Application of asiatic toddalia root lactone in preparation of medicine for promoting angiogenesis

Abstract

The invention provides application of asiatic toddalia root lactone in preparation of a medicine for promoting angiogenesis, relates to the technical field of biomedicine, and finds that the asiatic toddalia root lactone can promote angiogenesis of zebra fish after ischemia for the first time and is expected to provide a new theoretical basis and a potential intervention target for treatment of angiogenesis after ischemia. Experiments prove that after the Asiatic toddalia root lactone intervenes in the zebrafish of the microvascular deletion model, the Asiatic toddalia root lactone has the effect of promoting the Meanwhile, research on endothelial cells finds that after the asiatic toddalia root lactone intervenes an endothelial cell glucose-deficiency and hypoxia model, the activity of the asiatic toddalia root lactone is obviously improved; scratch experiments show that the asiatic toddalia root lactone can obviously promote cell migration. In MCAO model rats, the asiatic toddalia root lactone can increase the local cerebral blood flow perfusion amount of the rats and reduce the TTC staining infarction rate, which indicates that the asiatic toddalia root lactone resists cerebral ischemia by promoting the revascularization; in diabetic wounded rats, the asiatic toddalia root lactone can promote wound healing of the diabetic rats.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to an application of physalis lactone in the preparation of a drug for promoting angiogenesis. Background Art

[0002] Angiogenesis refers to the formation of new blood vessels from existing capillaries or post-capillary veins. Angiogenesis can improve tissue blood flow, increase brain tissue perfusion, promote nerve function recovery, and remove necrotic tissue. It plays an important role in tissue homeostasis and various physiological and pathological processes, such as cancer, diabetic microvascular complications and other vascular diseases. Therefore, regulating angiogenesis is considered an important strategy for cancer and vascular diseases. Feilongzhang hemolactone, also known as nematocystin, belongs to the coumarin class of compounds. This type of compound has good anti-HIV, anti-cancer, anti-inflammatory, and anticoagulant activities. It is a unique component of nematocystin and Feilongzhang hemolactone. At present, there are few studies on Feilongzhang hemolactone at home and abroad. There are only some reports on anti-inflammatory and analgesic effects, and no research on angiogenesis has been found.

[0003] In view of this, the present invention is proposed. Summary of the invention

[0004] One of the purposes of the present invention is to provide an application of phytomycin lactone in the preparation of a drug for promoting angiogenesis, and to propose for the first time that phytomycin lactone can promote angiogenesis, thereby providing a new theoretical basis and potential intervention target for angiogenesis treatment.

[0005] The second purpose of the present invention is to provide an application of the hemolactone of the phytomycin palm in the preparation of a medicine for treating vascular damage or treating diseases caused by vascular damage.

[0006] The third purpose of the present invention is to provide an application of the hemolactone of the physalis in the preparation of medicines for treating skin ulcers caused by diabetes.

[0007] A fourth object of the present invention is to provide a medicine.

[0008] In a first aspect, the present invention provides the use of Psoralea corylifolia hemolactone in the preparation of a drug for promoting angiogenesis.

[0009] Furthermore, the blood vessels include blood vessels located in the head, neck, trunk, limbs or skin.

[0010] In a second aspect, the present invention provides the use of Pteris fasciata hemolactone in the preparation of a drug for treating vascular damage or treating diseases caused by vascular damage.

[0011] Furthermore, the disease includes ischemic stroke, retinal ischemia, vascular dementia, lower limb ischemic lesions, myocardial infarction, myocardial ischemia, chronic heart failure, hypoxic pulmonary hypertension, acute lung injury or acute respiratory distress syndrome.

[0012] In a third aspect, the present invention provides the use of the hemolactone of the physalis in the preparation of medicines for treating skin ulcers caused by diabetes.

[0013] In a fourth aspect, the present invention provides a medicine comprising the phytomycin lactone described in any one of the aforementioned embodiments.

[0014] Furthermore, the dosage form of the drug includes a dosage form for gastrointestinal administration or a dosage form for parenteral administration.

[0015] Furthermore, the gastrointestinal tract administration dosage form includes powder or liquid;

[0016] Preferably, the parenteral dosage form comprises powder, liquid or gas.

[0017] Furthermore, the gastrointestinal administration method includes enteral administration, oral administration or rectal administration;

[0018] Preferably, the parenteral administration method includes intramuscular administration, subcutaneous administration, nasal administration, oral mucosal administration, skin administration or intraperitoneal administration.

[0019] Furthermore, it also includes a pharmaceutically acceptable carrier;

[0020] Preferably, the carrier comprises at least one of a diluent, a binder, an absorbent, a disintegrant, a dispersant, a wetting agent, a solubilizer, a buffer or a surfactant.

[0021] The application of the phylong palm blood lactone provided by the present invention in the preparation of a drug for promoting angiogenesis is the first to find that phylong palm blood lactone can promote angiogenesis after zebrafish ischemia, and is expected to provide a new theoretical basis and potential intervention target for the treatment of angiogenesis after ischemia. Experiments have shown that after phylong palm blood lactone intervened in the microvascular deficiency model zebrafish, the intestinal blood vessel area of ​​the vascular deficiency model zebrafish was significantly increased, the number of intestinal blood vessel branches increased, and the number of complete intersegmental blood vessels increased, which has the effect of promoting angiogenesis. At the same time, in the study of endothelial cells, it was found that after phylong palm blood lactone intervened in the endothelial cell hypoglycemia and hypoxia model, its vitality was significantly improved; the scratch experiment showed that phylong palm blood lactone can significantly promote cell migration. In MCAO model rats, phylong palm blood lactone can increase the local cerebral blood perfusion of rats and reduce the TTC staining infarction rate, indicating that phylong palm blood lactone resists cerebral ischemia by promoting angiogenesis; in diabetic trauma rats, phylong palm blood lactone can promote wound healing in diabetic rats. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 A typical diagram showing the effects of different concentrations of physalis lactone on the area and branches of the subintestinal blood vessels of zebrafish provided in Example 1 of the present invention;

