Application of corydalis saxicola bunting in anti-angiogenesis drugs

Abstract

The invention provides application of corydalis saxicola bunting in preparation of anti-angiogenesis drugs. The invention provides application of a corydalis saxicola bunting plant, a traditional Chinese medicinal material of the corydalis saxicola bunting plant or a corydalis saxicola bunting plant extract in preparation of anti-angiogenesis drugs. According to the present invention, the in vivo pharmacodynamic experiment performed by using the zebra fish angiogenesis model for the first time proves that the corydalis saxicola bunting extract can significantly inhibit the angiogenesis of zebra fish, such that the corydalis saxicola bunting extract has the anti-angiogenesis activity, and can be used as the angiogenesis inhibitor, the compound can be applied to treatment of neovascularization-dependent and neovascularization-related diseases such as tumors, arthritis, skin and ophthalmic diseases, atherosclerosis, vascular dementia, endometriosis and the like.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of Coptis chinensis in the preparation of anti-angiogenesis drugs. Background Art

[0002] Angiogenesis refers to the process by which new capillaries sprout from existing large blood vessels. Initially, angiogenesis begins with the dedifferentiation of endothelial cells. When conditions are met, the vascular basement membrane thins or disappears, and endothelial cells activate, proliferate, and migrate. A new basement membrane forms, covering the endothelial cells and vascular smooth muscle cells, ultimately forming new blood vessels.

[0003] Angiogenesis is highly correlated with many major human diseases, such as malignant tumors, atherosclerosis, arthritis, skin and eye diseases (wet age-related macular degeneration, diabetic retinopathy, vascular glaucoma, neonatal retinal vascular disease), vascular dementia, endometriosis, etc.

[0004] Therefore, there is an urgent need in the art to develop an anti-angiogenic drug that is safe to use, has excellent effects, is easily affordable, and / or is not prone to drug resistance. Summary of the Invention

[0005] The present invention provides an anti-angiogenesis drug with the advantages of safe use, low price, excellent effect, etc.

[0006] In a first aspect of the present invention, there is provided a use of a Coptis chinensis plant, a Chinese medicinal material of the Coptis chinensis plant, or a Coptis chinensis plant extract for preparing anti-angiogenic drugs.

[0007] In another preferred embodiment, the Coptis chinensis plant extract includes extracts of roots, stems, branches, leaves, and / or flowers of the Coptis chinensis plant, or extracts of the whole plant (such as extracts of the whole herb).

[0008] In another preferred embodiment, the extract is prepared by a method selected from the group consisting of solvent extraction, supercritical carbon dioxide extraction, extraction, chromatography, or a combination thereof.

[0009] In another preferred embodiment, the Coptis chinensis plant extract is an extract extracted using the following solvents: alcohol, water, or a mixed solvent of alcohol and water.

[0010] In another preferred embodiment, the extract is a water extract, an alcohol-soluble extract and / or a water-soluble extract.

[0011] In another preferred embodiment, the extract is an alcohol-soluble extract.

[0012] In another preferred embodiment, the alcohol is a C1-C3 lower alkanol.

[0013] In another preferred embodiment, the alcohol is methanol, ethanol, propanol or a combination thereof.

[0014] In another preferred embodiment, the alcohol is methanol.

[0015] In another preferred embodiment, the alcohol content in the alcohol-water mixed solvent is 5% to 100%, preferably 30% to 100%, and more preferably 50% to 95%, where the percentages are weight percentage (wt%) or volume percentage (v / v%).

[0016] In another preferred embodiment, the alcohol content in the alcohol-water mixed solvent is 5 wt% to 100 wt%, preferably 30 wt% to 100 wt%, and more preferably 50 wt% to 95 wt%.

[0017] In another preferred embodiment, the extract contains (but is not limited to) one or more active ingredients selected from the following groups: Coptis chinensis alkaloids (including Coptis chinensis total alkaloids), Coptis chinensis steroids, Coptis chinensis flavonoids (such as Coptis chinensis total flavonoids), Coptis chinensis alkanes, Coptis chinensis uracil or a combination thereof.

[0018] In another preferred embodiment, the extract contains active ingredients selected from the following group: total alkaloids of Coptis chinensis, steroids of Coptis chinensis, total flavonoids of Coptis chinensis, Coptis chinensis alkanes, Coptis chinensis uracil, or a combination thereof.

[0019] In another preferred embodiment, the composition includes a pharmaceutical composition, a food composition or a health product composition.

[0020] In another preferred embodiment, the drug further comprises an additional component selected from the group consisting of everolimus, lenalidomide, pomalidomide, ibrutinib, osimertinib, dasatinib, bevacizumab, aflibercept, and rosuvastatin.

