecting the first filtrate, and adding water to the filter residue and decocting again as above, filtering, and collecting the second filtrate; mixing the two collected filtrates to obtain the traditional Chinese medicine compound decoction.

[0036] The Chinese herbal compound decoction is vacuum freeze-dried into a powder, and pharmaceutically acceptable excipients are added to the powder and prepared according to conventional processes to prepare a drug-decoction formulation other than a decoction. The decoction formulation can be administered orally or parenterally to a patient in need of such treatment. Examples include tablets, granules, capsules, powders, injections, pills, inhalants, sublingual formulations, syrups, gels, ointments, suppositories, and pills. Tablets, capsules, pills, and pills are preferred. These formulations can be prepared by conventional methods by adding pharmaceutical carriers such as excipients, binders, humectants, disintegrants, and thickeners. The pharmaceutical carrier is selected from microcrystalline cellulose, powdered cellulose, mannitol, starch, lactose, gelatin, methylcellulose, dextrin, pregelatinized starch, micropowdered silica, hydroxypropyl methylcellulose, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, polyethylene glycol, xylitol, lactitol, glucose, sucrose, stevioside, potassium sorbate, glycine, mannitol, tartaric acid, silicon dioxide, calcium stearate, magnesium stearate, talc, etc.

[0037] Example 1

[0038] The raw materials of the Chinese herbal compound include 15g of Astragalus, 9g of stir-fried Scutellaria, 9g of Angelica dahurica, 6g of Xanthium sibiricum, 6g of Magnolia florid, 15g of Chrysanthemum indicum, 12g of stir-fried Gardenia jasminoides, 6g of Angelica sinensis, 6g of Carthamus tinctorius, and 9g of Menthol.

[0039] Example 2

[0040] The Chinese herbal compound decoction is prepared according to the following steps:

[0041] The raw materials of Example 1 were crushed and mixed to obtain a drug mixture; cold water equivalent to 4 times the total weight of the drug mixture was added, soaked for 30 minutes, boiled over high heat, and then simmered over low heat for 45 minutes, filtered, and the first filtrate was collected. The filter residue was added with water and simmered again as above (soaked for 30 minutes, boiled over high heat, and then simmered over low heat for 45 minutes), filtered, and the second filtrate was collected; the two collected filtrates were mixed to obtain a Chinese herbal compound decoction. It can be taken directly.

[0042] Example 3

[0043] The decoction of Example 2 was vacuum freeze-dried to prepare a Chinese medicine compound powder.

[0044] Example 4

[0045] The Chinese medicine compound tablets are prepared according to the following steps:

[0046] Take the traditional Chinese medicine compound powder prepared in Example 3, add 0.2-0.4 times the amount of pregelatinized starch and 0.1-0.2 times the amount of microcrystalline cellulose, mix well, granulate with ethanol, dry, and shape the granules. Then add 3% sodium carboxymethyl starch and 1% silicon dioxide, mix well, compress into tablets, and film-coat to obtain traditional Chinese medicine compound tablets.

[0047] Example 5

[0048] The Chinese medicine compound dripping pills are prepared according to the following steps:

[0049] Take polyethylene glycol-6000, heat and melt it, add the traditional Chinese medicine compound powder prepared in Example 3, mix well, and drop it into the condensing agent to make dripping pills, thereby obtaining the traditional Chinese medicine compound dripping pills.

[0050] Example 6

[0051] The Chinese medicine compound capsules are prepared according to the following steps:

[0052] Take the traditional Chinese medicine compound powder prepared according to Example 3, add 1.0-2.0 times the mass of starch or dextrin, mix well, make granules with 90% ethanol, dry, add magnesium stearate, mix well, and put into capsules to obtain traditional Chinese medicine compound capsules.

[0053] Example 7

[0054] Prepare the preparation of Chinese medicine compound pills (concentrated honey pills) according to the following steps:

[0055] Take the Chinese medicinal compound powder prepared in Example 3, add refined honey, and prepare concentrated honey pills.