[0024] Figure 2 A typical diagram of the effect of the hemolactone provided in Example 1 of the present invention on the number of intact intersegmental blood vessels in zebrafish;

[0025] Figure 3 A statistical graph of HUVEC survival rates under treatment with different concentrations of physostigmine provided in Example 2 of the present invention;

[0026] Figure 4 A statistical graph of HUVEC survival rates under treatment with a sodium dithionite low-sugar solution provided in Example 2 of the present invention;

[0027] Figure 5 Example 2 of the present invention provides a statistical graph of the survival rate of HUVECs injured by OGD under treatment with different concentrations of physalis lactone;

[0028] Figure 6 The statistical graph of HUVEC mobility under treatment with different concentrations of Psoralea corylifolia lactone provided in Example 2 of the present invention;

[0029] Figure 7 Inverted microscope observation diagram of promoting HUVEC angiogenesis under treatment with different concentrations of physalis lactone provided in Example 3 of the present invention;

[0030] Figure 8 Statistical graph of the number of tubes, the number of tube branches, and the number of tube nodes in promoting HUVEC angiogenesis under treatment with different concentrations of Psoralea corylifolia lactone provided in Example 3 of the present invention;

[0031] Fig. 9 Western Blot gel imaging of VEGF protein expression in a glucose-oxygen deprived HUVEC cell model treated with different concentrations of physalis lactone provided in Example 4 of the present invention;

[0032] Fig.10 A statistical graph of VEGF protein expression in a glucose-oxygen deprived HUVEC cell model treated with different concentrations of physalis lactone provided in Example 4 of the present invention;

[0033] Fig.11 This is a statistical graph of behavioral scores of MCAO rats treated with different concentrations of cyperus rotundus lactone provided in Example 5 of the present invention;

[0034] Fig.12 A statistical graph showing the change rate of local cerebral blood flow in MCAO rats 12 hours after surgery treated with different concentrations of cypermethrin provided in Example 5 of the present invention;

[0035] Fig.13 A statistical graph showing the cerebral infarction rate of MCAO rats treated with different concentrations of cyperus rotundus lactone provided in Example 5 of the present invention;

[0036] Fig.14 This is a statistical graph of the wound healing rate of diabetic rats treated with Echinocarp hemolactone provided in Example 6 of the present invention. DETAILED DESCRIPTION

[0037] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0038] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0039] The lycopodii hemolactone of the present invention is also known as lycopodii nitidin, belongs to the coumarin class of compounds, and is a unique component of lycopodii nitidin and lycopodii nitidin. The structural formula of the lycopodii hemolactone is shown in Formula I, and the acquisition route is sufficient from Chengdu Aifa Biotechnology Co., Ltd.

[0040]

[0041] In one aspect, the present invention provides the use of physalis lactone in the preparation of a drug for promoting angiogenesis.

[0042] Experiments have shown that after the intervention of feilongzhang hemolactone in the zebrafish model of microvascular deficiency, the area of ​​the intestinal blood vessels in the zebrafish model of vascular deficiency was significantly increased, the number of intestinal blood vessel branches increased, and the number of intact intersegmental blood vessels increased, which has the effect of promoting vascular regeneration; at the same time, in the study of endothelial cells, it was found that after feilongzhang hemolactone intervened in the endothelial cell hypoxia and glucose hypoxia model, the activity of endothelial cells was significantly improved; the scratch experiment showed that feilongzhang hemolactone could significantly promote cell migration. In the MCAO model rats, feilongzhang hemolactone can increase the local cerebral blood perfusion of rats and reduce the TTC staining infarction rate, indicating that feilongzhang hemolactone can resist cerebral ischemia by promoting vascular regeneration; in diabetic trauma rats, feilongzhang hemolactone can promote angiogenesis at the wound of diabetic rats, improve blood supply, and promote wound healing. It is the first time that feilongzhang hemolactone has been found to promote angiogenesis, which is expected to provide a new theoretical basis and potential intervention target for the treatment of angiogenesis after ischemia.

[0043] Experiments have shown that physalis lactone can effectively promote the migration of human umbilical vein endothelial cells, induce and promote intestinal angiogenesis in zebrafish with vascular deficiency, promote wound healing in diabetic rats, and promote angiogenesis in vivo and in vitro. In some specific embodiments, the blood vessels include blood vessels located in the head, neck, trunk, limbs or skin.

[0044] According to another aspect of the present invention, there is also provided the use of the hemolactone of the smilax china in the preparation of a medicine for treating vascular damage or treating a disease caused by vascular damage.

[0045] The present invention proves that the hemolactone of Phyllanthus avicularis can promote angiogenesis, and can be used for vascular deficiency to promote angiogenesis; or for inducing cerebral angiogenesis to resist vascular damage such as cerebral ischemia or diseases caused by vascular damage.

[0046] Wherein, the vascular injury includes vascular loss or vascular damage. Vascular loss includes vascular dysfunction, atrophy or loss due to non-mechanical collision. Vascular damage includes reversible vascular damage caused by external force collision, such as fracture healing, which can be accelerated by promoting angiogenesis, improving blood supply.

[0047] Ischemic stroke, also known as cerebral infarction or ischemic stroke, is mainly caused by obstruction of brain blood supply, which leads to ischemia and hypoxia of brain tissue, resulting in necrosis or softening. In MCAO model rats, cypermethrin can increase the local cerebral blood perfusion and reduce the TTC staining infarction rate, indicating that cypermethrin can resist the obstruction of local blood supply in the body, leading to local tissue ischemia, hypoxia, necrosis or softening by promoting vascular regeneration.

