Traditional Chinese medicine composition containing osmundone and application of traditional Chinese medicine composition in preparation of medicine for preventing and treating diabetic complications

Abstract

The invention relates to the technical field of traditional Chinese medicines, in particular to a traditional Chinese medicine composition containing osmundone and application of the traditional Chinese medicine composition to preparation of medicines for preventing and treating diabetic complications. The invention provides a traditional Chinese medicine composition and an effect of osmundone serving as an active ingredient of the traditional Chinese medicine composition in preventing and treating diabetic complications caused by oxidative stress and / or abnormal proliferation of renal tubular cells. Experiments show that the osmundone or the traditional Chinese medicine composition can reduce microvascular endothelial cell injury caused by oxidative stress injury and inhibit abnormal proliferation of renal tubular cells.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicines, and in particular to a traditional Chinese medicine composition containing porphyrin and application thereof in preparing medicines for preventing and treating diabetic complications. Background Art

[0002] Diabetic complications refer to a series of serious and complex complications that occur during the course of diabetes, when the patient is in a state of high blood sugar for a long time, causing continuous damage to various body systems. Generally speaking, they can be mainly divided into two categories: microvascular disease and macrovascular disease.

[0003] Microvessels are distributed throughout many important organs and tissues of the human body, such as the retina and kidneys. Long-term high blood sugar levels can cause abnormal changes in the structure and function of microvessels, such as thickening of the blood vessel walls and narrowing of the lumen, which in turn affect the normal functioning of the corresponding organs. This may lead to serious consequences such as retinopathy affecting vision, or kidney disease threatening kidney function, posing a great challenge to the patient's quality of life and physical health. Clinically, microvascular disease symptoms are common in diabetic retinopathy, diabetic nephropathy, diabetic foot disease, and diabetic neuropathy, with a high incidence rate and no targeted treatment options, and the pathogenesis is still unclear.

[0004] At present, the widely accepted mechanisms leading to microvascular disease include non-enzymatic glycosylation theory, polyol pathway theory, protein kinase C system theory, hexosamine pathway theory and oxidative stress theory. Oxidative stress usually has different degrees of influence in multiple disease links and is also an important factor leading to endothelial cell structural damage and dysfunction. Oxidative stress is usually accompanied by the production of inflammatory responses, which enhance microvascular disease. Under high sugar conditions, cells produce oxidative stress, leading to increased reactive oxygen and free radicals, the formation of glycation end products, and weakened anti-oxidative stress mechanisms. Oxidative stress reactions can activate almost all signal pathways related to diabetic microvascular disease, including polyol pathways, AGEs formation, PKC pathways, and hexosamine pathways. Therefore, seeking safe and effective anti-oxidative stress drugs is beneficial to the prevention and treatment of diabetic complications. Summary of the invention

[0005] 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 medicine for preventing and treating diabetic complications.

[0006] The invention provides application of porphyrin in preparing medicine for preventing and treating diabetic complications.

[0007] The present invention analyzes the monomer components of 258 kinds of traditional Chinese medicines, most of which are from polyphenols. The results show that purpurogenol has the strongest activating effect on antioxidant enzymes, which is superior to other common polyphenols such as quercetin, chlorogenic acid and catechins. After further verification, purpurogenol can protect microvascular endothelial cells, especially human retinal microvascular endothelial cells, under oxidative stress conditions. In addition, diabetes is an important risk factor for renal fibrosis, and the present invention detects important molecular markers of fibrosis and finds that purpurogenol can inhibit the expression level of fibrosis-related genes. Therefore, it is proved that purpurogenol has the effect of preventing and treating diabetic complications.

[0008] In diabetic patients, when in a high sugar state for a long time, the body will produce an oxidative stress response. Excessive free radical generation, imbalance of the antioxidant defense system, and then damage to cells, blood vessels, etc., involving multiple organs such as eyes, kidneys, and heart, so oxidative stress under high sugar conditions is the main cause of various complications of diabetes. The experiment of the present invention shows that osfera ketone can protect microvascular endothelial cells under oxidative stress conditions. Therefore, in the present invention, the diabetic complications are diabetic complications caused by oxidative stress under high sugar conditions.

