Traditional Chinese medicine composition and application thereof in preparation of medicine for treating diseases related to abnormal proliferation of epithelial cells

Abstract

The present application relates to the technical field of traditional Chinese medicine, and particularly relates to a traditional Chinese medicine composition and application thereof in preparation of a medicine for treating diseases related to abnormal proliferation of epithelial cells. The present application provides a prevention and treatment effect of aspidin on diseases related to abnormal proliferation of epithelial cells, and provides a composition for treating abnormal proliferation of epithelial cells and a preparation method and application thereof. Experiments show that the aspidin or the traditional Chinese medicine composition can reduce oxidative stress damage and inhibit abnormal cell proliferation, thereby improving fibrosis from two important pathogenesis mechanisms of fibrosis.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, and in particular to traditional Chinese medicine compositions and their application in the preparation of drugs for treating diseases related to abnormal proliferation of epithelial cells. Background Technology

[0002] Epithelial cells are cells located on the surface of human skin or cavities. They cover the outer surface of the body and the inner surface of various internal cavities and sacs, playing important roles in protection, secretion, and absorption. For example, skin epithelial cells can block the invasion of external pathogens and harmful substances; gastrointestinal epithelial cells participate in the absorption of nutrients.

[0003] Epithelial cells play a crucial role in various tissues and organs of the human body. Normal epithelial cell proliferation maintains the body's physiological homeostasis. However, abnormal proliferation often leads to a series of diseases, such as fibrosis and some tumors. The causes of abnormal epithelial cell proliferation are multifaceted. First, genetic factors cannot be ignored. Certain gene mutations can disrupt the normal proliferation regulation mechanisms within cells, leading to uncontrolled cell division and proliferation. For example, hereditary tumor syndromes involve specific gene defects that make epithelial cells prone to abnormal growth. Second, environmental factors have a significant impact. Long-term exposure to chemical carcinogens, such as polycyclic aromatic hydrocarbons in tobacco smoke, can damage epithelial cell DNA and induce abnormal proliferation; ionizing radiation can also damage cellular genetic material and initiate abnormal proliferation programs. Furthermore, chronic inflammation can also easily trigger abnormal proliferation. The persistent inflammatory factors in an inflammatory environment continuously stimulate epithelial cells, prompting them to constantly renew and divide, which over time can lead to uncontrolled proliferation.

[0004] Treatment methods for diseases caused by abnormal proliferation of epithelial cells are diverse. Clinically, Western medicine is the primary approach: for fibrotic diseases such as pulmonary fibrosis and liver fibrosis, commonly used drugs include immunosuppressants such as glucocorticoids, which can inhibit the inflammatory response, reduce further abnormal proliferation of epithelial cells caused by inflammatory stimulation, and slow the progression of fibrosis. For some tumors caused by abnormal proliferation of epithelial cells, surgical resection and radiotherapy / chemotherapy are the main treatment methods. Western medicine treatments for abnormal epithelial cell proliferation have significant side effects; immunosuppressants such as glucocorticoids used to treat fibrosis may cause metabolic disorders, while radiotherapy and chemotherapy used to treat tumors can lead to severe side effects.

[0005] Physical therapy and traditional Chinese medicine (TCM) play crucial roles in treating abnormal epithelial cell proliferation. For example, cryotherapy and laser therapy can be effective in treating superficial epithelial proliferative lesions. TCM has unique advantages in intervening in diseases caused by abnormal epithelial cell proliferation. Many TCM compound formulas or single herbs have been found to have anti-fibrotic and anti-tumor effects. Through the combination of multiple herbs, the body can be comprehensively regulated, improving the patient's constitution, correcting internal environmental imbalances, and fundamentally reducing the inducing factors of abnormal epithelial cell proliferation, achieving both symptomatic and root-cause treatment.

[0006] However, the existing compositions that inhibit epithelial cell proliferation have unclear mechanisms, insufficient efficacy, and unstable pharmacodynamics. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a traditional Chinese medicine composition and its application in the preparation of a medicament for treating fibrosis.

[0008] This invention provides the application of styraxone in the preparation of drugs for the prevention and treatment of diseases related to abnormal proliferation of epithelial cells.