[0048] Retinal ischemia is caused by a variety of reasons, including vasospasm, embolism, changes in arterial walls, endothelial damage and necrosis. These factors can lead to reduced or interrupted retinal blood flow, causing retinal tissue ischemia and hypoxia, which may eventually lead to retinal dysfunction or decreased vision. The published "Effect of Inhibition of Transient Receptor Potential Vanilloid Receptor Subfamily Type IV Channel on Apoptosis and Angiogenesis in Rats after Retinal Ischemia-Reperfusion Injury" (Guangxi Medicine, 2021, 43(15):1841-1846.) illustrates the correlation between angiogenesis and retinal ischemia and can be used to treat retinal ischemia.

[0049] Vascular dementia is a cognitive disorder caused by the destruction of brain tissue due to reduced or blocked blood supply to the brain. The published article "Exploring the mechanism of acupuncture intervention for vascular dementia based on the theory of copper death and angiogenesis" (Journal of Clinical Acupuncture, 2023, 39(06):1-6) explains the correlation between angiogenesis and retinal ischemia, which can be used to treat vascular dementia.

[0050] Lower limb ischemia is caused by insufficient blood supply due to lower limb thrombosis, atherosclerosis or vascular inflammation. The published study "Study on the Effect and Molecular Mechanism of Vascular Regeneration and Repair of Diabetic Lower Limb Ischemia Mice by Sogliflozin" (Chongqing University, 2022) illustrates the correlation between angiogenesis and lower limb ischemia. It can be used to treat lower limb ischemia.

[0051] Myocardial infarction is mainly caused by acute blockage of the coronary artery, which leads to myocardial necrosis due to lack of blood supply, resulting in impaired heart function and is an acute disease that may endanger life. The published study "Study on the Effect of CMTM5 Gene on Angiogenesis after Myocardial Infarction" (Chinese Journal of Medical Science, 2024, 14(04): 47-50.) illustrates the correlation between angiogenesis and myocardial infarction. It can be used to treat myocardial infarction.

[0052] Myocardial ischemia is caused by a variety of reasons, mainly involving reduced blood flow in the coronary arteries, which leads to insufficient myocardial oxygen and nutrient supply and reduced clearance of metabolites. The published "Protective effect of Danshen Tongluo Jiedu Decoction on rats with myocardial ischemia-reperfusion injury and its effects on autophagy and angiogenic factors" (Lizhen Traditional Chinese Medicine, 2024, 35(02): 293-296.) illustrates the correlation between angiogenesis and myocardial ischemia. It can be used to treat myocardial ischemia.

[0053] Chronic heart failure (CHF) is a heart disease caused by a variety of reasons, which leads to a decline in the heart's pumping function and an inability to meet the body's demand for blood. The published article "Exosomal microRNA-1246 from human umbilical cord mesenchymal stem cells potentiates myocardial angiogenesis inchronic heart failure" (Cell biology international, 2021, 45(11): 2211-2225.) illustrates the correlation between angiogenesis and chronic heart failure and can be used to treat chronic heart failure.

[0054] Hypoxic pulmonary hypertension is a pulmonary vascular disease caused by a long-term hypoxic environment. The published study "Effects and Mechanisms of Platelet-Derived Growth Factor-BB on Pulmonary Vascular Remodeling in Neonatal Rats with Hypoxic Pulmonary Hypertension" (Chinese Journal of Contemporary Pediatrics, 2023, 25(04): 407-414.) illustrates the correlation between angiogenesis and hypoxic pulmonary hypertension, which can be used to treat hypoxic pulmonary hypertension.

[0055] Acute lung injury and acute respiratory distress syndrome are mainly manifested by damage to alveolar epithelial cells and pulmonary capillary endothelial cells, leading to diffuse pulmonary interstitial and alveolar edema, thereby causing acute hypoxic respiratory insufficiency or respiratory failure. The published article "Stanniocalcin-1 Promotes Angiogenesis During the Resolution of Hyperoxia Induced Acute Lung Injury Through TLR4" (AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE, 2018, 197) illustrates the correlation between angiogenesis and acute lung injury, which can be used to treat acute lung injury and acute respiratory distress syndrome.

[0056] In some specific embodiments, the disease includes ischemic stroke, retinal ischemia, vascular dementia, lower limb ischemic disease, myocardial infarction, myocardial ischemia, chronic heart failure, hypoxic pulmonary hypertension, acute lung injury or acute respiratory distress syndrome.

[0057] According to another aspect of the present invention, there is also provided the use of the hemolactone of the smilax china in the preparation of medicines for treating skin ulcers caused by diabetes.

[0058] Under the condition of hyperglycemia, vascular endothelial cells are damaged, resulting in impaired endothelial barrier function. In addition, due to high blood sugar, insulin resistance and increased oxidative stress levels, wound angiogenesis and cell proliferation are reduced, which in turn affects the normal healing of wounds and causes skin ulcers. Feilongzhang hemolactone accelerates the wound healing rate in rats and improves the wound healing rate in diabetic rats. This lays the foundation for the application of Feilongzhang hemolactone in the treatment of skin ulcers caused by diabetes.

[0059] According to another aspect of the present invention, a medicine is provided, comprising the above-mentioned Fructus Achyranthis Bidentatae hemolactone.

[0060] The medicine prepared with the hemolactone of the palm of hand can be used to promote angiogenesis, treat vascular damage or diseases caused by vascular damage, or treat skin ulcers caused by diabetes.

[0061] In some specific embodiments, the dosage form of the drug includes a dosage form for enteral administration or a dosage form for parenteral administration.

