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

Abstract

The invention relates to the technical field of traditional Chinese medicines, in particular to a traditional Chinese medicine composition and application thereof in preparation of medicines for treating diseases related to abnormal proliferation of epithelial cells. The invention provides a prevention and treatment effect of osmundone on epithelial cell abnormal proliferation related diseases, and provides a composition for treating epithelial cell abnormal proliferation and a preparation method and application thereof. Experiments show that the osmundone or the traditional Chinese medicine composition can reduce oxidative stress injury and inhibit abnormal cell proliferation, and fibrosis is improved from two important pathogenesis corresponding to fibrosis.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicines, and in particular to a traditional Chinese medicine composition and its application in preparing medicines for treating diseases related to abnormal proliferation of epithelial cells. Background Art

[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 cavities and cysts in the body, playing important roles such as protection, secretion, and absorption. For example, epithelial cells of the skin can block the invasion of external pathogens and harmful substances; epithelial cells of the gastrointestinal tract participate in the absorption of nutrients.

[0003] Epithelial cells play a key role in various tissues and organs of the human body. Normal epithelial cell proliferation maintains the physiological homeostasis of the body. However, when abnormal proliferation occurs, it often causes a series of diseases, such as fibrosis and some tumors. There are many reasons for abnormal proliferation of epithelial cells. First, genetic factors cannot be ignored. Certain gene mutations can make the normal proliferation regulation mechanism in cells ineffective, causing cells to divide and proliferate uncontrollably. Like hereditary tumor syndrome, there are 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 during smoking, can damage the DNA of epithelial cells and induce abnormal proliferation; ionizing radiation can also destroy the genetic material of cells and start abnormal proliferation programs. Furthermore, chronic inflammatory stimulation is also prone to abnormal proliferation. The inflammatory factors that persist in the inflammatory environment constantly stimulate epithelial cells, prompting them to continuously renew and divide. Over time, uncontrolled proliferation may occur.

[0004] There are various treatment methods for diseases caused by abnormal proliferation of epithelial cells. Western medicine is the main treatment in clinical practice: For fibrotic diseases, such as pulmonary fibrosis and liver fibrosis, commonly used drugs in clinical practice include immunosuppressants such as glucocorticoids, which can inhibit inflammatory responses, reduce further abnormal proliferation of epithelial cells caused by inflammatory stimulation, and delay the process of fibrosis. For some tumor diseases caused by abnormal proliferation of epithelial cells, surgical resection and radiotherapy and chemotherapy are the main treatment methods. Western medicine has large side effects in treating abnormal proliferation of epithelial cells. Immunosuppressants such as glucocorticoids for treating fibrosis may cause metabolic disorders, and radiotherapy and chemotherapy for treating tumors can cause serious side effects.

[0005] Physical therapy and Chinese medicine treatment also play a vital role in the treatment of abnormal proliferation of epithelial cells. For example, in some superficial epithelial abnormal proliferation lesions, physical means such as cryotherapy and laser therapy can also play a role. Chinese medicine has unique advantages in intervening in diseases caused by abnormal proliferation of epithelial cells. Many Chinese herbal compound prescriptions or single herbs have been found to have anti-fibrosis, anti-tumor and other effects. Through the combination of multiple herbs, the body can be comprehensively conditioned, the patient's physical condition can be improved, the internal environment disorder in the body can be corrected, and the inducement of abnormal proliferation of epithelial cells can be fundamentally reduced, achieving the effect of treating both the symptoms and the root causes.

[0006] However, the existing compositions that inhibit epithelial cell proliferation do not have clear mechanisms, significant effects, stable efficacy, and so on. Summary of the invention

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

[0008] The invention provides application of porphyrin in preparing medicine for preventing and treating diseases related to abnormal proliferation of epithelial cells.

[0009] The present invention analyzes the monomer components of 258 kinds of traditional Chinese medicines, most of which are from polyphenols. The results show that osfera ketone has the strongest activation effect on antioxidant enzymes, which is better than other common polyphenols such as quercetin, chlorogenic acid and catechins. After further verification, osfera ketone can protect epithelial cells under oxidative stress conditions and can inhibit the abnormal proliferation of epithelial cells. That is, in the present invention, the prevention and treatment of diseases related to abnormal proliferation of epithelial cells includes anti-oxidative stress damage to epithelial cells.