[0009] This invention analyzes the monomeric components of 258 kinds of traditional Chinese medicines, most of which are polyphenols. The results show that osmidrone has the strongest activating effect against antioxidant enzymes, superior to other common polyphenols such as quercetin, chlorogenic acid, and catechins. Further verification shows that osmidrone can protect epithelial cells under oxidative stress and inhibit abnormal epithelial cell proliferation. Therefore, the prevention and treatment of epithelial cell abnormal proliferation-related diseases in this invention includes combating oxidative stress-induced damage to epithelial cells.

[0010] Both COL1A1 (type I collagen α1 chain) and FN1 (fibronectin 1) are closely related to epithelial cell proliferation. In pathological conditions, such as fibrotic diseases (liver fibrosis, pulmonary fibrosis, etc.), fibroblasts become overactivated due to inflammatory stimulation and cytokine imbalance, leading to excessive synthesis and abnormal deposition of COL1A1. FN1 participates in regulating epithelial cell behavior; in tissue fibrosis caused by chronic inflammation, FN1 also shows abnormal expression. The experiments in this invention show that scutellarin can inhibit the expression levels of COL1A1 and / or FN1 in epithelial cells, especially TGF-β1-induced abnormally proliferating epithelial cells.

[0011] The epithelial cells are widely distributed: stratified squamous epithelial cells on the skin surface provide protection; they are also found on the inner surfaces of the digestive system (e.g., stomach, intestines); in the respiratory system (trachea, alveoli); in the urinary system (bladder); and in the reproductive system. By inhibiting the expression of COL1A1 and / or FN1, scutellarin can inhibit the abnormal proliferation of epithelial cells from various sources. In this embodiment of the invention, the epithelial cells are lung epithelial cells. As a feasible example, the diseases associated with the abnormal proliferation of these epithelial cells include at least one of pulmonary fibrosis, lung cancer, pulmonary nodules, chronic obstructive pulmonary disease, or asthma.

[0012] Ostreone is found in a variety of plants. This invention extracts ostreone-rich plants and then combines them to obtain a composition from multiple formulations. The composition of each component is reasonably matched and can achieve a better effect of inhibiting epithelial cell proliferation compared with other formulations.

[0013] The present invention also provides a composition comprising Osmanthus fragrans, Brassica oleracea, Phellinus linteus, Chaga mushroom and Elsholtzia ciliata.

[0014] The composition of the present invention comprises the following components in parts by weight: 3-30 parts of Osmanthus fragrans, 2-30 parts of Bracken fern, 2-50 parts of Phellinus linteus, 5-50 parts of Chaga mushroom, and 1-30 parts of Elsholtzia splendens.

[0015] In some specific embodiments, the mass parts of each component in the composition are: 1 part of Osmanthus fragrans, 1 part of Bracken fern, 1 part of Phellinus linteus, 1 part of Chaga mushroom, and 1 part of Elsholtzia ciliata.

[0016] Although *Dryopteris crassirhizoma*, *Vitis thunbergii*, *Phellinus linteus*, *Chaga mushroom*, and *Elsholtzia ciliata* all contain osmidrone, experiments show that the effect of the combination is not entirely positively correlated with the osmidrone content, possibly due to other components in the extract. The combination of *Dryopteris crassirhizoma*, *Vitis thunbergii*, *Phellinus linteus*, *Chaga mushroom*, and *Elsholtzia ciliata* is currently the most effective formulation obtained through screening.

[0017] Furthermore, the present invention also provides an extract obtained by extraction from the composition as described above.

[0018] The present invention does not limit the preparation method of the extract, and any extraction method known in the art can be used to prepare the extract of the present invention.

[0019] Furthermore, the present invention also provides a method for preparing the extract as described above, which includes refluxing Osmanthus fragrans, Bletilla striata, Phellinus linteus, Chaga mushroom and Elsholtzia ciliata with an ethanol aqueous solution, concentrating the extracts separately and then mixing them to obtain the extract.

[0020] In this embodiment of the invention, the volume fraction of ethanol in the aqueous ethanol solution is 20%–70%, preferably 30%–60%, and more preferably 45%–55%. For example, the volume fraction of ethanol in the aqueous ethanol solution of the extraction step is 45%, 50%, or 55%.