[0062] Among them, the gastrointestinal dosage form includes powder or liquid; the non-gastrointestinal dosage form includes powder, liquid or gas; the gastrointestinal administration method includes enteral administration, oral or rectal administration; the non-gastrointestinal administration method includes intramuscular administration, subcutaneous administration, nasal administration, oral mucosal administration, skin administration or abdominal administration.

[0063] In some specific embodiments, a pharmaceutically acceptable carrier is also included.

[0064] Wherein, the carrier includes at least one of a diluent, a binder, an absorbent, a disintegrant, a dispersant, a wetting agent, a solubilizer, a buffer or a surfactant.

[0065] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0066] MTC: maximum detectable concentration;

[0067] HUVEC: human umbilical vein endothelial cells;

[0068] EC 50 : The half effective concentration, after standardizing the efficacy of the hemolactone in the dragon palm, was calculated using SPSS26.0 software 50 ;

[0069] TOD: Flying Dragon Palm Blood Lactone, 483-90-9;

[0070] DMEM: basic medium for mammalian cell culture;

[0071] CCK-8: water-soluble tetrazolium salt of WST-8;

[0072] TTC: 2,3,5-triphenyltetrazolium chloride;

[0073] STZ: streptozotocin;

[0074] Transgenic vascular green fluorescent Fli-1 strain zebrafish: The animals are from the Huante Biological Innovation Experimental Center, and the experimental animal use license number is: SYXK(Zhejiang)2022-0004.

[0075] Example 1 Study on the effect of physalis lactone on promoting angiogenesis in the lower intestine of zebrafish

[0076] (I) Maximum detectable concentration (MTC) determination

[0077] Transgenic vascular green fluorescent Fli-1 strain zebrafish at 1 day post-fertilization (1 dpf) were randomly selected in a 6-well plate, and 30 zebrafish were treated in each well (experimental group). Simvastatin was induced for 3 hours to establish a zebrafish microvascular deficiency model. After the inducer was washed off, each well was given 62.5μg / mL, 125μg / mL, 250μg / mL, 500μg / mL, 1000μg / mL and 2000μg / mL of pyralidone in water, respectively. A normal control group and a model control group were set up at the same time, and the volume of each well was 3mL. After 2 days of treatment at 28℃, the MTC of the sample on the model zebrafish was measured. The specific results are shown in Table 1 (n=30).

[0078] Table 1 Experimental results on the maximum detection concentration (MTC) of the angiogenesis-promoting effect of the phytosporin

[0079]

[0080] As shown in Table 1, the maximum detection concentration (MTC) of the angiogenesis-promoting effect of the hemolactone of the physalis is 125 μg / mL.

[0081] (II) Determination of the area and number of intestinal vascular branches

[0082] 1dpf transgenic vascular green fluorescent Fli-1 strain zebrafish were randomly selected in a 6-well plate, and 30 zebrafish were treated in each well. Simvastatin was induced in water for 3 hours to establish a zebrafish microvascular deficiency model. After the inducer was washed off, each well was given 8.53μg / mL, 7.81μg / mL, 15.6μg / mL, 31.2μg / mL, 62.5μg / mL and 125μg / mL of physalis lactone in water, and the positive control was 8.53μg / mL of calycosin. A normal control group and a model control group were set up at the same time, and the volume of each well was 3mL. After 2 days of treatment at 28℃, 10 zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope (such as Figure 1 As shown, a is a normal control group, b is a model control group, c is a positive control group, and d to h are experimental groups with 8.53 μg / mL, 7.81 μg / mL, 15.6 μg / mL, 31.2 μg / mL, 62.5 μg / mL and 125 μg / mL of physalis lactone, respectively. The data were analyzed and collected using NIS-Elements D3.20 advanced image processing software. The area of ​​the subintestinal blood vessels and the number of subintestinal blood vessel branches of zebrafish were analyzed. The statistical analysis results of the above indicators were used to evaluate the efficacy of the samples in promoting angiogenesis. The statistical processing results are expressed as mean ± SE. SPSS 26.0 software was used for statistical analysis, and p < 0.05 indicated that the difference was statistically significant. The specific results are shown in Tables 2 and 3 (n = 10).

[0083] Table 2 Experimental results of evaluation of the angiogenesis effect of physalis lactone (intestinal vascular area)

[0084]

[0085] Table 3 Experimental results of evaluation of angiogenesis (number of intestinal vascular branches)

[0086]

[0087] Among them, compared with the model control group, *p<0.05, **p<0.01, ***p<0.001.

[0088] It can be seen from Tables 2 and 3 that, compared with the model control group, different concentrations of Flying Dragon Palm Blood Lactone can increase the intestinal vascular area and the number of intestinal vascular branches. When the concentration of Flying Dragon Palm Blood Lactone reaches 125 μg / mL, the intestinal vascular area and the number of intestinal vascular branches increase significantly.

[0089] The EC50 of the intestinal vascular area was increased to 12.0μg / mL; the EC50 of the intestinal vascular branches was increased to 17.3μg / mL.

[0090] 3. Determination of the number of intact intersegmental vessels

[0091] 1dpf transgenic vascular green fluorescent Fli-1 strain zebrafish were randomly selected in a 6-well plate, and 30 zebrafish were treated in each well (experimental group). Simvastatin was induced for 3 hours in water to establish a zebrafish microvascular deficiency model. After the inducer was washed off, each well was given 8.53μg / mL, 7.81μg / mL, 15.6μg / mL, 31.2μg / mL, 62.5μg / mL and 125μg / mL of physalis lactone in water, respectively, and the positive control was 8.53μg / mL of calycosin. At the same time, a normal control group and a model control group were set up, and the volume of each well was 3mL. After 2 days of treatment at 28℃, 10 zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope (such as Figure 2 As shown, a is a normal control group, b is a model control group, c is a positive control group, and d to h are experimental groups with 8.53μg / mL, 7.81μg / mL, 15.6μg / mL, 31.2μg / mL, 62.5μg / mL and 125μg / mL of physalis lactone, respectively. The data were analyzed and collected using NIS-ElementsD3.20 advanced image processing software, and the number of intact intersegmental blood vessels in zebrafish was analyzed. The statistical analysis results of the above indicators were used to evaluate the efficacy of the samples in promoting angiogenesis. The statistical processing results are expressed as mean±SE. SPSS26.0 software was used for statistical analysis, and p<0.05 indicated that the difference was statistically significant. The specific results are shown in Table 4.