[0010] COL1A1 (type I collagen α1 chain) and FN1 (fibronectin 1) are closely related to epithelial cell proliferation. Under pathological conditions, such as in fibrotic diseases (liver fibrosis, pulmonary fibrosis, etc.), due to inflammatory stimulation, cytokine imbalance and other factors, fibroblasts will be overactivated, leading to the massive synthesis and abnormal deposition of COL1A1. FN1 is involved in regulating the behavior of epithelial cells. In tissue fibrosis caused by chronic inflammation, FN1 will also be abnormally expressed. The experiments of the present invention show that purpurogenol can inhibit the expression level of COL1A1 and / or FN1 in epithelial cells, especially in abnormally proliferating epithelial cells induced by TGF-β1.

[0011] The epithelial cells are widely distributed: the skin on the body surface has stratified squamous epithelial cells, which play a protective role; the digestive system, such as the stomach and the inner surface of the intestine; the respiratory system trachea, alveoli; the urinary system bladder and other parts, as well as the reproductive system, all have epithelial cells. By inhibiting the expression of COL1A1 and / or FN1, osfera ketone can inhibit the abnormal proliferation of epithelial cells from various sources. In an embodiment of the present invention, the epithelial cells are lung epithelial cells. As a feasibility case, the disease related to the abnormal proliferation of epithelial cells is at least one of pulmonary fibrosis, lung cancer, pulmonary nodules, chronic obstructive pulmonary disease or asthma.

[0012] Osfera ketone exists in many plants. The present invention extracts plants rich in osfera ketone and then compounds them, and obtains a composite material from multiple formulas. The compatibility of each component is reasonable, and compared with other formulas, a better effect of inhibiting epithelial cell proliferation can be obtained.

[0013] The invention also provides a composition, which consists of Osmanthus fragrans, Osmanthus chinensis, Phellinus linteus, Chaga and Elsholtzia ciliata.

[0014] In the composition of the present invention, the weight parts of each component are: 3-30 parts of Osmanthus fragrans, 2-30 parts of Osmanthus fragrans, 2-50 parts of Phellinus linteus, 5-50 parts of Chaga, and 1-30 parts of Elsholtzia ciliata.

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

[0016] Although Osmanthus fructus, Osmanthus chinensis, Phellinus linteus, Chaga and Elsholtzia ciliata all contain osmanthone, the experiment shows that the effect of the combination is not completely positively correlated with the content of osmanthone, which may be related to other components in the extract. The combination of Osmanthus fructus, Osmanthus chinensis, Phellinus linteus, Chaga and Elsholtzia ciliata is the best combination screened so far.

[0017] Furthermore, the present invention also provides an extract obtained by extracting 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 comprises refluxing the extracts of Osmanthus fragrans, Osmanthus chinensis, Phellinus linteus, Chaga and Elsholtzia ciliata with ethanol aqueous solution, respectively, and concentrating the extracts respectively and then mixing them to obtain the extract.

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

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

[0022] In the embodiment of the present invention, in the extraction step, the reflux extraction condition is reflux at 40-60°C for 0.5-2h, and reflux extraction is performed 2-5 times. Preferably, the reflux extraction condition is reflux at 50°C for 1h, and reflux extraction is performed 3 times.

[0023] In the embodiment of the present invention, the ratio of each extract when mixed is 3-30 parts of Osmanthus fragrans, 2-30 parts of Osmanthus chinensis, 2-50 parts of Phellinus linteus, 5-50 parts of Chaga, and 1-30 parts of Elsholtzia ciliata. Preferably, the ratio of each extract when mixed is 1 part of Osmanthus fragrans, 1 part of Osmanthus fragrans, 1 part of Phellinus linteus, 1 part of Chaga, and 1 part of Elsholtzia ciliata.

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

[0025] The medicine of the present invention also includes pharmaceutically acceptable adjuvants or additives.

[0026] In the medicine described in 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, glycerol, propylene glycol, Tween 80, sodium benzoate, citric acid, tartaric acid, sodium bicarbonate, and sodium carbonate.

[0027] In the embodiment of the present invention, the dosage form of the drug is tablets, capsules, pills, powders, granules, syrups, solutions, suspensions, emulsions, tinctures, suppositories, ointments, creams, gels, patches, aerosols, powder sprays, injections, eye drops, nasal drops, ear drops, lotions, liniments, coatings, gargles, enemas, films, pellets, microcapsules or liposomes.