[0009] Long-term high blood sugar environment can stimulate abnormal proliferation of renal tubular cells, causing changes in renal structure and function. This will not only lead to diabetic nephropathy, but may also indirectly affect nerve and vascular function due to metabolic disorders, and accelerate the development of other diabetic complications. The experiment of the present invention shows that purpurogenol can inhibit the abnormal proliferation of renal tubular cells. Therefore, in the present invention, the diabetic complications also include diabetic complications caused by abnormal proliferation of renal tubular cells.

[0010] In the present invention, the prevention and treatment includes:

[0011] I) inhibiting cell damage caused by oxidative stress;

[0012] II), inhibiting the expression of fibrosis-related genes FN and / or COL1A1;

[0013] III) inhibit the abnormal proliferation of renal tubular cells.

[0014] The cell damage caused by oxidative stress includes damage to renal tubular cells, retinal microvascular endothelial cells and / or pulmonary microvascular endothelial cells caused by oxidative stress.

[0015] The present invention can achieve good prevention and / or treatment effects on various complications of diabetes by protecting cells and inhibiting fibrosis. For example, the complications of diabetes are diabetic retinopathy, diabetic nephropathy, diabetic foot and / or diabetic neuropathy.

[0016] There are many plants that naturally contain porphyrin. The present invention extracts plants rich in porphyrin and then compounds them, and obtains a composite material from multiple formulas. The compatibility of each component is reasonable. Compared with other formulas, better inhibition of cell damage and fibrosis caused by oxidative stress can be achieved, thereby preventing and treating diabetic complications.

[0017] The present invention also provides a composition for preventing and treating diabetic complications, which is composed of the following components in parts by weight:

[0018] 3 to 30 parts of Radix Cynomorii Fructus;

[0019] 2 to 50 parts of Phellinus igniarius;

[0020] 5 to 50 portions of Chaga.

[0021] In the composition of the present invention, the weight parts of each component are:

[0022] 5 parts of Osmanthus fragrans, 5 parts of Phellinus linteus and 5 parts of Chaga.

[0023] Compared with the scheme of using only porphyrin, the composition provided by the present invention has more advantages in preventing and treating diabetic complications, which may be related to other components in the extract. The combination of porphyrin, mulberry and chaga is the best combination currently screened.

[0024] Furthermore, the present invention also provides an extract for preventing and treating diabetic complications, which is obtained by extracting the composition with an ethanol aqueous solution.

[0025] The extracts described in the present invention are respectively obtained by extracting Osmanthus flavescentis, Phellinus linteus and Chaga. The extracts are total extracts, covering but not limited to components obtained by other modern separation technologies such as extraction with different polar solvents and components obtained by elution with macroporous adsorption resin column chromatography, silica gel column chromatography and gel column chromatography, and contain the component porphyrin.

[0026] Preferably, the preparation method of the extract of the present invention comprises refluxing extraction of Osmanthus fragrans, Phellinus linteus and Chaga with ethanol aqueous solution respectively, concentrating the extracts respectively, and then mixing them to obtain the extract.

[0027] 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%;

[0028] In the 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.

[0029] 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.

[0030] Furthermore, the present invention also provides a drug for preventing and treating diabetic complications, which comprises porphyrin, the above-mentioned composition, the above-mentioned extract or the extract prepared by the above-mentioned preparation method.

[0031] The medicine of the present invention also includes pharmaceutically acceptable adjuvants or additives.

[0032] In the medicine described in the present invention, the excipients include at least one of lactose, starch, microcrystalline cellulose, sodium carboxymethyl starch, magnesium stearate, hydroxypropyl methylcellulose, povidone, poloxamer, mannitol, sorbitol, glycerol, propylene glycol, polyethylene glycol, Tween 80, sodium lauryl sulfate, ethyl cellulose, shellac, gelatin, gum arabic, sucrose, dextrin, pre-coated starch, calcium hydrogen phosphate, silicon dioxide, talc, chitosan, sodium alginate, β-cyclodextrin, citric acid or tartaric acid.