[0056] Example 8

[0057] Effect of the Traditional Chinese Medicine Compound Prepared in Example 2 on the Cell Cycle of Normal Human Nasal Mucosal Epithelial Cells HNEpC

[0058] Human nasal epithelial cells (HNEpC) in the logarithmic growth phase were obtained and digested with trypsin to prepare a single-cell suspension. The cell concentration was adjusted to 1.5×105 cells / mL and 2 mL was inoculated into each well of a 6-well plate. After 24 hours, drugs (0, 0.1, 0.5, and 1 mg / mL, respectively) were added and cultured for 24 hours. All cells were collected and approximately 1×106 cells were transferred to a centrifuge tube. The cells were centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded and the cells were washed once with PBS solution. About 0.2 mL of PBS was left in the centrifuge tube. 1 mL of 75% ice ethanol was added to mix and fix the cells. The cells were placed at 4°C for more than 4 hours. During the detection, the ethanol was removed by centrifugation, and the cells were washed once with PBS. 1 mL of PBS was left in the centrifuge tube to break up the cell clumps. 5 μL of RNase and 500 μL of PI (100 μg / mL) staining solution were added. The cells were stained at 37°C in the dark for 30 minutes. 10,000 cells were counted by flow cytometry, and the cell cycle distribution and apoptosis were analyzed using FlowJo software.

[0059] The results are as follows Figure 1 As shown in the results, with the increase of drug concentration, the ratio of HNEpC cell cycle in S phase and G2 / M phase did not change significantly. This study shows that the Chinese herbal compound of the present invention has no significant effect on normal nasal mucosal cells.

[0060] Example 9

[0061] Effect of the Traditional Chinese Medicine Compound Prepared in Example 2 on Tumor Cell Apoptosis

[0062] Human nasal epithelial (HNEpC) cells were plated in 6-well plates and cultured overnight. Culture medium containing 0, 0.1, 0.5, or 1 mg / mL of the traditional Chinese medicine compound was then added for an additional 24 hours. After 24 hours of incubation, apoptosis was assessed using the Annexin-V FITC / PI apoptosis detection kit. The assay procedure was as follows: 1) Cell suspension was harvested by trypsinization and centrifuged at 1500 rpm for 5 minutes; 2) Cells were washed twice with PBS; 3) Supernatant was removed and cells were resuspended in 300 μL of staining buffer; 4) 5 μL of Annexin-V FITC antibody was added and mixed, followed by 5 μL of PI staining solution, which was gently mixed. The cells were incubated at room temperature in the dark for 5-10 minutes; 5) Flow cytometry was performed using Annexin-V FITC single-positive tubes and PI single-positive tubes for fluorescence compensation. Data were analyzed using FlowJo software.

[0063] The results are as follows Figure 2 As shown, the Chinese herbal compound did not significantly induce apoptosis in HNEpC cells. This study indicates that the Chinese herbal compound of the present invention has no toxic effect on normal cells.

[0064] Example 10

[0065] Experimental study on the invasion of human monocytic lymphoma cells into human umbilical vein endothelial cells by the Chinese herbal compound prepared in Example 2

[0066] The Transwell assay protocol is briefly described as follows: 1) Matrix Coating: Mix the frozen-thawed Matrigel matrix using a pre-chilled pipette tip. Dilute the Matrigel with pre-chilled serum-free medium at a 4:1 volume ratio. Place an 8μm pore size Transwell chamber in a matching 24-well plate. Coat the bottom filter membrane of the Transwell chamber with the diluted Matrigel and incubate at room temperature for 1 hour. After coating, remove any Matrigel matrix not bound to the filter membrane and gently rinse twice with serum-free medium. 2) Add a suspension of digested and resuspended human umbilical vein endothelial cells to the upper chamber. After the cells form a confluent cell layer, stimulate with TNF-α (10 ng / mL) for 4 hours. 3) Cell Treatment: 250 μL of human monocytic lymphoma U937 cells (cultured in serum-free medium) were digested and plated on the upper layer of the matrix-coated Transwell chamber. Treatment was then performed with various concentrations of the traditional Chinese medicine compound (0, 0.1, 0.25, and 0.5 mg / mL). 4) Invasion culture: Add 750 μL of culture medium containing 20% FBS to the culture well in the lower chamber. FBS serves as a chemoattractant for the lower chamber. Place the culture plate in a cell culture incubator and continue culturing for 24 hours. 5) Staining: After culturing for 24 hours, remove the culture plate and remove the culture medium in the culture well. Wash twice with PBS and place the chamber in a 4% paraformaldehyde solution for fixation for 30 minutes. Then wash twice with PBS, wipe off the cells on the upper side of the chamber with a cotton swab, and wash with PBS until all the cells on the upper side of the chamber are washed away. Place the chamber in 0.1% crystal violet staining working solution for staining for 30 minutes. After staining, wash three times with PBS, take pictures with a microscope, and calculate the differences in the invasion ability of U937 cells in different groups. Invasion inhibition rate = (1-number of invasive cells in the experimental group / number of invasive cells in the control group) × 100%.