[0021] In another preferred embodiment, the composition or medicine contains the Coptis chinensis plant extract and a pharmaceutically acceptable carrier or excipient.

[0022] In another preferred embodiment, the composition or medicine includes: an oral preparation and a parenteral preparation.

[0023] In another preferred embodiment, the preparation includes: capsules, tablets, granules, suspensions, microcapsules, injections, suppositories, powders, sprays, patches or ointments.

[0024] In another preferred embodiment, the drug is administered to a subject.

[0025] In another preferred embodiment, the subject is a human or non-human mammal, or a fish (such as zebrafish).

[0026] In another preferred embodiment, the subject is a non-tumor patient.

[0027] In another preferred embodiment, the angiogenesis is non-tumor (or non-tumor-induced) angiogenesis.

[0028] In another preferred embodiment, the anti-angiogenic drug is used to treat one or more diseases selected from the following group:

[0029] (a) Inhibit angiogenesis in psoriatic lesion tissues;

[0030] (b) inhibiting angiogenesis in Paget's disease;

[0031] (c) inhibiting angiogenesis in benign vascular proliferative diseases;

[0032] (d) inhibiting angiogenesis in arthritis lesions;

[0033] (e) inhibiting angiogenesis in neovascular eye diseases;

[0034] (f) inhibiting angiogenesis in atherosclerotic lesions;

[0035] (g) Inhibit angiogenesis in endometriosis lesion tissue.

[0036] In another preferred embodiment, the anti-angiogenic drugs are also used to inhibit vascular dementia.

[0037] In another preferred embodiment, the Coptis chinensis plant extract is prepared by an extraction method comprising the following steps:

[0038] (i) providing Coptis chinensis medicinal material powder;

[0039] (ii) extracting the Coptis chinensis medicinal material powder to obtain the Coptis chinensis plant extract.

[0040] In another preferred embodiment, the extraction method is solvent extraction (such as extraction after soaking with a solvent at a certain temperature (such as 4° C. to reflux temperature, preferably 10-90° C.)).

[0041] In another preferred embodiment, in the extraction method, the mass volume ratio (w / v) of the medicinal material powder to the solvent is (0.5-2):(5-15), wherein the mass volume ratio is calculated as g:ml.

[0042] In another preferred embodiment, the mass volume ratio (w / v) of the medicinal material powder to water is 0.5-1.5:8.

[0043] In another preferred embodiment, the mass volume ratio (w / v) of the medicinal material powder to water is 0.5-1.5:6.

[0044] In another preferred embodiment, the medicinal material is pulverized into coarse powder using a grinder, the coarse powder is soaked in a solvent, and filtered with filter paper;

[0045] The filtrate was concentrated to dryness under reduced pressure using a rotary evaporator to obtain a concentrate;

[0046] The concentrate is dried using a freeze dryer to obtain an extract, which is then frozen and stored for future use.

[0047] In another preferred embodiment, the soaking time is 12-48 hours, preferably 16-32 hours.

[0048] In another preferred embodiment, the freezing condition is -25°C to -18°C, preferably about -20°C.

[0049] In another preferred embodiment, the Coptis chinensis plant extract is obtained by extraction with a solvent selected from the group consisting of alcohol, water, or a mixed solvent of alcohol and water.

[0050] In another preferred embodiment, the alcohol is a C1-C3 lower alkanol.

[0051] In another preferred embodiment, the alcohol is methanol, ethanol, propanol or a combination thereof.

[0052] In another preferred embodiment, the alcohol is methanol.

[0053] In another preferred embodiment, the alcohol content in the alcohol-water mixed solvent is 5% to 100%, preferably 30% to 100%, and more preferably 50% to 95%, where the percentages are weight percentage (wt%) or volume percentage (v / v%).

[0054] In a second aspect of the present invention, a composition for preparing an anti-angiogenic drug is provided, the composition comprising:

[0055] (i) the Coptis chinensis plant, a Chinese medicinal material of the Coptis chinensis plant, or an extract of the Coptis chinensis plant; and

[0056] (ii) pharmaceutically acceptable carriers or excipients.

[0057] In another preferred embodiment, the extract is prepared by a method selected from the group consisting of solvent extraction, supercritical carbon dioxide extraction, extraction, chromatography, or a combination thereof.

[0058] In another preferred embodiment, the Coptis chinensis plant extract is extracted using a solvent selected from the group consisting of alcohol, water, or a mixed solvent of alcohol and water.

[0059] In another preferred embodiment, the alcohol is a C1-C3 lower alkanol.

[0060] In another preferred embodiment, the alcohol is methanol, ethanol, propanol, or a combination thereof.