[0092] Table 4 Experimental results of evaluation of the effect of physalis palmatum hemolactone on promoting angiogenesis (intersegmental blood vessels)

[0093]

[0094] Among them, **p<0.01, ***p<0.001.

[0095] As shown in Table 4, compared with the model control group, different concentrations of physalis lactone can increase the number of intact intersegmental vessels, and the number of intact intersegmental vessels gradually increases with the increase of physalis lactone concentration. When the physalis lactone concentration reaches 31.2, the number of intact intersegmental vessels increases significantly. Calculate EC 50 It is 11.9μg / mL.

[0096] Example 2 Study on the effect of physalis lactone on protecting hypoxic endothelial cells and promoting cell migration (I) CCK-8 detection of the effect of physalis lactone on the cell viability of normal HUVEC

[0097] When the cells are in good condition and the density reaches about 80%, they can be plated. Add 2 mL of trypsin and digest for 2 to 3 minutes. Observe under a microscope that most cells are round and not spindle-shaped. Add 3 mL of complete culture medium and pipette. Centrifuge and resuspend to count cells. Adjust the cell concentration to 8×104 Use 96-well plates for plating, add 100 μL of cell solution to each well, add 100 μL of D-PBS to the edge wells, and culture in a cell culture incubator overnight to allow the cells to adhere to the wall.

[0098] When the cell density is greater than 70%, the drug is added. The culture medium is discarded, and the TOD gradient is divided into 0, 3.125μM, 6.25μM, 12.5μM, 25μM, 50μM, 100μM, 200μM and 400μM; the blank group is a cell-free group, with 6 replicate wells in each group, 100μL / well, and cultured in a constant temperature incubator for 24 hours. Add CCK-8 solution (10μL / well) in a dark place, wrap the 96-well plate tightly with tin foil, incubate in a constant temperature incubator for 45 to 60 minutes, and detect the OD value at 450nm / 650nm. Calculate cell viability according to the following formula:

[0099]

[0100] The more the cells proliferate, the stronger the absorbance and the higher the activity; the greater the cytotoxicity, the weaker the absorbance and the lower the activity.

[0101] Specific results such as Figure 3 As shown in the figure, compared with the TOD concentration of 0, the HUVEC activity decreased significantly when the TOD concentration was 200μM and 400μM. There was no significant difference in the effect of physalis lactone on the activity of HUVEC in other groups. Therefore, within 24 hours of drug intervention, 0-100μM is within the safe dose range of physalis lactone.

[0102] (II) Preparation of HUVEC OGD model

[0103] Plate the cells according to the experimental method in (I). Use low-glucose culture medium to simulate a sugar-deficient environment, and Na2S2O4 to simulate an oxygen-deficient environment. Discard the culture medium, and dilute the Na2S2O4 mother solution to 32mM, 16mM, 8mM, 4mM, 2mM, and 1mM with low-glucose DMEM to prepare low-glucose oxygen-deficient culture medium. The blank group is a cell-free group; the control group is a normal cell group; each group has 6 replicate wells, 100μL / well, and cultured in a constant temperature incubator for 24h. Add CCK-8 to calculate cell viability.

[0104] Specific results such as Figure 4 As shown in the figure (con is the control group), the effect of Na2S2O4 hypoglycemia (OGD) model on HUVEC activity. After 24h of cell culture, compared with the control group, 2-32mM Na2S2O4 low-glucose solution significantly decreased the activity of HUVEC, and there was a dose-dependent relationship. When the concentration of Na2S2O4 low-glucose solution was 4.00mmol·L -1 It is suitable for subsequent experimental model preparation.

[0105] (III) CCK-8 assay to detect the effect of phytosporin on the activity of HUVECs damaged by OGD model

[0106] The cells were plated according to the experimental method in (I). A control group was given normal culture medium, a model group was given culture medium containing 4.00 mM sodium dithionite solution, and the experimental group was divided into 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM and 1.563 μM concentration groups, and a final concentration of 4.00 mM sodium dithionite solution was given. Each group had 6 replicate wells and was cultured in a cell culture incubator for 24 hours. CCK-8 was added to calculate cell viability.

[0107] Specific data such as Figure 5 As shown (con is the control group and m is the model group). After 24 hours of cell culture, the HUVEC activity of the model group was significantly decreased compared with the control group (P<0.01); compared with the model group, when the concentration of physalis hemolactone was 25-100 μM, the cell activity was significantly improved, but 100 μM was lower than 50 μM, so the optimal intervention concentration of physalis hemolactone was determined to be 12.5, 25, and 50 μM, which can be used for later experiments.

[0108] (IV) Scratch assay to detect the effect of phytosporin on HUVEC migration

[0109] When the cells are in good condition and the density reaches about 80%, they can be plated. Adjust the cell density to 5×10 6 / mL. Use a 6-well plate for plating, add 2mL of cell solution to each well, and culture in a cell culture incubator overnight to allow it to adhere to the wall. Take out the 6-well plate and scratch the cells in each well evenly. Rinse off the cells in the scratched area, aspirate the original culture medium, add D-PBS to wash twice, add pre-prepared low-glucose DMEM containing 12.5μM, 25μM and 50μM TOD and Na2S2O4 (where low sugar refers to a glucose concentration of 1.0g / L), take pictures under a microscope at 0h, 12h and 24h to record the migration status, and use PS software for analysis.