[0021] In this embodiment of the invention, in the extraction step, the material-to-liquid ratio of the medicinal material to the ethanol-water solution is 1:(1-10). Preferably, the material-to-liquid ratio is 1:(3-7). More preferably, the material-to-liquid ratio is 1:(4-6). For example, in the extraction step, the material-to-liquid ratio of the medicinal material to the ethanol-water solution is 1:4, 1:5, or 1:6.

[0022] In this embodiment of the invention, the reflux extraction conditions in the extraction step are reflux at 40–60°C for 0.5–2 hours, and reflux extraction 2–5 times. Preferably, the reflux extraction conditions are reflux at 50°C for 1 hour, and reflux extraction 3 times.

[0023] In this embodiment of the invention, the mixing ratio of each extract is 3-30 parts of *Dryopteris crassirhizoma*, 2-30 parts of *Vitis vinifera*, 2-50 parts of *Phellinus linteus*, 5-50 parts of *Chaga mushroom*, and 1-30 parts of *Elsholtzia ciliata*. Preferably, the mixing ratio of each extract is 1 part of *Dryopteris crassirhizoma*, 1 part of *Vitis vinifera*, 1 part of *Phellinus linteus*, 1 part of *Chaga mushroom*, and 1 part of *Elsholtzia ciliata*.

[0024] Furthermore, the present invention also provides a medicament for preventing and treating diseases related to abnormal proliferation of epithelial cells, comprising scutellarin, the composition as described above, the extract as described above, or the extract prepared by the method described above.

[0025] The drug described in this invention also includes pharmaceutically acceptable excipients or additives.

[0026] In the drug of the present invention, the excipients include at least one of lactose, starch, microcrystalline cellulose, sodium carboxymethyl starch, hydroxypropyl methylcellulose, povidone, magnesium stearate, talc, silicon dioxide, mannitol, sorbitol, sucrose, glucose, dextrin, pregelatinized starch, ethyl cellulose, methyl cellulose, poloxamer, polyethylene glycol, sodium lauryl sulfate, lecithin, glycerin, propylene glycol, Tween 80, sodium benzoate, citric acid, tartaric acid, sodium bicarbonate, and sodium carbonate.

[0027] In this embodiment of the invention, the dosage form of the drug is tablet, capsule, pill, powder, granule, syrup, solution, suspension, emulsion, tincture, suppository, ointment, cream, gel, patch, aerosol, powder spray, injection, eye drops, nasal drops, ear drops, lotion, liniment, film, mouthwash, enema, film, microcapsule, microencapsulation, or liposome.

[0028] Furthermore, the present invention also provides a method for preventing and treating diseases related to abnormal proliferation of epithelial cells, comprising administering the drug as described above.

[0029] In this invention, the administration methods include: oral, rectal, topical application, patch, inhalation, injection, eye drops, nasal drops, ear drops, external irrigation, external wiping, oral gargling, enema, sublingual administration, transdermal administration, vaginal administration, urethral administration, nasal spray, ear spray, oral spray, implantation, intraperitoneal administration, intrathecal administration, intra-articular administration, local administration, transdermal absorption, oral mucosal administration, pharyngeal administration, gastric administration, or duodenal administration.

[0030] In this invention, the diseases for preventing and treating abnormal proliferation of epithelial cells include at least one of pulmonary fibrosis, lung cancer, pulmonary nodules, chronic obstructive pulmonary disease, or asthma. It may also involve diseases caused by abnormal proliferation of epithelial cells in other locations, such as liver fibrosis, kidney fibrosis, myocardial fibrosis, pancreatic fibrosis, or skin fibrosis.

[0031] In this invention, the target of prevention and control is a human or mammal, including bovines, equines, sheep, pigs, canines, felines, rodents, and primates.