[0033] In an embodiment of the present invention, the dosage form of the drug is tablets, capsules, pills, powders, granules, syrups, oral solutions, suspensions, emulsions, patches, suppositories, ointments, creams, gels, aerosols, sprays, powder aerosols, injections, fluid extracts, extracts, sustained-release tablets or controlled-release tablets.

[0034] Furthermore, the present invention also provides a method for preventing and treating diabetic complications, which comprises administering the above-mentioned drug.

[0035] In the present invention, the administration method includes: oral administration, sublingual administration, buccal mucosal administration, rectal administration, vaginal administration, eye administration, ear administration, nasal administration, skin administration, injection (including subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, intrathecal injection, intraarticular injection, arterial injection), inhalation administration, transdermal administration, implantation administration, spray administration, infusion administration, perfusion administration, local application administration, gargle administration, flushing administration, ion introduction administration, acupoint administration, sublingual administration, rectal perfusion administration, vaginal packing administration, nasal spray administration, eye drops administration, ear drops administration, skin patch administration or subcutaneous implantation administration.

[0036] In the present invention, the object of prevention and treatment is a human or a mammal, and the mammal includes Canidae (such as dogs, wolves), Felidae (such as cats, tigers, lions), Bovidae (such as cattle, goats, sheep), Muridae (such as mice), Leporidae (such as rabbits), Ursidae (such as black bears, brown bears), Primates (such as macaques), Cervidae (such as sika deer), Suidae (such as pigs), Elephantidae (such as Asian elephants), Equine (such as horses), Camelidae (such as camels) or Pongidae (such as gorillas).

[0037] The present invention provides a Chinese medicine composition and its active ingredient osfera ketone in preventing and treating diabetic complications caused by oxidative stress and / or abnormal proliferation of renal tubular cells. Experiments show that the osfera ketone or the Chinese medicine composition can reduce microvascular endothelial cell damage caused by oxidative stress damage and inhibit abnormal proliferation of renal tubular cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The relative luciferase activity of 258 monomer components of traditional Chinese medicine;

[0039] Figure 2 This is a high performance liquid chromatography identification chart showing that the extracts of Ospermum officinale, Phellinus linteus and Chaga contain porphyrinone in Example 2;

[0040] Figure 3 This is a data diagram showing the effect of porphyrin on the activity of hRMEC cells in Example 3;

[0041] Figure 4 A is the data of Example 3 on the effect of porphyrinone-containing Osmanthus extract on the activity of hRMEC cells; B is the data of Example 3 on the effect of porphyrinone-containing Phellinus linteus extract on the activity of hRMEC cells; C is the data of Example 2 on the effect of porphyrinone-containing Chaga extract on the activity of hRMEC cells;

[0042] Figure 5 This is a data diagram showing the effect of osfera ketone in the osfera extract on HMVEC cell activity in Example 3;

[0043] Figure 6 A is a data graph showing the effect of porphyrinone-containing Osmanthus fuchsia extract on HMVEC cell activity in Example 3; B is a data graph showing the effect of porphyrinone-containing Phellinus linteus extract on HMVEC cell activity in Example 3; C is a data graph showing the effect of porphyrinone-containing Chaga mushroom extract on HMVEC cell activity in Example 3;

[0044] Figure 7 The figure shows the effects of compositions 1 to 2 on the activity of hRMEC cells relative to control group A without adding drugs;

[0045] Figure 8This is a data graph showing the protective effect of odoriferone, a characteristic component in the odorifera extract, on tert-butyl hydroperoxide-induced oxidative stress injury in hRMEC cells in Example 4;

[0046] Fig. 9 A is a data graph showing the protective effect of the Osmanthus fragrans extract on oxidative stress injury of hRMEC cells induced by tert-butyl hydroperoxide in Example 4; B is a data graph showing the protective effect of the Phellinus linteus extract on oxidative stress injury of hRMEC cells induced by tert-butyl hydroperoxide in Example 4; C is a data graph showing the protective effect of the Chaga extract on oxidative stress injury of hRMEC cells induced by tert-butyl hydroperoxide in Example 4;