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

[0029] In the present invention, the administration method includes: oral, rectal administration, external application, application, inhalation, injection, eye drops, nose drops, ear drops, external irrigation, external wiping, oral gargle, 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, throat administration, gastric administration or duodenal administration.

[0030] In the present 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. Diseases caused by abnormal proliferation of epithelial cells in other locations may also be involved, for example, liver fibrosis, kidney fibrosis, myocardial fibrosis, pancreatic fibrosis or skin fibrosis.

[0031] In the present invention, the object of prevention and treatment is a human or a mammal, and the mammal includes bovines, equines, ovines, porcines, canines, felines, rodents, and primates.

[0032] The present invention provides the preventive and therapeutic effects of osidone 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 osidone or the Chinese medicine composition can reduce oxidative stress damage and inhibit abnormal cell proliferation, thereby improving fibrosis from two important pathogenesis mechanisms corresponding to fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Relative luciferase activity of 258 monomer components of traditional Chinese medicine;

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

[0035] Figure 3 This is a high performance liquid chromatography identification chart of porphyrin in the composition of Example 2;

[0036] Figure 4 This is a data graph showing the effect of porphyrin on A549 cell activity after 24 hours of treatment;

[0037] Figure 5 This is a data graph showing the effect of the full-form combination on the activity of A549 cells for 24 hours;

[0038] Figure 6 This is a data graph showing the effect of porphyrin on the activity of A549 cells after 48 hours of treatment;

[0039] Figure 7 This is a data graph showing the effect of the whole composition on the activity of A549 cells after 48 hours of action;

[0040] Figure 8 This is a data graph showing the effects of the whole composition and compositions 1 to 8 on the activity of A549 cells after 48 hours of action;

[0041] Fig. 9 The data graph shows the protective effect of porphyrin on tert-butyl hydroperoxide-induced oxidative stress injury in A549 cells;

[0042] Fig.10 This is a data graph showing the protective effect of the whole combination on tert-butyl hydroperoxide-induced oxidative stress damage in A549 cells;

[0043] Fig.11 The data graph shows the inhibitory effect of the whole formula of the combination and purpurogenol on abnormal proliferation of A549 cells induced by TGF-β1 by reducing the mRNA expression level of COL1A1;

[0044] Fig.12 The graph shows the data of the inhibitory effect of the whole formula of the combination and purpurogenol on abnormal proliferation of A549 cells induced by TGF-β1 by reducing the mRNA expression level of FN1. DETAILED DESCRIPTION

[0045] The present invention provides a Chinese medicine composition and its use in preparing a drug for treating fibrosis. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It should be particularly noted that:

[0046] All similar substitutions and modifications are obvious to those skilled in the art, and they are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0047] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings understood by those of ordinary skill in the art.

[0048] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

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

[0050] In this application, "include", "comprising" and "having" are used interchangeably, and are intended to indicate the inclusiveness of the solution, meaning that the solution may have other elements besides the listed elements. At the same time, it should be understood that the use of "include", "comprising" and "having" descriptions in this article also provides a "consisting of..." solution.

[0051] In this application, "prevention and treatment" refers to the prevention and / or treatment of diseases. Specifically, it includes: through drug intervention, developing and using various drugs with targeted pharmacological effects, starting from regulating the physiological functions of the human body, relying on the preventive efficacy of drugs, enhancing the body's immunity, changing the pathogenic microenvironment in the body before the disease occurs, so as to reduce the risk of disease occurrence and achieve the purpose of preventing the disease; and when the disease has already occurred, according to the type, symptoms and development stage of the disease, select a suitable drug combination or specific therapeutic means, such as chemical drug therapy, to use drug ingredients to accurately act on the lesion site, inhibit the growth and reproduction of pathogens, alleviate symptoms or repair damaged body tissues, etc., to achieve effective treatment of the disease.

[0052] In this application, osmundacetone, CAS No.: 37079-84-8. English name: OsMundacetone. It is a simple phenolic compound with anti-inflammatory and antioxidant pharmacological activities, and has been proven to be able to treat neurodegenerative diseases such as Alzheimer's disease.

[0053] Osmunda japonica is the dried rhizome and petiole residues of the fern plant Osmunda japonica of the Osmundaaceae family and the genus Osmunda. It enters the lung, stomach and liver meridians and has the effects of clearing away heat, detoxifying, removing blood stasis and stopping bleeding.