[0032] This invention provides the preventive and therapeutic effects of osmidrone on diseases related to abnormal epithelial cell proliferation, and provides a composition for treating abnormal epithelial cell proliferation, its preparation method, and its application. Experiments show that the osmidrone or traditional Chinese medicine composition can reduce oxidative stress damage and inhibit abnormal cell proliferation, thereby improving fibrosis by addressing two important pathogenic mechanisms corresponding to fibrosis. Attached Figure Description

[0033] Figure 1 The relative luciferase activity of 258 monomeric components of traditional Chinese medicine;

[0034] Figure 2 This is the concentration-dose curve of osmidrone;

[0035] Figure 3 This is a high-performance liquid chromatography (HPLC) image of the component osmidrone contained in the composition of Example 2;

[0036] Figure 4 A graph showing the effect of styraxone treatment on the viability of A549 cells after 24 hours;

[0037] Figure 5 A graph showing the effect of the full-formula composition on the activity of A549 cells after 24 hours of action;

[0038] Figure 6 A graph showing the effect of styraxone treatment on the viability of A549 cells after 48 hours;

[0039] Figure 7 A graph showing the effect of the full-formula composition on the activity of A549 cells after 48 hours of action;

[0040] Figure 8 Graphs showing the effect of the full formula of the composition and compositions 1-8 on the activity of A549 cells after 48 hours of treatment;

[0041] Figure 9 A graph showing the protective effect of styraxone against tert-butyl hydrogen peroxide-induced oxidative stress damage in A549 cells;

[0042] Figure 10 The graph shows the protective effect of the entire composition against tert-butyl hydrogen peroxide-induced oxidative stress damage in A549 cells.

[0043] Figure 11 The image shows the data on the inhibitory effect of the whole formula and osmanthus on TGF-β1-induced abnormal proliferation of A549 cells by reducing the mRNA expression level of COL1A1.

[0044] Figure 12 This is a graph showing the inhibitory effect of the whole formula and osmanthus on TGF-β1-induced abnormal proliferation of A549 cells by reducing the mRNA expression level of FN1. Detailed Implementation

[0045] This invention provides a traditional Chinese medicine composition and its application in the preparation of a drug for treating fibrosis. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that...

[0046] All similar substitutions and modifications will be obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art will be able to make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit and scope of this invention to implement and apply the technology of this invention.

[0047] Unless otherwise defined in this invention, the scientific and technical terms associated with this invention shall have the meanings understood by one of ordinary skill in the art.

[0048] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural.

[0049] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0050] In this application, the terms "comprising," "including," and "having" are used interchangeably to indicate the inclusiveness of a solution, meaning that the solution may contain elements other than those listed. It should also be understood that the use of "comprising," "including," and "having" herein also provides for solutions "consisting of...".

[0051] In this application, "prevention and treatment" refers to the prevention and / or treatment of diseases. Specifically, this includes: developing and utilizing various drugs with targeted pharmacological effects through drug intervention, starting with regulating human physiological functions, to enhance the body's immunity and alter the pathogenic microenvironment within the body before the disease occurs, thereby reducing the risk of disease and achieving the goal of disease prevention; and, in the event that a disease has already occurred, selecting appropriate drug combinations or specific therapeutic methods, such as chemotherapy, based on the type, symptoms, and stage of the disease, to utilize drug components to precisely target the lesion site, inhibiting the growth and reproduction of pathogens, alleviating symptoms, or repairing damaged body tissues, thereby achieving effective treatment of the disease.

[0052] In this application, OsMundacetone (CAS No. 37079-84-8) is mentioned. It is a structurally simple phenolic compound with anti-inflammatory and antioxidant pharmacological activities, and has been shown to treat neurodegenerative diseases such as Alzheimer's disease.

[0053] Osmunda japonica is the dried rhizome and petiole remnants of Osmunda japonica, a fern belonging to the Osmunaceae family and the Osmunda genus. It enters the lung, stomach, and liver meridians and has the effects of clearing heat and detoxifying, removing blood stasis and stopping bleeding.

[0054] Bracken fern, also known as fern shoots, is the tender leaves and stems of the sporophyte of Dryopteris crassirhizoma. It is edible and commonly called "bracken fern," "fern," or "ox hair bracken." It is rich in vitamins, trace elements, protein, and essential amino acids.

[0055] Phellinus igniarius is the fruiting body of the fungus Phellinus igniarius in the Polyporaceae family. It is distributed in North China and Northwest China and has the effects of promoting blood circulation, stopping bleeding, resolving phlegm, and stopping diarrhea.

[0056] Chaga mushroom, also known as Inonotus obliquus, is a medicinal fungus belonging to the genus Inonotus of the family Inonotaceae in the class Agaricales of the phylum Basidiomycota. It grows on the bark of birch trees and other trees and has various pharmacological activities such as anti-oxidation, hypoglycemia, and immunomodulation.