[0047] Fig.10 This is a data diagram showing the protective effect of porphyrin on tert-butyl hydroperoxide-induced oxidative stress injury in HMVEC cells in Example 4;

[0048] Fig.11 A is a data graph showing the protective effect of the Phellinus linteus extract on oxidative stress injury of hRMEC cells induced by tert-butyl hydroperoxide in Example 4; B is a data graph showing the protective effect of the Chaga mushroom extract on oxidative stress injury of hRMEC cells induced by tert-butyl hydroperoxide in Example 4;

[0049] Fig.12 The effects of compositions 1 to 3 on HK2 cell activity are shown relative to control group A without drug addition;

[0050] Fig.13 The graph shows the effects of porphyrin and compositions 1 and 2 on the fibrosis markers FN and COL1A1, relative to the control group A without drug addition and the group M with TGFβ addition. DETAILED DESCRIPTION

[0051] The present invention provides a Chinese medicine composition and its use in the preparation of a medicine for preventing and treating diabetic complications. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0052] 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.

[0053] 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 may represent: A exists alone, A and B exist at the same time, and B exists alone. A and B may be singular or plural.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] In the present application, osmundacetone, CAS number: 37079-84-8 or 123694-03-1. English name: Osmundacetone. It is a phenolic compound with a simple structure, which has pharmacological activities such as anti-inflammatory and antioxidant, and has been proven to be able to treat neurodegenerative diseases such as Alzheimer's disease. The osmundacetone and the drug combination derived from it provided by the present invention have been shown by experimental studies to have a significant effect in preventing and treating diabetic complications, especially for diseases caused by oxidative stress damaging microvascular endothelial cells.

[0058] Osmunda japonica is the dried rhizome and petiole residues of Osmunda japonica, a fern plant 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.

[0059] Phellinusi gniarius is the fruiting body of the Polyporaceae fungus Phellinus gniarius, which is distributed in North China, Northwest China, Heilongjiang, Jilin, Taiwan, Guangdong, Sichuan, Yunnan, Tibet and other places. It has the effects of promoting blood circulation, stopping bleeding, resolving fluid and stopping diarrhea.

[0060] 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.

[0061] 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%.

[0062] It should be understood that in various embodiments of the present application, the size of the sequence number of each process does not mean the order of execution, some or all of the steps can be executed in parallel or in sequence, 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. 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:

[0063] Example 1 Preparation of Osmanthus fuchsia Extract and Identification Experiment of Osmanthus fuchsia Extract

[0064] In order to confirm that the osmanthus extract contains the characteristic component osmanthus ketone, the present invention firstly weighs an appropriate amount of osmanthus ketone standard, uses methanol to prepare a working solution with a concentration of 0.2 mL / kg, filters it through a 0.22 μm microporous filter head, injects it into an injection bottle, and detects it through a high performance liquid chromatography.

[0065] Then, dried Osmanthus fragrans, Phellinus linteus and Chaga were extracted by reflux method using ethanol aqueous solution. The extraction temperature was set at 50°C, the reflux time was 1h, the solid-liquid ratio was 1:5, refluxed 3 times, and the three reflux extracts were combined. The combined extract was concentrated by rotary evaporator under reduced pressure to recover ethanol, and freeze-dried to obtain the extract powder of each raw material drug.

[0066] Take appropriate amounts of Osmanthus fragrans, Phellinus linteus, and Chaga extracts, dilute with methanol, and filter using a 0.22 μm microporous filter to obtain each test sample, inject into a sample injection bottle, and detect by high performance liquid chromatography. In order to ensure that the standard and the osmanthone contained in the sample correspond to each other, the above analytical chromatographic conditions are consistent, and the specific settings are as follows:

[0067] (1) A Z0RBAX SB-C18 column (5 μm, 4.6 min × 250 mm) was used;

[0068] (2) Using water (A)-acetonitrile (B) as the mobile phase, and adding 0.1% formic acid to A;

[0069] (3) Column temperature was 25 °C, injection volume was 10 μL, and flow rate was 1 mL / min;

[0070] (4) The elution procedure was as follows: 0-25 min 15%-20% B, 25-26 min 20%-95% B, 26-35 min 95% B.