[0061] In another preferred embodiment, the alcohol is methanol.

[0062] In another preferred embodiment, the alcohol content in the alcohol-water mixed solvent is 5% to 100%, preferably 30% to 100%, and more preferably 50% to 95%, where the percentages are weight percentage (wt%) or volume percentage (v / v%).

[0063] In the third aspect of the present invention, a use of a composition is provided for preparing anti-angiogenic drugs, the composition comprising: (a) a Coptis chinensis plant, a Chinese medicinal material of the Coptis chinensis plant, or a Coptis chinensis plant extract; and (b) a carrier or excipient acceptable in pharmaceuticals, food, or health products.

[0064] In another preferred embodiment, the composition includes a pharmaceutical composition, a food composition or a health product composition.

[0065] In another preferred embodiment, the composition is a pharmaceutical composition.

[0066] In another preferred embodiment, the extract is prepared by a method selected from the group consisting of solvent extraction, supercritical carbon dioxide extraction, extraction, chromatography, or a combination thereof.

[0067] In another preferred embodiment, the Coptis chinensis plant extract is prepared by a method comprising the following steps:

[0068] (i) Providing Coptis chinensis medicinal material powder

[0069] (ii) performing solvent extraction on the Coptis chinensis medicinal material powder to obtain the Coptis chinensis extract.

[0070] In a fourth aspect of the present invention, an in vitro non-therapeutic method for inhibiting angiogenesis is provided, comprising the steps of administering a Coptis chinensis plant extract to a subject in need thereof.

[0071] In another preferred embodiment, the extract is prepared by a method selected from the group consisting of solvent extraction, supercritical carbon dioxide extraction, extraction, chromatography, or a combination thereof.

[0072] In another preferred embodiment, the Coptis chinensis plant extract is extracted using a solvent selected from the group consisting of alcohol, water, or a mixed solvent of alcohol and water.

[0073] In another preferred embodiment, the alcohol is a C1-C3 lower alkanol.

[0074] In another preferred embodiment, the alcohol is methanol, ethanol, propanol, or a combination thereof.

[0075] In another preferred embodiment, the alcohol is methanol.

[0076] In another preferred embodiment, the alcohol content in the alcohol-water mixed solvent is 5% to 100%, preferably 30% to 100%, more preferably 50% to 95%, where the percentages are weight percentage (wt%) or volume percentage (v / v%).

[0077] In another preferred embodiment, when the Coptis chinensis plant extract is an alcohol extract, the administration concentration of the alcohol extract is 10-200 μg / mL, preferably 20-150 μg / mL, and more preferably 25-100 μg / mL.

[0078] In another preferred embodiment, the alcohol is methanol.

[0079] In another preferred embodiment, the object is selected from the following group: zebrafish, mouse corneal pocket (for Corneal Micropocket Assay), chicken chorioallantoic membrane (for Chick Chorioallantoic Membrane Assay), mouse arterial ring (for Mouse Aortic Ring Assay) and human umbilical vein endothelial cell tube formation (for HUVECs tubeformation assay).

[0080] In another preferred embodiment, the object is an ex vivo tissue.

[0081] In a fifth aspect of the present invention, a use of a Coptis chinensis plant extract is provided for preparing an experimental reagent, wherein the experimental reagent is used as an inhibitor of a zebrafish intersegmental angiogenesis (ISV) model.

[0082] In another preferred embodiment, the extract is prepared by a method selected from the group consisting of solvent extraction, supercritical carbon dioxide extraction, extraction, chromatography, or a combination thereof.

[0083] In another preferred embodiment, the Coptis chinensis plant extract is extracted using a solvent selected from the group consisting of alcohol, water, or a mixed solvent of alcohol and water.

[0084] In another preferred embodiment, the alcohol is a C1-C3 lower alkanol.

[0085] In another preferred embodiment, the alcohol is methanol, ethanol, propanol, or a combination thereof.

[0086] In another preferred embodiment, the alcohol is methanol.

[0087] In another preferred embodiment, the alcohol content in the alcohol-water mixed solvent is 5% to 100%, preferably 30% to 100%, more preferably 50% to 95%, where the percentages are weight percentage (wt%) or volume percentage (v / v%).

[0088] In another preferred embodiment, when the Coptis chinensis plant extract is an alcohol extract, the administration concentration of the alcohol extract is 10-200 μg / mL, preferably 20-150 μg / mL, and more preferably 25-100 μg / mL.

[0089] In another preferred embodiment, the alcohol is methanol.

[0090] In another preferred embodiment, the zebrafish is a vascular transgenic fluorescent zebrafish 2-3 days after fertilization.