[0057] Mosla chinensis is the dried aerial part of the plant Mosla chinensis or Mosla spp. in the Lamiaceae family. It enters the lung and stomach meridians and has the functions of inducing sweating and relieving exterior syndromes, resolving dampness and harmonizing the middle jiao.

[0058] The combination of scutellarin and its derived drugs provided by this invention has been experimentally proven to have a significant therapeutic effect on pulmonary fibrosis, and has the potential to treat other lung diseases related to oxidative stress damage and abnormal cell proliferation, such as lung injury, pulmonary nodules, lung cancer, asthma, and chronic obstructive pulmonary disease.

[0059] The numerical ranges and parameters involved in this invention have been presented as precisely as possible in the specific embodiments. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise explicitly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have a reasonable deviation within a certain range, such as ±10%, ±5%, ±1%, or ±0.5%.

[0060] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0061] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0062] Example 1: High-throughput screening of compounds with antioxidant effects

[0063] The Keap1 / Nrf2 / ARE pathway is a crucial pathway for oxidative stress. The antioxidant response element (ARE) is a key gene regulatory sequence that regulates phase II metabolic enzymes, and its activation leads to the production of various antioxidant enzymes such as heme oxygenase 1 (HO-1) and glutathione (GSH). High-throughput drug screening models at the cellular and molecular level are widely used, and the ARE antioxidant response element is an important gene regulatory sequence that can activate the expression of various antioxidant enzymes. Therefore, this section first tandemly integrates ARE with the Luciferace reporter gene to construct a stable ARE overexpression cell line, thereby screening for effective antioxidant components from a traditional Chinese medicine compound library.

[0064] Using the Hek293-ARE cell line, 258 monomeric components of traditional Chinese medicine from the compound library were treated at a concentration of 10 μM. After incubation in an incubator for 24 h, the activity of luciferase in the cells was detected, and the relative luciferase activity of the compounds was calculated. The screening results are as follows: Figure 1 As shown.

[0065] The results showed that osmidrone had a good luciferase-inducing effect on Hek293-ARE cells, with an activation rate of 22.6%, which was superior to other common polyphenols such as quercetin (11.3%), chlorogenic acid (10.1%), and catechin (9.8%). To further screen the activity of osmidrone, it was repeatedly diluted 3-fold, and its concentration-dose curve was plotted. Its EC50 value was calculated, and the results are as follows: Figure 2 As shown, the results indicate that zisperidone not only has a good luciferase-inducing effect on Hek293-ARE cells, but also exhibits a significant dose-response relationship.

[0066] Example 2: Preparation of the composition and detection experiment of scutellarin in it

[0067] To confirm the presence of osmidrone in the composition, this invention first weighed an appropriate amount of osmidrone standard, prepared a working solution with methanol at a concentration of 1 mL / kg, filtered through a 0.22 μm microporous filter, injected into a sample vial, and detected by high-performance liquid chromatography (HPLC). Then, *Osmidrone sibiricum*, *Vitis thunbergii*, *Sanghuang*, *Chaenomeles speciosa*, and *Elsholtzia ciliata* were refluxed with 50% dilute ethanol at 50°C for 1 h, with a solid-liquid ratio of 1:5, and refluxed three times. The extracts from the three refluxes were combined. The combined extracts were concentrated under reduced pressure using a rotary evaporator to recover ethanol, and then freeze-dried to obtain powders of each active ingredient. These powders were then reconstituted in methanol or DMSO (samples for HPLC analysis were dissolved in methanol, and samples for cell experiments were dissolved in DMSO) to achieve a uniform crude drug concentration of 100 mg / mL, and mixed at a volume ratio of 1:1 to obtain the composition.

[0068] The extract of the composition was detected by high-performance liquid chromatography (HPLC). To ensure that the scutellarin contained in the standard and the sample corresponded, the above analytical chromatographic conditions were kept consistent, and the specific settings were as follows:

[0069] (1) A Z0RBAX SB-C18 column (5μm, 4.6mm×250mm) was used;

[0070] (2) Using water (A)-acetonitrile (B) as the mobile phase, and adding 0.1% formic acid to A;

[0071] (3) The column temperature is 25℃, the injection volume is 10μL, and the flow rate is 1mL / min;

[0072] (4) The elution program is as follows: 0-30 min 5%-95% B, 30-40 min 95% B.