[0110] Specific results such as Figure 6 As shown in the data (where control is the control group and model is the model group), after 12h and 24h of cell culture, compared with the control group, 12.5, 25, and 50μM of Fructus Achyranthis Bidentatae hemolactone significantly promoted the migration ability of HUVEC (P<0.05), and there was a dose-dependent relationship.

[0111] Example 3 Tube formation method to detect the effect of physalis lactone on HUVEC angiogenesis

[0112] (i) Pre-cool the matrix gel at 4°C overnight. Pre-cool the 1 mL and 200 μL pipette tips and 96-well plates in an ice box. Store the matrix gel in ice when in use.

[0113] (ii) Prepare drug-containing culture medium in advance, where the drug is TOD, and the drug concentration and Na2S2O4 concentration are twice the final concentration in the 96-well plate. Place the pre-cooled 96-well plate on an ice box, and use a pre-cooled pipette tip to add 50 μL / well matrix gel to the 96-well plate, taking care not to create bubbles. After adding, place it in a 37°C incubator for 30 minutes to solidify. After the gel solidifies, add 50 μL of low-glucose DMEM containing 2 times the final concentration of the drug to the well; digest the centrifuged cells, resuspend them with drug-free culture medium, and adjust the cell number to 4×10 5 50 μL was added to each well of a 96-well plate, so that the final concentration of TOD added to the 96-well plate was the final concentration and the number of cells was 2×10 5 / ml, incubated for 12h. The TOD concentrations in the 96-well plate were 12.5μM, 25μM and 50μM respectively.

[0114] Take pictures of the middle, top, bottom, left and right fields of view under an inverted microscope. Figure 7 The Angiogenesis plug-in in Image J was used to count the number of tubes, the number of tube branches, and the number of tube nodes in each field of view. The specific results are shown in the figure below. Figure 8 As shown (wherein control is the control group, the control group is normal cells, model is the model group, the model group is 4 mM sodium dithionite solution without medication, AS-IV is the 10 μM astragaloside group, TOD-L is the 12.5 μM TOD group, TOD-M is the 25 μM TOD group, and TOD-H is the 50 μM TOD group).

[0115] Depend on Figure 7 and Figure 8 It can be seen that after 24 hours of cell culture, compared with the control group, 12.5μM, 25μM and 50μM of Todendron odoratum lactone (TOD) can promote angiogenesis in a dose-dependent manner.

[0116] Example 4 Western Blot detection of VEGF protein expression in the glucose-oxygen deprivation HUVEC cell model with phytomycin

[0117] (I) Digest centrifuged cells and culture the model cells at 1×10 6 / ml density was inoculated into a 6-well plate, and the drug-containing culture medium prepared in Example 3 was added to the 6-well plate in advance, and the TOD concentrations were 12.5μM, 25μM and 50μM, respectively. At the same time, the same control group, model group and 10μM astragaloside group as in Example 3 were set. Take out the 6-well plate, remove the drug-containing culture medium, and wash it 3 times with pre-cooled PBS. Prepare RIPA lysis solution containing 1mM PMSF, add 100μL to each well, and shake the well plate to cover the cell surface with the lysis solution. Place the well plate on ice and shake for 10min. Scrape the cells with a 1ml gun tip, collect the cell fragments and lysis solution into a 1.5mL EP tube, centrifuge at 14000g and 4℃ for 15min, and the supernatant is the cell protein stock solution.

[0118] Use the BCA method to detect the concentration of cell protein. Use protein loading buffer and PBS to calibrate the protein to 1 mg / mL and then perform boiling denaturation on the protein. Select the concentration of separation gel according to the molecular weight of the target protein and the instructions. After preparing the gel, you can proceed to the next step of loading or put the gel into a self-sealing bag containing water at 4°C for standby use. It can be stored for up to one week. Use a rack to clamp the gel, place it in the electrophoresis tank, fill it with 1× electrophoresis solution, add a volume of 25μg of protein sample to the lane, first perform electrophoresis at a voltage of 80V for about 30min, and after the protein marker is separated, increase the voltage to 120V and continue electrophoresis for about 1h.

[0119] Cut a PVDF membrane of appropriate size, soak it in methanol for 1 minute, transfer the membrane to filter paper, wet the filter paper with 1× fast transfer solution, gently place the cut gel on the membrane, and cover it with a layer of filter paper to form a "sandwich" structure. Place it in the transfer tank, align it with the electrode, and transfer the membrane at 200mA on ice for about 2 hours.

[0120] Block with 5% skim milk at room temperature on a shaker for 2 hours, then wash with TBST solution 3 times, 5 minutes each time; prepare the corresponding primary antibody with TBST solution according to the recommended antibody concentration of the target protein, and incubate the band in the "primary antibody" of VEGF at 4°C overnight; wash with TBST solution 3 times, 5 minutes each time, and incubate in the corresponding secondary antibody (1:5000) at room temperature on a shaker for 30 minutes;

[0121] Wash with TBST solution for 3 times, 5 min each time, prepare ECL colorimetric solution with A solution: B solution in a ratio of 1:1, evenly cover the target band, develop under chemiluminescent gel imaging system, and export the image (such as Fig. 9 ) and performed statistical analysis on the images using Image Lab software. The results are shown in Fig.10As shown. Among them, control is the control group, which is normal cells, model is the model group, which uses pure water instead of TOD, AS-IV is the 10μM astragaloside group, tl is the 12.5μM TOD group, tm is the 25μM TOD group, and th is the 50μM TOD group.