[0054] Osmanthus fern is the spore body, young leaves and stems of the Osmanthus fern, which is edible. It is commonly known as "Osmanthus fern", "fern" and "Niumaoguang". It is rich in various nutrients such as vitamins, trace elements, protein and essential amino acids for the human body.

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

[0056] Chaga, also known as Inonotus obliquus, is a medicinal fungus of the genus Inonotus, belonging to the phylum Basidiomycetes, class Agaricaceae, order Folium Folium Folium, family Folium Folium. It grows on the bark of birch trees and trees and has multiple pharmacological activities such as antioxidant, hypoglycemic and immunomodulatory.

[0057] Elsholtzia (Mosla chinensis) is the dried aerial part of Mosla chinensis or Mosla chinensis of the genus Elsholtzia of the family Lamiaceae. It belongs to the lung and stomach meridians and has the functions of diaphoresis, dehumidification and diuresis.

[0058] The porphyrin ketone and the drug combination derived from it provided by the present invention have been shown by experimental studies to have a significant effect in treating pulmonary fibrosis, and have the potential to treat other related lung diseases caused by oxidative stress damage and abnormal cell proliferation, such as lung injury, lung nodules, lung cancer, asthma, chronic obstructive pulmonary disease, etc.

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

[0060] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and 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 the present application.

[0061] The test materials used in the present invention are all common commercial products and can be purchased on the market. The present invention is further described below in conjunction with the embodiments:

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

[0063] The Keap1 / Nrf2 / ARE pathway is an important pathway of oxidative stress. The antioxidant response element ARE is a key gene regulatory sequence that regulates phase II metabolic enzymes. After ARE is activated, it will produce a variety of antioxidant enzymes such as heme oxygenase 1 (HO-1) and glutathione (GSH). The high-throughput screening model of drugs at the cellular molecular level is widely used. The ARE antioxidant response element is an important gene regulatory sequence that can activate the expression of a variety of antioxidant enzymes. Therefore, this part first concatenates ARE with the Luciferace reporter gene to construct an ARE overexpression stable cell line, so as to screen out effective antioxidant components in the traditional Chinese medicine compound library.

[0064] The Hek293-ARE cell line was used to treat the 258 Chinese medicine monomer components in the compound library at a concentration of 10 μM. After incubation in the incubator for 24 hours, the luciferase activity in the cells was detected and the relative luciferase activity of the compounds was calculated. The screening results are as follows: Figure 1 shown.

[0065] The results showed that osidone had a good luciferase induction effect on Hek293-ARE cells, and its activation effect was 22.6%, which was better than other common polyphenols such as quercetin (11.3%), chlorogenic acid (10.1%) and catechins (9.8%). In order to re-screen the activity of osidone, it was diluted 3 times in sequence, its concentration dose curve was made, and its EC50 value was calculated. The results are as follows Figure 2 As shown, the results showed that purpurogenol not only had a good luciferase induction effect on Hek293-ARE cells but also had an obvious dose-effect relationship.

[0066] Example 2 Preparation of the composition and the detection experiment of porphyrinone therein

[0067] In order to confirm that the composition contains osfera ketone, the present invention first weighs an appropriate amount of osfera ketone standard, uses methanol to prepare a working solution with a concentration of 1 mL / kg, filters through a 0.22 μm microporous filter head, injects into a sample injection bottle, and detects by high performance liquid chromatography. Then, osfera cyrtonema, osmanthus, phellinus, chaga and elsholtzia are refluxed at 50°C for 1h with a solid-liquid ratio of 1:5, refluxed 3 times, and the three reflux extracts are combined. The combined extract is concentrated under reduced pressure by a rotary evaporator to recover ethanol, and each raw drug extract powder is obtained by freeze drying. Redissolved with methanol or DMSO (the sample for HPLC analysis is dissolved in methanol, and the sample for cell experiment is dissolved in DMSO), the concentration of the raw drug is unified to 100 mg / ml, and mixed by volume 1:1 to obtain a composition.