[0071] The experimental results are as follows Figure 2 As shown, each extract has a peak within the peak range of the standard substance of porphyrinone. This result indicates that the porphyrinone extract, phellinus linteus extract and Chaga extract described in the present invention all contain porphyrinone.

[0072] Example 2 Preparation of Composition 1

[0073] The purslane root, mulberry and chaga were extracted 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 reflux was performed 3 times. The three reflux extracts of the three drugs were combined, 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 1.

[0074] Example 3 Preparation of Composition 2

[0075] The Aster fern and Phellinus linteus 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 under reduced pressure on a rotary evaporator and dried into powder to obtain extracts of the two drugs. The two extracts were mixed in equal mass proportions to obtain composition 2.

[0076] Example 4 Preparation of Composition 3

[0077] The Osmanthus fragrans 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 two drugs were combined, 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 3.

[0078] Example 5 High-throughput screening of antioxidant compounds

[0079] 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.

[0080] 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 (the solvent was DMSO). After incubation in an 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.

[0081] The results showed that purpurogenol had a good luciferase induction effect on Hek293-ARE cells, with an activation effect of 22.6%, which was better than other common polyphenols such as quercetin (11.3%), chlorogenic acid (10.1%) and catechins (9.8%).

[0082] Example 6 Study on the effects of porphyrinone and porphyrinone-containing porphyrin extract, phellinus linteus extract and Chaga on the activity of hRMEC and HMVEC cells

[0083] hRMEC and HMVEC cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. When each cell grew to 85%-90%, the cells were passaged, the old culture medium was discarded, 1-2mL PBS was used for washing, 1-2mL trypsin was used for digestion for 1min, 2-3mL fresh complete culture 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 an appropriate amount of fresh complete culture medium was used for resuspending, and the cells were passaged at a ratio of 1:3. The cells were cultured continuously in a 37℃, 5% CO2 incubator, and the cell status was observed regularly and the medium was changed in time.

[0084] Take cells in the logarithmic growth phase and dilute them quantitatively 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:

[0085] The control group was added with pure culture medium containing 0.25% DMSO;

[0086] The drug administration group was added with porphyrin solution or compositions 1 to 3 diluted two-fold in pure culture medium, and 3 parallel wells were set for each concentration.

[0087] After 24 hours in the incubator, the drug-containing medium was discarded, and 100 μL of MTT solution with a mass concentration of 0.5 mg·mL-1 was added to each well. After 4 hours of incubation, the supernatant was discarded, and 150 μL of DMSO was added to each well. The crystals were fully dissolved by shaking for 10 minutes. 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 each drug on the cell survival rate.

[0088] Survival rate = (A drug group - A blank group) / (A control group - A blank group) × 100%.

[0089] The experimental results are as follows Figure 3-Figure 7 As shown, in hRMEC cells, the maximum non-toxic concentration of asterone is 25μM, and the maximum non-toxic concentrations of aster extract, phellinus extract and Chaga extract are 62.5μg / mL, 31.3μg / mL, and 18.8μg / mL, respectively; in HMVEC cells, the maximum non-toxic concentration of asterone is 50μM, and the maximum non-toxic concentrations of aster extract, phellinus extract and Chaga extract are 125μg / mL, 32.5μg / mL, and 37.5μg / mL, respectively. In hRMEC cells, when the same concentration combination is given for treatment, the safety of the combination of three drugs is greater than that of the combination of two drugs.

[0090] Example 7

[0091] A method similar to that of Example 6 was used to detect the effects of porphyrinone and porphyrinone-containing porphyrinone extract, phellinus linteus extract, and Chaga on HK2 cell activity.