[0073] Experimental results are as follows Figure 3 As shown, in the high-performance liquid chromatogram of the Osmanthus fragrans extract, an elution peak corresponding to the osmanthus ketone standard was observed at a retention time of 12.6 min. This result indicates that the Osmanthus fragrans extract described in this invention does indeed contain the characteristic component osmanthus ketone.

[0074] Example 3: Preparation of the complete formula of the composition

[0075] The five herbs, namely, *Dryopteris crassirhizoma*, *Vitis vinifera*, *Elsholtzia ciliata*, *Phellinus linteus*, and *Chaga mushroom*, were extracted by reflux with an ethanol-water solution at a temperature of 50°C for 1 hour, with a material-to-liquid ratio of 1:5. The reflux was repeated three times. The extracts from the three refluxes of the five herbs were combined. The extracts were then concentrated under reduced pressure using a rotary evaporator and dried into powder to obtain extracts of the five herbs. The five extracts were then mixed in equal mass ratios to obtain the complete formula.

[0076] Example 4 Preparation of Composition 1

[0077] Ostreae stolonifera, Brassica rapa, Elsholtzia ciliata, and Phellinus linteus were extracted separately by reflux with an ethanol-water solution. The extraction temperature was set at 50℃, the reflux time was 1h, the material-to-liquid ratio was 1:5, and the reflux was repeated 3 times. The three reflux extracts of the four drugs were combined. The extracts were concentrated under reduced pressure using a rotary evaporator and dried into powder to obtain extracts of the four drugs. The four extracts were mixed in equal mass ratios to obtain composition 1.

[0078] Example 5 Preparation of Composition 2

[0079] Osmanthus fragrans, Brassica oleracea, Elsholtzia ciliata, and Chaga mushroom were extracted by reflux with ethanol-water solution at a temperature of 50°C for 1 hour, with a material-to-liquid ratio of 1:5. The reflux was repeated three times. The extracts from the three refluxes of the four drugs were combined. The extracts were concentrated under reduced pressure using a rotary evaporator and dried into powder to obtain extracts of the four drugs. The four extracts were mixed in equal mass ratios to obtain composition 2.

[0080] Example 6 Preparation of Composition 3

[0081] Osmanthus fragrans, Brassica oleracea, and Elsholtzia ciliata were extracted separately by reflux with an ethanol-water solution. The extraction temperature was set at 50℃, the reflux time was 1h, the material-to-liquid ratio was 1:5, and the reflux was repeated 3 times. The three reflux extracts of the three drugs were combined. The extracts were concentrated under reduced pressure using a rotary evaporator and dried into powder to obtain extracts of the three drugs. The three extracts were mixed in equal mass ratios to obtain composition 3.

[0082] Example 7 Preparation of Composition 4

[0083] Bracken fern, Elsholtzia ciliata, and Chaga mushroom were extracted separately by reflux with an ethanol-water solution. The extraction temperature was set at 50℃, the reflux time was 1h, the material-to-liquid ratio was 1:5, and the reflux was repeated 3 times. The three reflux extracts of the three drugs were combined. The extracts were concentrated under reduced pressure using a rotary evaporator and dried into powder to obtain extracts of the three drugs. The three extracts were mixed in equal mass ratios to obtain composition 4.

[0084] Example 8 Preparation of Composition 5

[0085] Ostreae stolonifera, Brassica oleracea, and Phellinus linteus were extracted by reflux with ethanol-water solution at a temperature of 50°C for 1 hour, a material-to-liquid ratio of 1:5, and refluxed three times. The three reflux extracts of the three drugs were combined and concentrated under reduced pressure using a rotary evaporator and dried into powder to obtain extracts of the three drugs. The three extracts were then mixed in equal mass ratios to obtain composition 5.

[0086] Example 9 Preparation of Composition 6

[0087] Elsholtzia ciliata and Chaga mushroom were extracted separately by reflux with ethanol-water solution. The extraction temperature was set at 50℃, the reflux time was 1h, the material-to-liquid ratio was 1:5, and the reflux was repeated 3 times. The three reflux extracts of the two drugs were combined. The extracts were concentrated under reduced pressure by rotary evaporator and dried into powder to obtain extracts of the two drugs. The two extracts were mixed in equal mass ratio to obtain composition 6.