[0068] The extract of the composition was detected by high performance liquid chromatography. In order to ensure that the standard product and the purpurogenol contained in the sample correspond to each other, the above analytical chromatographic conditions were kept consistent, and the specific settings were as follows:

[0069] (1) Using a Z0RBAX SB-C18 column (5 μm, 4.6 mm × 250 mm);

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

[0071] (3) Column temperature was 25 °C, injection volume was 10 μL, and flow rate was 1 mL / min;

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

[0073] The experimental results are as follows Figure 3 As shown, in the HPLC chromatogram of the Osmanthus fuchsia extract, a peak corresponding to the standard substance of porphyrinone was observed at a retention time of 12.6 min. This result indicates that the Osmanthus fuchsia extract described in the present invention does contain the characteristic component porphyrinone.

[0074] Example 3 Preparation of the whole composition

[0075] The extracts of Osmanthus fragrans, Osmunda japonica, Elsholtzia ciliata, Phellinus linteus and Chaga were respectively extracted by reflux with ethanol aqueous solution, the extraction temperature was set to 50°C, the reflux time was 1h, the solid-liquid ratio was 1:5, and the extraction was repeated 3 times. The three reflux extracts of the five drugs were respectively combined, and the extracts were respectively concentrated by rotary evaporator under reduced pressure and dried into powder to obtain extracts of the five drugs. The five extracts were mixed in equal mass proportions to obtain the whole formula of the composition.

[0076] Example 4 Preparation of Composition 1

[0077] The Osmanthus fragrans, Osmanthus chinensis, Elsholtzia ciliata and Phellinus linteus were respectively extracted by reflux with ethanol aqueous solution, the extraction temperature was set to 50°C, the reflux time was 1h, the solid-liquid ratio was 1:5, and the extraction was refluxed 3 times. The three reflux extracts of the four drugs were respectively combined, and the extracts were respectively concentrated by rotary evaporator under reduced pressure and dried into powder to obtain extracts of the four drugs. The four extracts were mixed in equal mass proportions to obtain composition 1.

[0078] Example 5 Preparation of Composition 2

[0079] Osmanthus fragrans, Osmunda oxyphylla, Elsholtzia ciliata and Chaga were extracted by reflux with ethanol aqueous solution, the extraction temperature was set to 50°C, the reflux time was 1h, the solid-liquid ratio was 1:5, and the extraction was refluxed 3 times. The three reflux extracts of the four drugs were combined respectively, and the extracts were concentrated by rotary evaporator under reduced pressure and dried into powder to obtain extracts of the four drugs. The four extracts were mixed in equal mass proportions to obtain composition 2.

[0080] Example 6 Preparation of Composition 3

[0081] Osmanthus fragrans, Osmunda cyperus and Elsholtzia ciliata were respectively extracted by reflux with ethanol aqueous solution, the extraction temperature was set to 50°C, the reflux time was 1h, the solid-liquid ratio was 1:5, and reflux was performed 3 times. The three reflux extracts of the three drugs were combined respectively, and the extracts were concentrated under reduced pressure on a rotary evaporator and dried to powder to obtain extracts of the three drugs. The three extracts were mixed in equal mass proportions to obtain composition 3.

[0082] Example 7 Preparation of Composition 4

[0083] Osmunda japonica, Elsholtzia ciliata and Chaga were respectively extracted by reflux with ethanol aqueous solution, the extraction temperature was set to 50°C, the reflux time was 1h, the solid-liquid ratio was 1:5, and the extraction was refluxed 3 times. The three reflux extracts of the three drugs were combined respectively, and the extracts were concentrated by rotary evaporator under reduced pressure and dried into powder to obtain extracts of the three drugs. The three extracts were mixed in equal mass proportions to obtain composition 4.

[0084] Example 8 Preparation of Composition 5

[0085] The Osmanthus fragrans, Osmanthus chinensis and Phellinus igniarius were extracted by reflux with ethanol aqueous solution, the extraction temperature was set to 50°C, the reflux time was 1h, the solid-liquid ratio was 1:5, and the extraction was refluxed 3 times. The three reflux extracts of the three drugs were combined respectively, and the extracts were concentrated by rotary evaporator under reduced pressure and dried into powder to obtain extracts of the three drugs. The three extracts were mixed in equal mass proportions to obtain composition 5.

[0086] Example 9 Preparation of Composition 6

[0087] Elsholtzia ciliata and Chaga were extracted with ethanol aqueous solution by reflux, the extraction temperature was set to 50°C, the reflux time was 1h, the solid-liquid ratio was 1:5, and the extraction was refluxed 3 times. The three reflux extracts of the two drugs were combined respectively, and the extracts were concentrated under reduced pressure on a rotary evaporator and dried to powder to obtain extracts of the two drugs. The two extracts were mixed in equal mass proportions to obtain composition 6.