[0067] The results are as follows Figure 3 As shown in the figure, with the increase of drug concentration, the number of U937 cells invading into the lower layer was significantly reduced. This study shows that the Chinese herbal compound of the present invention has the effect of inhibiting the transendothelial migration of inflammatory cells such as U973.

[0068] Example 11

[0069] Experimental study on the adhesion of human monocytic lymphoma cells to human umbilical vein endothelial cells using the traditional Chinese medicine compound prepared in Example 2

[0070] Umbilical vein endothelial cells in the logarithmic phase were inoculated into a 24-well plate covered with gelatin. After the cells covered the bottom of the bottle, the stimulating factor TNF-α (10ng / ml) was added for 4 hours, and different concentrations of drugs were added to make the final concentrations of 0, 0.1, 0.25, and 0.5mg / mL respectively. After culturing for 12 hours, U937 cells stained with rhodamine-123 were configured into 105 A single-cell suspension of 100 μg / mL was prepared. The culture medium in the 24-well plate was aspirated, and the cells were washed three times with PBS. 500 μL of the single-cell suspension was added to each well. The plates were incubated at 37°C, 5% CO₂ for 1 hour. After washing three times with PBS, 500 μL of serum-free and phenol red-free culture medium was added. Images were then taken under a fluorescence microscope, and the adhesion inhibition rates of the different groups of U937 cells under the influence of the traditional Chinese medicine compound of the present invention were calculated.

[0071] The results are as follows Figure 4 As shown, the adhesion rate between inflammatory cells and endothelial cells was in a dose-effect relationship with drug concentration. Compared with the TNF-α group, as the drug concentration increased, the adhesion ability of inflammatory cells on the endothelial cell surface decreased.

[0072] Example 12

[0073] Analysis of the inhibition of the expression of adhesion molecules ICAM-1, VCAM-1 and E-selectin on human umbilical vein endothelial cells by the traditional Chinese medicine compound prepared in Example 2

[0074] Human umbilical vein endothelial cells in the logarithmic phase were selected and digested with trypsin. The cell concentration was adjusted to 2×10 5 Cells were seeded with 2 mL of the drug per well at 37°C (5% CO) per well and incubated in a 37°C, 5% CO2 incubator for 24 hours. The old culture medium was discarded, and different concentrations of the drug were added to achieve final concentrations of 0, 0.1, 0.25, 0.5, and 1 mg / mL, respectively. The cells were incubated in a 37°C, 5% CO2 incubator for 20 hours. The stimulatory factor TNF-α (10 ng / mL) was added for a further 4 hours, and a blank control group was established. The cell suspension was then harvested by trypsinization and centrifuged at 1500 rpm for 5 minutes. The supernatant was carefully aspirated, and the cells were resuspended in approximately 100 μL of ice-cold PBS. Antibodies to be tested were added to the blank and drug-treated groups, and the corresponding isotype control was added to the control group. After incubation at 4°C for 30 minutes, the expression of ICAM-1, VCAM-1, and E-selectin on the endothelial cell surface was determined by flow cytometry.