[0122] Depend on Fig. 9 and Fig.10 It can be seen that compared with the Control group, the VEGF expression in the model group was significantly decreased; compared with the model group, the VEGF expression level after TOD administration increased.

[0123] Example 5 The therapeutic effect of physalis lactone on MCAO rats

[0124] (I) Establishment of rat middle cerebral artery occlusion (MCAO) model

[0125] The rats were fasted for 12 hours, with free access to water, and weighed to calculate the amount of anesthesia and medication. The positive control group was given 20 mg / kg cinepazide maleate injection, and the experimental groups were given 5 mg / kg and 20 mg / kg of cypermethrin, respectively. Each group was intraperitoneally injected 15 minutes before surgery.

[0126] Rats were anesthetized by intraperitoneal injection of sodium pentobarbital solution at a volume of 45 mg / 100 g, and the rat middle artery occlusion (MCAO) model was used. The sham operation group was not occluded, but the blood vessels were separated and sutured after being exposed to air for 10 minutes; the other groups were all subjected to MCAO model.

[0127] Divide the cotton thread that blocks the artery into segments and soak them in saline. When the righting reflex of the anesthetized rat disappears, fix the rat's limbs and head, tie it to the dissection board, and use a shaver to shave the hair on the rat's neck and disinfect it. Cut the skin in the middle of the rat's neck, bluntly separate the subcutaneous tissue and fat, slowly separate various muscles, expose the common carotid artery, and thread the thread to block the blood flow of the common carotid artery with a slipknot. Continue upward, separate the common carotid artery, internal carotid artery and external carotid artery, ligate the proximal end of the common carotid artery, expose the internal carotid artery below, and insert the ligature from the cut at the lower edge of the three-way intersection; after inserting about 1.8 cm, ligate the ligature, suture the wound, sprinkle a small amount of antibiotics on the suture, put the rat in a cage, raise its head and wait for it to wake up.

[0128] (II) Neurobehavioral scoring

[0129] The rats were scored for neurological function using a five-level scoring method before surgery, 6 hours after surgery, and 12 hours after surgery. The specific operation was as follows: lift the rat's tail about 1 foot above the ground and observe the condition of the rat's two forelimbs; place the rat on a horizontal ground, push the shoulders, and observe whether there is any difference in the resistance of the rat's two limbs; observe the rat's walking on a horizontal ground. The higher the score, the more severe the rat's neurobehavioral damage. All the scoring processes for rats with unknown groups were completed by the same experimenter.

[0130] Specific scoring rules: If the behavior is completely normal, 0 points will be recorded; if the rat's tail is lifted about 1 foot from the ground and the forelimb on the opposite side of the operation is internally rotated and adducted, 1 point will be recorded; if the rat is placed on a horizontal ground and its resistance is checked by squeezing both sides with hands, if the resistance on the opposite side of the operation decreases, 2 points will be recorded; if the rat is placed on a horizontal ground and its walking is observed, if it circles around the opposite side of the operation, 3 points will be recorded; if the rat is extremely seriously injured and can no longer move independently, 4 points will be recorded.

[0131] Specific results such as Fig.11 As shown in the figure (where sham is the sham operation group, y is the positive control group, model is the model group, tl is 5 mg / kg of phytomycin, and th is 20 mg / kg of phytomycin), behavioral scores were performed on the rats at 6h and 12h after surgery. Compared with the model group, the symptoms of tl and th in the experimental group were milder and the scores were lower.

[0132] 3. Regional cerebral blood flow (rCBF)

[0133] After the rats were anesthetized, 15 minutes before the MCAO surgery, the head hair was shaved, and the skin from the midpoint of the two ears to the top of the skull was cut to completely expose the skull. The rats were transferred to the laser, and the position of the skull was adjusted to 21 cm from the scanning head. The detection data was recorded and photographed, and the monitoring value of stable blood flow was taken as the local cerebral blood perfusion before modeling (TOI before modeling).

[0134] After the MCAO surgery, the rats were placed under the laser speckle analyzer again, and the position of the rat skull was adjusted to 21 cm from the scanning head. The detection data were recorded and photos were taken. The monitoring value of stable blood flow was taken as the local cerebral blood perfusion after modeling (TOI after modeling).

[0135] The scalp was sutured and disinfected with alcohol 12 hours after surgery. The sutures were removed, the scalp was incised, and the rat was placed under the laser speckle analyzer. The position of the rat skull was adjusted to 21 cm from the scanning head. The test data was recorded and photographed. The monitoring value of stable blood flow was taken as the local cerebral blood perfusion volume (TOI after drug administration) after 12 hours. The test data was processed according to the following formula:

[0136]

[0137] Specific results such as Fig.12 As shown in the figure (where sham is the sham operation group, y is the positive control group, model is the model group, tl is 5 mg / kg of phytomycin, and th is 20 mg / kg of phytomycin), compared with the model group, the local cerebral blood perfusion of tl and th in the experimental group 12 hours after surgery was significantly increased, and the blood flow of tl in the experimental group recovered more than that of th in the experimental group.

[0138] (IV) TTC staining

[0139] Prepare 2% TTC solution in advance and store in a 4°C refrigerator away from light. 12 hours after surgery, anesthetize the rats by intraperitoneal injection of 10% chloral hydrate solution at a volume of 0.3 mL / 100 g. The rats were killed, and the cerebellum and olfactory bulb were removed. They were quickly frozen at -20°C for 20 minutes and then placed in a mold. The slices were sliced ​​every 2 mm and transferred to a 6-well plate containing 2% TTC. They were incubated at 37°C for 15 minutes. During this period, the brain slices were turned over to make them evenly contact with the TTC solution. The white part is the infarcted part. The infarct area was calculated using PS software, and the total area of ​​the brain slice was recorded as V, and the infarct area was recorded as V1. The infarction rate was calculated according to the following formula:

[0140]

[0141] Specific results such as Fig.13 As shown, the rats were killed 12 hours after surgery and TTC staining was performed on the rat brains. Compared with the sham operation group, the infarction rate in the model group was significantly increased, while the infarction rates in the experimental groups tl and th were reduced, among which the experimental group tl was better than the experimental group th.