[0088] Example 10 Preparation of Composition 7

[0089] Bracken fern and Phellinus linteus were extracted separately by reflux with an ethanol-water solution. The extraction temperature was set at 50℃, the reflux time was 1h, the material-to-liquid ratio was 1:5, and the reflux was repeated 3 times. The three reflux extracts of the two drugs were combined. The extracts were concentrated under reduced pressure using a rotary evaporator and dried into powder to obtain extracts of the two drugs. The two extracts were mixed in equal mass ratios to obtain composition 7.

[0090] Example 11 Preparation of Composition 8

[0091] Ostreae stolonifera and Elsholtzia ciliata were extracted by reflux with ethanol-water solution. The extraction temperature was set at 50℃, the reflux time was 1h, the material-liquid ratio was 1:5, and the reflux was repeated 3 times. The three reflux extracts of the two drugs were combined. The extracts were concentrated under reduced pressure by rotary evaporator and dried into powder to obtain extracts of the two drugs. The two extracts were mixed in equal mass ratio to obtain composition 7.

[0092] Example 12 Study on the effects of scutellarin and its composition on lung epithelial cell activity

[0093] A549 cells were cultured in 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. When cells reached 85%–90% confluence, they were passaged, the old medium was discarded, and the cells were washed with 1–2 mL of PBS, digested with 1–2 mL of trypsin for 2 minutes, and gently pipetted with 2–3 mL of fresh complete medium. The cell suspension was transferred to 15 mL centrifuge tubes, centrifuged, the supernatant was discarded, and the cells were resuspended in an appropriate amount of fresh complete medium. The cells were then passaged 1:3. Cells were continued to culture at 37°C in a 5% CO2 incubator, with regular monitoring of cell status and medium changes as needed.

[0094] Cells in the logarithmic growth phase were quantitatively diluted with complete culture medium and seeded into 96-well cell culture plates, 100 μL per well. After culturing for 24 h until the cells adhered, the original culture medium was discarded, and fresh culture medium was added according to the groups: the control group was given pure culture medium containing 0.25% DMSO; the drug-treated group was given either styraxone solution (maximum working concentration 100 μM) or a serially diluted complete solution of the composition from Example 3 (maximum working concentration 250 μg / mL), with 3 parallel wells for each concentration. After incubation for 24 h, the drug-containing culture medium was discarded, and 100 μL of a 0.5 mg / mL solution was added to each well. -1 After culturing in MTT solution for 4 hours, the supernatant was discarded, and 150 μL of DMSO was added to each well. The mixture was shaken for 10 minutes to fully dissolve the crystals. The absorbance (A) of each well was measured using a microplate reader at a wavelength of 490 nm. Cell viability was calculated from the measured absorbance, and cell viability reflects the effect of the drug on cell survival.

[0095] Survival rate = (A 药物组 -A 空白组 ) / (A 对照组 -A 空白组 )×100%.

[0096] Experimental results are as follows Figures 4-8 As shown, in A549 cells, the maximum non-toxic concentration (NDC) of osmidrone after 24 hours of treatment was 50 μM, and after 48 hours of treatment, it was 100 μM; the maximum NDC of the entire composition after 24 / 48 hours of treatment was 31.3 μg / mL. Furthermore, based on this, cell safety was tested by unifying the concentration of compositions 1-8 to 31.3 μg / mL. Figure 9 It is evident that the entire composition exhibits the highest cellular safety, therefore it was selected as the subject of subsequent research for efficacy validation.

[0097] Example 13: Pharmacodynamic experiment on the protective effect of strychnine and its composition against tert-butyl hydroperoxide (t-BHP)-induced oxidative stress damage in A549 cells.