[0092] like Fig.12 As shown, in HK2 cells, when the same concentration of the composition was administered for treatment, the results showed that the safety concentrations of composition 1 and 2 were higher than that of composition 3, so the efficacy evaluation of composition 3 was temporarily not investigated in the future.

[0093] like Fig.13 As shown, in human renal tubular cells HK2, a cell model consistent with the pathological characteristics of fibrosis in diabetic nephropathy was constructed by TGF-β1, and when the same concentration of the composition and its common component purpurogenol was given for treatment, the expression levels of fibrosis indicators FN and COL1A1 were detected by PCR. The results showed that compositions 1 and 2 and purpurogenol were able to inhibit the abnormal proliferation of renal tubular cells and had significant therapeutic effects.

[0094] Example 8

[0095] Efficacy experimental method for the protective effect of osmatone and osmatone-containing osmatone extract, phellinus linteus extract and chaga extract on tert-butyl hydroperoxide-induced oxidative stress injury of hRMEC and HMVEC cells:

[0096] Based on the cytotoxic concentration of osfera ketone and osfera extracts containing osfera ketone, phellinus linteus extracts and Chaga extracts on hRMEC and HMVEC, and taking cell viability as an indicator, the antioxidant stress damage activity of osfera ketone and three kinds of Chinese herbal extracts containing osfera ketone was detected. hRMEC or HMVEC 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 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 were added with pure culture medium containing drug solvents; the drug group was added with osfera ketone solution or extracts 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 culturing in the incubator for 24 hours, tert-butyl hydroperoxide (modeling dose of 300 μM and 200 μM, respectively) was added to the model group and drug-treated group of hRMEC and HMVEC cells to establish an oxidative stress injury model, and the normal group was added with an equal amount of culture medium. After culturing for 4 hours and 3 hours, CCK-8 was used to detect cell viability.

[0097] The experimental results are as follows Figure 8-Figure 11 As shown in the data, at safe concentrations, Osmanthus fuchsia extract and Osmanthus fuchsia extract containing porphyrinone, Phellinus linteus extract and Chaga extract can effectively improve the survival rate of hRMEC and HMVEC cells induced by tert-butyl hydroperoxide-induced oxidative stress damage, among which the antioxidant activity of porphyrinone is more significant than that of Osmanthus fuchsia extract.

[0098] It can be seen from the above examples that osfera ketone and osfera ketone-containing osfera ketone extract, phellinus linteus extract and chaga extract can improve diabetic complications by reducing oxidative stress damage.

[0099] 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. Application of porphyrin in the preparation of drugs for preventing and treating diabetic complications.

2. The use according to claim 1, characterized in that: The diabetic complications are diabetic complications caused by oxidative stress and / or abnormal proliferation of renal tubular cells.

3. The use according to claim 2, characterized in that: The diabetic complications are diabetic retinopathy, diabetic nephropathy, diabetic foot and / or diabetic neuropathy.

4. The use according to any one of claims 1 to 3, characterized in that: The control includes: I) inhibiting cell damage caused by oxidative stress; II), inhibiting the expression of fibrosis-related genes FN and / or COL1A1; III) inhibit the abnormal proliferation of renal tubular cells.

5. A composition for preventing and treating diabetic complications, characterized in that: It is composed of the following components by mass: 3 to 30 parts of Rhizoma Cyperi; Phellinus linteus 2 to 50 parts; 5 to 50 portions of Chaga.

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

7. An extract for preventing and treating diabetic complications, characterized in that: The composition according to claim 5 or 6 is extracted with an ethanol aqueous solution to obtain the composition.

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, Phellinus linteus and Chaga with ethanol aqueous solution respectively, concentrating the extracts respectively and mixing them to obtain the extract.

9. The preparation method according to claim 8, characterized in that: The solid-liquid ratio of the medicinal material to the ethanol aqueous solution is 1:5; The volume fraction of ethanol in the ethanol aqueous solution is 50%; 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 diabetic complications, characterized in that: The invention comprises porphyrin, the composition according to claim 5 or 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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