[0142] Example 6 Effect of Psoralea corylifolia Lactone on Wound Healing in Diabetic Rats

[0143] 1. Wound healing experiment in diabetic rats

[0144] The rats in the modeling group were fed with a high-fat diet for 2 weeks. The day before modeling, the amount of STZ and buffer was estimated according to the fasting weight of all rats, and the buffer was prepared. On the day of modeling, STZ was weighed and packaged, and the sealed bags were labeled and wrapped with two layers of tin foil. The outermost layer was covered with a black plastic bag to prevent moisture and light. Rats were fasted for 12 hours overnight, but water was not forbidden. On the day of modeling, the fasting weight of all rats was weighed, and each modeling rat was intraperitoneally injected with 35 mg / kg STZ. STZ was prepared and used immediately. The NC group was intraperitoneally injected with an equal volume of buffer. 72 hours after STZ injection, blood was taken from the tail vein of each rat to test random blood sugar (to ensure that blood sugar is close to the true value, this and subsequent tail vein blood sugar measurements are based on the second drop of blood received by the blood sugar test strip). Blood sugar ≥11.1mmol, accompanied by polydipsia, polyphagia, and loose and yellow hair, was considered a successful modeling of diabetic rats.

[0145] After one week of adaptive feeding, all rats can be included in the animal modeling stage if there are no abnormalities in mental state, behavioral activities, diet, etc. After the rats are anesthetized, the middle part of the upper lumbar spine of the rats is selected as the skin preparation area, and the back skin hair is mechanically removed with an electric shaver. Select instruments such as 10mm skin punchers, scalpels, tweezers, and surgical scissors. Prepare the skin defect wound under sterile conditions, and remove the full layer of skin including the epidermis, dermis, and subcutaneous fascia tissue. If there is bleeding during the operation, sterile gauze is used to apply pressure to control the bleeding. All the above surgical steps are performed under sterile conditions. After surgery, the rats were kept in single cages, and the wound and surrounding skin were routinely disinfected with iodine.

[0146] On the day of image acquisition (0th, 3rd, 7th and 10th day), with a white mouse board as the background, a measuring ruler was placed at the lower edge of the wound for scale comparison, and the same photographer took the photo perpendicular to the wound. The high-definition images were uploaded to the computer, and the Imageproplus6.0 software was opened. The 20.0mm scribe line was drawn using the line drawing tool in the software. The software can automatically analyze the pixel density, and then the brush tool is used to carefully outline the outline of the wound edge in the image. The software automatically calculates the wound area of ​​a rat at the corresponding time point, and the wound healing at each time point is calculated and analyzed according to the following formula.

[0147]

[0148] Specific results such as Fig.14 As shown in the figure (where NC is normal rats, DM is diabetic rats, TOD is diabetic rats with a wound surface treated with 25 mg / kg TOD, and Puerarin is diabetic rats with a wound surface treated with 80 mg / kg puerarin), compared with the NC group, the wound healing of the DM group was significantly delayed. On the 7th and 10th days after the wound was formed, the wound healing rate of the DM group was significantly lower than that of the NC group, and the difference was statistically significant (P<0.01); secondly, in the effect of TOD on diabetic wound healing, the results showed that although there was no significant difference in the wound healing rate between the TOD group and the DM group on the 3rd and 7th days after the wound was formed, on the 10th day after the wound was formed, the wound healing rate of the TOD (Feilongzhang hemolactone) group was significantly accelerated compared with the DM group, and the difference was statistically significant. In summary, Feilongzhang hemolactone improved the wound healing rate of diabetic rats.

[0149] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of Hemolactone of Phyllanthus truncatula in the preparation of drugs for promoting angiogenesis.

2. The use according to claim 1, characterized in that: The blood vessels include those located in the head, neck, trunk, limbs or skin.

3. Application of the hemolactone of Phyllanthus avicularis in the preparation of drugs for treating vascular damage or diseases caused by vascular damage.

4. The use according to claim 3, characterized in that: The diseases include ischemic stroke, retinal ischemia, vascular dementia, lower limb ischemic lesions, myocardial infarction, myocardial ischemia, chronic heart failure, hypoxic pulmonary hypertension, acute lung injury or acute respiratory distress syndrome.

5. Application of Hemolactone in the preparation of medicines for treating skin ulcers caused by diabetes.

6. A drug, characterized in that It comprises the hemolactone of any one of claims 1 to 5.

7. The drug according to claim 6, characterized in that The dosage form of the drug includes a dosage form for gastrointestinal administration or a dosage form for parenteral administration.

8. The drug according to claim 7, characterized in that The gastrointestinal tract administration dosage form includes powder or liquid; Preferably, the parenteral dosage form comprises powder, liquid or gas.

9. The drug according to claim 7, characterized in that The gastrointestinal administration method includes enteral administration, oral administration or rectal administration; Preferably, the parenteral administration method includes intramuscular administration, subcutaneous administration, nasal administration, oral mucosal administration, skin administration or intraperitoneal administration.

10. The drug according to any one of claims 6 to 9, characterized in that Also includes a pharmaceutically acceptable carrier; Preferably, the carrier comprises at least one of a diluent, a binder, an absorbent, a disintegrant, a dispersant, a wetting agent, a solubilizer, a buffer or a surfactant.

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