[0088] Example 10 Preparation of Composition 7

[0089] Osmunda odorata and Phellinus igniarius were extracted by reflux with ethanol aqueous solution, the extraction temperature was set at 50°C, the reflux time was 1h, the solid-liquid ratio was 1:5, and the extraction was refluxed 3 times. The three reflux extracts of the two drugs were combined respectively, and the extracts were concentrated by rotary evaporator under reduced pressure and dried into powder to obtain extracts of the two drugs. The two extracts were mixed in equal mass proportions to obtain composition 7.

[0090] Example 11 Preparation of Composition 8

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

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

[0093] A549 cells were cultured in 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. When the cells grew to 85%-90%, the cells were passaged, the old medium was discarded, 1-2mL PBS was used for washing, 1-2mL trypsin was used for digestion for 2min, 2-3mL fresh complete medium was used to gently blow the cells, the cell suspension was transferred to a 15mL centrifuge tube, the supernatant was discarded after centrifugation, and the cell suspension was resuspended in an appropriate amount of fresh complete medium, and passaged at a ratio of 1:3. The cells were cultured continuously in a 37℃, 5% CO2 incubator, the cell status was observed regularly, and the medium was changed in time.

[0094] Take the cells in the logarithmic growth phase and quantitatively dilute them with complete medium and inoculate them in a 96-well cell culture plate, 100 μL per well. After culturing for 24 hours until the cells adhere to the wall, discard the original medium and replace it with fresh medium according to the grouping, that is, add pure medium containing 0.25% DMSO to the control group; add purpurogenol solution (maximum working concentration is 100 μM) or two-fold serial dilution of the whole solution of the composition of Example 3 (maximum working concentration is 250 μg / mL), and set up 3 parallel wells for each concentration. After being placed in the incubator for 24 hours, discard the drug-containing medium, add 100 μL of 0.5 mg·mL -1 MTT solution was added, and the supernatant was discarded after 4 hours of incubation. 150 μL DMSO was added to each well, and the cells were shaken for 10 minutes to fully dissolve the crystals. The absorbance (A) of each well was measured by an ELISA instrument, and the detection wavelength was 490 nm. The cell viability was calculated from the measured absorbance, and the cell viability reflected the effect of the drug on the cell survival rate.

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

[0096] The experimental results are as follows Figure 4-Figure 8 As shown, in A549 cells, the maximum non-toxic concentration of osfera ketone for 24 hours was 50μM, and the maximum non-toxic concentration of osfera ketone for 48 hours was 100μM; the maximum non-toxic concentration of the whole combination for 24 / 48 hours was 31.3μg / mL. In addition, on this basis, the concentration of combination 1-8 was unified to 31.3μg / mL for cell safety testing ( Fig. 9 ), it can be seen that the cell safety of the whole combination is the highest, so it was selected as the research object for subsequent efficacy verification.

[0097] Example 13: Pharmacological efficacy experiment of the protective effect of porphyrin and its combination on t-butyl hydroperoxide (t-BHP)-induced oxidative stress damage in A549 cells

[0098] Based on the cytotoxic concentration of osfera ketone and the combination acting on A549 cells for 24 hours, and taking cell viability as an indicator, the anti-oxidative stress damage activity of the whole combination and osfera ketone was detected. A549 cells in the logarithmic growth phase were quantitatively diluted with complete medium and inoculated in 96-well cell culture plates, 100 μL per well. After culturing for 24 hours until the cells adhered to the wall, the original culture medium was discarded, and the cells cultured in the 96-well plate were divided into a control group, a model group and a drug group. Fresh culture medium was replaced according to the grouping, that is, the control group and the model group added pure culture medium containing drug solvent; the drug group added the whole combination solution or osfera ketone solution diluted twice in pure culture medium (the maximum concentration was the non-toxic concentration of each), and 3 parallel wells were set for each concentration. After being placed in an incubator for 24 hours, tert-butyl hydroperoxide (modeling dose of 500 μM) was added to the model group and the drug group of A549 cells to establish an oxidative stress damage model, and the normal group added an equal amount of culture medium. After culturing for 2 h, CCK-8 was used to detect cell viability.