[0075] The results are as follows Figure 5 As shown, after incubation of HUVECs with different concentrations of the Chinese herbal compound, the cell surface adhesion molecules on the HUVECs cell surface that increased with TNF-α stimulation were significantly inhibited, indicating that the Chinese herbal compound of the present invention has a dose-dependent inhibitory effect on the expression of ICAM-1, VCAM-1, and E-selectin induced by TNF-α.

[0076] Example 13

[0077] Effect of the Traditional Chinese Medicine Compound Prepared in Example 2 on the Expression Levels of NFKB Pathway-Related Proteins

[0078] Healthy human umbilical vein endothelial cells were plated evenly in 6-well plates. When the cell density reached approximately 60%, the old culture medium was aspirated and discarded. Different concentrations of drug-containing medium (0, 0.1, 0.25, 0.5, and 1 mg / mL) were added and incubated in a 37°C, 5% CO2 incubator for 20 hours. TNF-α (10 ng / mL) was added for an additional 4 hours. A blank control group was also established. The cells were washed with normal saline and lysed with RIPA lysis buffer (containing 1% protease and phosphatase inhibitors). The cell lysate was transferred to a centrifuge tube and allowed to lyse on ice for 30 minutes. The cells were then centrifuged at 12,000 g for 20 minutes at 4°C. The supernatants were collected. Protein concentration was determined using a BCA assay and adjusted to the same concentration using RIPA lysis buffer based on the calculated protein concentration. 5× protein loading buffer (25% protein volume) was then added and mixed thoroughly. The samples were denatured in a 100°C thermostatted metal bath for 10 minutes. After cooling, the samples were aliquoted and stored at -40°C. Western-Blot method was used for quantitative detection to observe the effect of the Chinese herbal compound of the present invention on the expression level of NFKB pathway-related proteins.

[0079] The results are as follows Figure 6 As shown in the figure, after incubation of HUVECs with different concentrations of the Chinese herbal compound, the phosphorylation levels of p-IKKα / β (Ser176 / 180), p-IKBα (Ser32) and p-NF-κB p65 (Ser536) of HUVECs cells decreased in a concentration-dependent manner, indicating that the Chinese herbal compound of the present invention has an inhibitory effect on the activation of the NF-κB signaling pathway.

[0080] Example 14

[0081] Effect of the Traditional Chinese Medicine Compound Prepared in Example 2 on Reactive Oxygen Species in Human Nasal Epithelial Cells HNEpC

[0082] Human nasal mucosal epithelial cells in the logarithmic phase were selected and digested with trypsin. The cell concentration was adjusted to 2 × 10 5Cells were plated at 2 mL / well in a 6-well plate and cultured in a 37°C, 5% CO2 incubator for 24 hours. The old culture medium was discarded and different concentrations of the Chinese herbal compound containing Erastin (10 μM) were added at concentrations of 0, 0, and 0.5 mg / mL, mixed evenly, and cultured for 24 hours. HNEpC cell pellets were collected by trypsin digestion without EDTA, washed once with PBS, and centrifuged to obtain a cell pellet; DCFH-DA was then diluted with serum-free DMEM medium to a final working solution concentration of 10 μM. 1 mL of DCFH-DA working solution was added and the cells were mixed evenly. Incubate at 37°C in the dark for 30 minutes. After incubation, the cells were washed once with PBS and centrifuged to obtain a cell pellet. 500 μL of PBS was added to resuspend the cells. Detection was performed using a flow cytometer and processed using FlowJo software.

[0083] The results are as follows Figure 7 As shown in the results, compared with the blank control group, the Erastin group can significantly increase the content of ROS in HNEpC cells; while the Chinese herbal compound of the present invention can significantly inhibit the level of ROS in cells and maintain it at a normal state, indicating that the Chinese herbal compound of the present invention can inhibit the production of reactive oxygen species and inhibit the production of ROS, thereby blocking the increase in lipid peroxidation level caused by ROS accumulation, thereby exerting an anti-ferroptosis effect.