[0098] Based on the cytotoxic concentration of scutellarin and its composition after 24 hours in A549 cells, and using cell viability as an indicator, the antioxidant stress damage activity of the complete composition and scutellarin was detected. Logarithmically growing A549 cells were quantitatively diluted with complete culture medium and seeded into 96-well cell culture plates, 100 μL per well. After 24 hours of culture and cell attachment, the original culture medium was discarded, and the cells in the 96-well plates were divided into control, model, and drug treatment groups. The culture medium was replaced according to the groups: the control and model groups were given pure culture medium containing the drug solvent; the drug treatment groups were given serially diluted (two-fold) solutions of the complete composition or scutellarin (maximum concentration being the non-toxic concentration for each), with three parallel wells for each concentration. After culturing for another 24 hours, tert-butyl hydroperoxide (500 μM) was added to the model and drug treatment groups to establish an oxidative stress damage model, while the normal group received an equal volume of culture medium. After 2 hours of culture, cell viability was assessed using a CCK-8 assay.

[0099] Experimental results are as follows Figures 9-10 As shown, at safe concentrations, both styraxone and the entire composition can effectively improve the survival rate of A549 cells damaged by tert-butyl hydrogen peroxide induced by oxidative stress.

[0100] Example 14: Pharmacodynamic experiment on the inhibitory effect of scutellarin and its composition on the abnormal proliferation of A549 cells induced by transforming growth factor-β1.

[0101] Based on the cytotoxic concentration of styraxone and its composition after 48 hours of treatment in A549 cells, the expression levels of fibrosis markers were detected by RT-qPCR, and the antifibrotic activity of the composition and styraxone was assessed. A549 cells in logarithmic growth phase were quantitatively diluted with complete culture medium and seeded into 12-well cell culture plates (1000 μL per well). After 24 hours of culture and cell attachment, the original culture medium was discarded, and the cells in the 12-well plates were divided into control, model, and treatment groups. The maximum non-toxic concentration of the drug was determined based on the MTT assay results. The drug was added using pure culture medium (or pure culture medium containing 10 ng / mL TGF-β1), and the cells were cultured for another 48 hours. After 48 hours, the cells were lysed, incubated overnight, and RNA was extracted. After reverse transcription, the expression levels of commonly used fibrosis markers COL1A1 and FN1 were detected by qPCR.

[0102] Experimental results are as follows Figures 11-12 As shown, scutellarin and the complete formula can inhibit the expression levels of COL1A1 and FN1 mRNAs associated with pulmonary fibrosis.

[0103] Conclusion: The whole formula of scutellarin and scutellarin-containing compounds can reduce oxidative stress damage and inhibit abnormal cell proliferation. The effect of the compounds is better than that of scutellarin, thus improving pulmonary fibrosis by addressing two important pathogenesis mechanisms of pulmonary fibrosis.

[0104] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of scutellarin in the preparation of drugs for the prevention and treatment of pulmonary fibrosis.

2. The application according to claim 1, characterized in that, The prevention and treatment of pulmonary fibrosis includes combating oxidative stress damage to epithelial cells; Or the prevention and treatment of pulmonary fibrosis may include inhibiting the expression levels of COL1A1 and / or FN1.

3. The composition, characterized in that, It consists of 3-30 parts of Osmanthus fragrans, 2-30 parts of Bracken fern, 2-50 parts of Phellinus linteus, 5-50 parts of Chaga mushroom, and 1-30 parts of Elsholtzia ciliata.

4. The composition according to claim 3, characterized in that, The mass fractions of each component are: One part of Osmanthus fragrans, one part of Bracken fern, one part of Phellinus linteus, one part of Chaga mushroom, and one part of Elsholtzia ciliata.

5. An extract, characterized in that, Obtained by extraction from the composition according to claim 3 or 4.

6. The method for preparing the extract according to claim 5, characterized in that, The method involves reflux extraction of Osmanthus fragrans, Brassica oleracea, Phellinus linteus, Chaga mushroom and Elsholtzia ciliata with ethanol aqueous solution, followed by concentration and drying of the extracts and mixing to obtain the extract.

7. The preparation method according to claim 6, characterized in that, The volume fraction of ethanol in the ethanol-water solution is 50%. The ratio of medicinal materials to the ethanol aqueous solution is 1:5; The reflux extraction conditions were reflux at 50°C for 1 hour, and reflux extraction was performed 3 times. The mixing involves mixing the extracts in equal proportions.

8. A medicament for the prevention and treatment of diseases related to abnormal proliferation of epithelial cells, comprising the composition of claim 3 or 4, the extract of claim 5, or the extract prepared by the method of claim 6 or 7.

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