[0099] The experimental results are as follows Figure 9-10 As shown, at a safe concentration, porphyrin and the whole combination can effectively improve the survival rate of A549 cells induced by tert-butyl hydroperoxide oxidative stress damage.

[0100] Example 14 Pharmacological Experiment on the Inhibitory Effect of Osiris ketone and Composition on Abnormal Proliferation of A549 Cells Induced by Transforming Growth Factor-β1

[0101] Based on the cytotoxic concentration of osfera ketone and the combination acting on A549 cells for 48 hours, the expression level of fibrosis indicators was detected by RT-qPCR, and the anti-fibrosis activity of the combination and osfera ketone was detected. A549 cells in the logarithmic growth phase were quantitatively diluted with complete medium and inoculated in a 12-well cell culture plate, 1000 μL per well. After culturing for 24 hours until the cells adhered to the wall, the original culture medium was discarded, and the cells cultured in the 12-well plate were divided into a control group, a model group, and a drug group. The maximum non-toxic concentration of the drug was determined according to the MTT results, and the drug was added using pure culture medium (or pure culture medium containing 10 ng / mL TGF-β1) and continued to be cultured for 48 hours. After 48 hours, the cells were lysed overnight, and the expression levels of COL1A1 and FN1, common indicators of fibrosis, were detected by qPCR after RNA extraction and reverse transcription.

[0102] The experimental results are as follows Figure 11-Figure 12 As shown in the results, osfera ketone and the whole combination can inhibit the mRNA expression levels of COL1A1 and FN1 related to pulmonary fibrosis.

[0103] Conclusion: Osfera ketone and the combination containing osfera ketone can reduce oxidative stress damage and inhibit abnormal cell proliferation, and the effect of the combination is better than that of osfera ketone, improving pulmonary fibrosis from the two important pathogenesis of pulmonary fibrosis.

[0104] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. The application of porphyrin in the preparation of drugs for preventing and treating diseases related to abnormal proliferation of epithelial cells.

2. The use according to claim 1, characterized in that: The prevention and treatment of diseases related to abnormal proliferation of epithelial cells includes resisting damage to epithelial cells caused by oxidative stress; Or the prevention and treatment of diseases related to abnormal epithelial cell proliferation includes inhibiting the expression level of COL1A1 and / or FN1.

3. The use according to claim 1 or 2, characterized in that: The epithelial cells are lung epithelial cells; the disease associated with abnormal proliferation of epithelial cells is at least one of pulmonary fibrosis, lung cancer, pulmonary nodules, chronic obstructive pulmonary disease or asthma.

4. A composition, characterized in that It is composed of Osmanthus fragrans, Osmanthus fragrans, Phellinus linteus, Chaga and Elsholtzia ciliata.

5. The composition according to claim 4, characterized in that The mass parts of each component are: 3-30 parts of Osmanthus fragrans, 2-30 parts of Osmanthus fragrans, 2-50 parts of Phellinus linteus, 5-50 parts of Chaga, and 1-30 parts of Elsholtzia ciliata.

6. The composition according to claim 5, characterized in that The mass parts of each component are: 1 part of Osmanthus fragrans, 1 part of Osmanthus fragrans, 1 part of Phellinus linteus, 1 part of Chaga, and 1 part of Elsholtzia ciliata.

7. An extract, characterized in that Obtained by extracting the composition according to any one of claims 4 to 6.

8. The method for preparing the extract according to claim 7, characterized in that: The method comprises the steps of refluxing extraction of Osmanthus fragrans, Osmanthus chinensis, Phellinus linteus, Chaga and Elsholtzia ciliata respectively with ethanol aqueous solution, and concentrating and drying the extracts respectively and then mixing them to obtain the extract.

9. The preparation method according to claim 8, characterized in that: The volume fraction of ethanol in the ethanol aqueous solution is 50%; The solid-liquid ratio of the medicinal material to the ethanol aqueous solution is 1:5; The reflux extraction conditions are reflux at 50°C for 1 hour and reflux extraction 3 times; The mixing is to mix the extracts in equal proportions.

10. A drug for preventing and treating diseases related to abnormal proliferation of epithelial cells, comprising porphyrin, the composition according to any one of claims 4 to 6, the extract according to claim 7, or the extract prepared by the preparation method according to claim 8 or 9.

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