[0084] Example 15

[0085] Effect of the Traditional Chinese Medicine Compound Prepared in Example 2 on Lipid Peroxidation in Human Nasal Epithelial Cells HNEpC

[0086] Human nasal mucosal epithelial cells in the logarithmic phase were selected and digested with trypsin. The cell concentration was adjusted to 2 × 10 5 The cells were washed twice with PBS, and 1 mL of 5 μM BODIPY C11 working solution was added to the cells. The cells were incubated at 37°C in a 5% CO2 incubator for 30 minutes. The cells were then washed twice with PBS, and 1 mL of serum-free DMEM medium was added. The cells were then observed by a live cell workstation.

[0087] The results are as follows Figure 8 As shown, the medication group can significantly alleviate the cellular lipid peroxidation induced by Erastin, indicating that the Chinese herbal compound of the present invention antagonizes the occurrence of ferroptosis by inhibiting lipid peroxidation.

[0088] Example 16

[0089] The Chinese herbal compound prepared in Example 2 inhibits mitochondrial deformation of ferroptotic cells

[0090] HNEpC cells were plated in a confocal microplate and allowed to reach a cell density of approximately 60%-70%. Different concentrations of BYT solution containing Erastin (10 μM) were added, at 0, 0, and 0.5 mg / mL, and cultured for 24 hours. The old culture medium was discarded, the cells were washed twice with PBS, and 1 mL of 100 nM Mito-Tracker Deep Red FM working solution prepared in DMEM was added. The cells were incubated at 37°C in the dark for 30 minutes. The cells were then washed twice with PBS, and 1× Hoechest 33342 live cell stain was added. The cells were incubated at room temperature in the dark for 10 minutes. The cells were then washed twice with PBS, and 1 mL of DMEM was added. The cells were then imaged and observed using a live cell imaging workstation.

[0091] The results are as follows Figure 9 As shown in the data, Erastin treatment caused the mitochondrial morphology in HNEpC cells to shrink and undergo obvious deformation; while the intervention of the Chinese herbal compound of the present invention could significantly improve the morphology of mitochondria, so that most mitochondria maintained normal thread-like or rod-like shapes, indicating that the Chinese herbal compound of the present invention can effectively alleviate the mitochondrial deformation caused by ferroptosis and maintain the normal functional state of mitochondria.

[0092] Example 17

[0093] Effect of the Traditional Chinese Medicine Compound Prepared in Example 2 on the Levels of Ferroptosis Pathway-Related Proteins in HNEpC Cells

[0094] Healthy human nasal epithelial cells were plated evenly in a 6-well plate. When the cell density reached approximately 60%, the culture medium was aspirated and different concentrations of BYT solution containing erastin (10 μM) were added (0, 0, and 0.5 mg / mL). The cells were then incubated at 37°C in a 5% CO2 incubator for 24 hours. The cells were washed with normal saline and lysed with RIPA lysis buffer (containing 1% protease and phosphatase inhibitors). The cell lysate was transferred to a centrifuge tube and allowed to lyse on ice for 30 minutes. The cells were then centrifuged at 12,000 g for 20 minutes at 4°C. The supernatant was collected. Protein concentration was determined using a BCA assay and adjusted to the same concentration using RIPA lysis buffer based on the calculated protein concentration. 5× protein loading buffer (25% protein volume) was then added and mixed thoroughly. The samples were denatured in a 100°C thermostatted metal bath for 10 minutes. After cooling, the samples were aliquoted and stored at -40°C. Western-Blot method was used for quantitative detection to observe the effect of the Chinese herbal compound of the present invention on the levels of proteins related to the ferroptosis pathway in HNEpC cells.

[0095] The results showed that Figure 10As shown in the data, after incubation of the Chinese herbal compound with HUVECs, the protein expressions of GPX4, SLC7A11 and FTH1 in the model group were significantly inhibited compared with the blank control group, while the Chinese herbal compound of the present invention could significantly upregulate the protein levels of GPX4, SLC7A11 and FTH1. This result indicates that the mechanism by which the Chinese herbal compound of the present invention inhibits ferroptosis is related to the upregulation of SLC7A11, FTH1 and GPX4 genes.

[0096] Example 18

[0097] The Chinese herbal compound prepared in Example 2 inhibits the number of inflammatory cells in the peripheral blood of CRS patients

[0098] Peripheral blood samples of CRS patients before and after treatment with the Chinese herbal compound of the present invention were collected and placed in blood collection tubes (containing sodium citrate anticoagulant) and gently inverted to mix. 10 μL of blood samples were taken in 1.5 mL centrifuge tubes, and appropriate amounts of CD8-FITC, CD16-APC, CD45-PerCP-Cy5.5 and CD3-PE antibodies were added and mixed, and then incubated at 4 ° C in the dark for 30 minutes. After the incubation, 1 mL of red blood cell lysis buffer was added and lysed in a 37 ° C water bath for 10 minutes. The cell pellet was then collected by centrifugation at 400g for 5 minutes. After washing the cell pellet twice with PBS, the cells were resuspended in 500 μL of PBS. Data were collected using flow cytometry. The ratio of CD3+CD8+ cells to CD45+ white blood cells was used to represent the distribution of cytotoxic T lymphocytes (Tc cells) within the white blood cell population, and changes in their proportion within total lymphocytes (LYM) were also analyzed. The ratio of CD16+ cells to CD45+ cells reflected the distribution of eosinophils (EOS) within the granulocyte (GRA) population. Changes in the levels of different inflammatory cells in the blood were analyzed based on the experimental results.

[0099] The results are as follows Figure 11 As shown, the number of eosinophils in the medication group was significantly less than that in the untreated group, indicating that the Chinese herbal compound of the present invention has the ability to regulate the activity of inflammatory cells to alleviate the inflammatory symptoms of CRS patients.

[0100] Example 19

[0101] Effect of the Traditional Chinese Medicine Compound Prepared in Example 2 on Inflammatory Factors in the Peripheral Blood of CRS Patients

[0102] Peripheral blood samples were collected from CRS patients before and after treatment with the herbal compound of the present invention. The samples were placed in blood collection tubes (containing sodium citrate as an anticoagulant) and gently inverted to mix. After centrifugation at 1000 g for 30 minutes, the samples were collected and stored in aliquots at -20°C. The reagents used were prepared according to the ELISA kit instructions and used for testing.

[0103] The results are as follows Figure 12 As shown in the results, the Chinese herbal compound of the present invention can significantly reduce the expression levels of inflammatory factors TNF-α and IFN-γ in peripheral blood, indicating that the Chinese herbal compound of the present invention can inhibit the expression of inflammatory factors and thus affect the activity of inflammatory cells, thereby improving the inflammatory response and promoting the repair of sinus mucosa.

[0104] Example 20

[0105] The Chinese medicinal compound prepared in Example 2 is clinically used to improve chronic sinusitis

[0106] Patients diagnosed with chronic sinusitis and suitable for functional endoscopic sinus surgery were divided into two groups (factors that could affect the results, such as other physical indicators, were considered during grouping, and balanced distribution was performed to ensure comparability between groups). These groups included a normal treatment group and a group treated with the Chinese herbal compound of the present invention. Preoperative and postoperative nasal endoscopic examinations were performed to observe sinus recovery.

[0107] The results are as follows Figure 13 As shown in the data, compared with the normal treatment group, the patients in the Chinese herbal compound treatment group (in addition to the same treatment as the normal treatment group, they also took the Chinese herbal compound prepared in Example 2) had no obvious sinus mucosal congestion under nasal endoscopy one month after surgery, and the range of the formed nasal mucosal vesicles was relatively limited and dissipated faster. This result shows that BYT can improve the inflammation after CRS surgery and promote the repair of sinus mucosa.

[0108] Example 21

[0109] Analysis of the synergistic effect of the chemical components in the traditional Chinese medicine compound prepared in Example 2

[0110] 0.1 g of the traditional Chinese medicine compound powder prepared in Example 3 was accurately weighed and thoroughly dissolved in 10 mL of methanol, and the mass was then reweighed. After ultrasonication for 30 min, the mass was reweighed again. After adding methanol to the weight, the sample solution was filtered through a 0.22 μm aqueous filter to obtain a sample solution. The sample solution was detected using UPLC-Q Exactive-Orbitrap MS technology and matched with the established data.

[0111] The results are as follows Figure 14 and Figure 15 As shown, 101 chemical components were preliminarily identified, among which some compounds showed good response signals in both positive and negative ion modes.

[0112] The Pubchem database was used to obtain the SMILES files of each compound, and then the SwissTargePrediction database was used to import each compound to obtain its target. Targets with "Probability ≥ 0" were summarized, and all targets were corrected using the UniProt database, and duplicates were removed to obtain a target database. The Metascape database and the Harmonizone database were used to summarize the targets of the NF-κB signaling pathway and the ferroptosis pathway, and target databases were established respectively. The compound targets of the traditional Chinese medicine compound of the present invention were mapped to the targets of the NF-κB signaling pathway and the ferroptosis pathway, and a "component-intersection target" network diagram was further constructed using Cytoscape3.8.1 software.

[0113] The results are as follows Figure 16 and Figure 17 The results showed that 60 compounds from the present invention's traditional Chinese medicine compound formula are associated with the NF-κB signaling pathway, including active ingredients such as leonurine, baicalin, and berberine. These compounds exert their pharmacological effects by interacting with 26 NF-κB targets (VAM-1, ICAM-1, TNF, and IκBκB, among others). A total of 57 compounds from the present invention's traditional Chinese medicine compound formula are associated with the ferroptosis pathway, including ryegrass lactone, piperine, and kaempferol. These active ingredients participate in regulating the ferroptosis process by interacting with 10 ferroptosis-related targets (such as HMOX1, ALOX15, and NOX4). These results suggest that the anti-inflammatory and anti-ferroptosis effects of the traditional Chinese medicine compound formula depend on the synergistic effects of its multiple potential active ingredients.

[0114] In the above Examples 8 to 21, unless otherwise specified, words such as medicine and BYT all refer to the Chinese herbal compound decoction prepared in Example 2.

[0115] Although the specific embodiments of the present invention are described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and are not intended to limit the scope of the present invention. Any modifications and changes made by those skilled in the art in accordance with the spirit of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A Chinese medicinal compound for reducing the recurrence of chronic sinusitis, characterized by: The Chinese medicine compound is prepared from the following raw materials by weight: Astragalus 14-16g, stir-fried Scutellaria baicalensis 8-10g, Angelica dahurica 8-10g, Xanthium sibiricum 5-7g, Magnolia florid 5-7g, Chrysanthemum indicum 14-16g, stir-fried Gardenia jasminoides 11-13g, Angelica sinensis 5-7g, Safflower 5-7g, and Menthol 8-10g.

2. A Chinese medicinal compound for reducing the recurrence of chronic sinusitis according to claim 1, characterized in that: The astragalus root 15g, the fried scutellaria root 9g, the angelica root 9g, the Xanthium sibiricum 6g, the magnolia flower 6g, the wild chrysanthemum 15g, the fried gardenia 12g, the angelica root 6g, the safflower 6g, and the mint 9g.

3. The method for preparing the Chinese medicinal compound according to claim 1, wherein: The method comprises the following steps: weighing each raw medicine, crushing and mixing them respectively to obtain a medicine mixture; adding cold water equivalent to 3-5 times the total weight of the medicine mixture, soaking for 30 minutes, boiling over high heat and then decocting over low heat for 30-60 minutes, filtering, collecting the first filtrate, adding water to the filter residue and decocting again as above, filtering, and collecting the second filtrate; and mixing the two collected filtrates to obtain a traditional Chinese medicine compound decoction.

4. The preparation method according to claim 3, wherein: The traditional Chinese medicine compound decoction is vacuum freeze-dried to obtain powder, pharmaceutically acceptable excipients are added to the powder, and a drug administration preparation other than the decoction is prepared according to conventional processes.

5. The preparation method according to claim 4, characterized in that: The administration preparation is tablet, pill, powder, granule, oral liquid or capsule.

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