[0091] In another preferred embodiment, the experimental reagent is used to prepare fish water: 0.3±0.03g sea salt is added to 1L reverse osmosis water, the pH value is controlled at 6.9-7.2, and the application concentration of the extract is 10-200μg / mL.

[0092] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] Figure 1 A 2-day postfertilization (2 dpf) zebrafish intersegmental vascular (ISV) model of vascular transgenic fluorescent zebrafish is shown.

[0094] Figure 2Figure 3 shows the inhibitory effect of Coptis chinensis ethanol extract on zebrafish intersegmental vessel (ISV) formation. Figures (ae) show zebrafish at 8 hpf (post-fertilization) (hpf) treated with the drug for 40 hours, with images taken at 52 hpf. (a) shows the solvent control (0.1% DMSO), (bd) show groups treated with different concentrations of Coptis chinensis ethanol extract, and (e) shows the positive control (PTK787). The red boxes in (ae) show magnified ISV sites in each experimental group (fj). Compared with the solvent control (0.1% DMSO), Coptis chinensis ethanol extract significantly inhibited ISV formation in zebrafish, as evidenced by narrowing of the ISVs, as indicated by the yellow arrows in (g). The dorsal long axis vessels (DLAVs) were missing and disrupted, with only a few sporadic endothelial cell sprouts observed in the dorsal aorta (DA), as indicated by the white asterisks in (hi). As the concentration increased, the inhibitory effect of the ethanol extract of Coptis chinensis on zebrafish intersegmental angiogenesis gradually increased, showing a dose-dependent effect, as shown in (k); when the concentration of the ethanol extract of Coptis chinensis was 100 μg / mL, the formation of zebrafish intersegmental angiogenesis was significantly inhibited; ***: There was a very significant difference compared with the control group (the group with the addition of solvent 0.1% DMSO) (p<0.0001).

[0095] Figure 3 The results show that the inhibition rate of Coptis chinensis ethanol extract on the formation of intersegmental vessels (ISV) in zebrafish increased gradually with the increase of concentration. The inhibition rates of Coptis chinensis ethanol extract at different concentrations on the formation of intersegmental vessels (ISV) in zebrafish were 25 μg / mL (11.96%), 50 μg / mL (20.29%), and 100 μg / mL (65.58%).

[0096] Figure 4 The results showed that the water extract of Coptis chinensis had an inhibitory effect on the generation of intersegmental blood vessels (ISV) in zebrafish: the inhibitory effect of the water extract of Coptis chinensis on the generation of intersegmental blood vessels (ISV) in zebrafish also increased gradually with the increase of concentration. DETAILED DESCRIPTION

[0097] After extensive and intensive research and extensive screening, the present inventors unexpectedly discovered for the first time that Coptis chinensis extracts can significantly inhibit angiogenesis. Experiments in the present invention demonstrate that Coptis chinensis extracts (water extracts, alcohol-soluble extracts, and / or water-soluble extracts) significantly inhibit intersegmental angiogenesis (ISV) in zebrafish. This is the basis for the present invention.

[0098] Coptis chinensis

[0099] Corydalis saxicola Bunting (CSB) is the dried whole herb of Corydalis saxicola Bunting, a plant of the genus Corydalis in the Papaveraceae family. Other names for this herb include Corydalis saxicola Bunting, Corydalis saxicola Bunting, Chrysanthemum Coptis chinensis, and Coptis chinensis. Corydalis saxicola Bunting has a bitter taste and cooling properties, and enters the stomach and large intestine meridians.

[0100] In China, Coptis chinensis is mainly distributed in Gansu, Hubei, Guangxi, Sichuan, Guizhou, Yunnan and other places.

[0101] Common effects include clearing away heat and detoxifying, promoting dampness, relieving pain, etc. It is often used to treat hepatitis, oral erosion, fire eyes, eye opacity, dysentery, diarrhea, abdominal pain, etc.

[0102] Modern pharmacological studies have shown that Coptis chinensis has a wide range of pharmacological effects, including liver protection, analgesia, anti-tumor and antibacterial effects, and has broad prospects for development and application.

[0103] As used herein, the term "Corydalis plant" refers to a plant belonging to the same genus Corydalis or the same species as Corydalis, for example, Corydalis saxicola Bunting, Corydalis thalictrifolia.

[0104] However, before the present invention, there was no report on the use of Coptis chinensis plants, medicinal materials or extracts thereof for inhibiting angiogenesis.

[0105] angiogenesis

[0106] Angiogenesis refers to the process by which new capillaries sprout from existing large blood vessels. Initially, angiogenesis begins with the dedifferentiation of endothelial cells. When conditions are met, the vascular basement membrane thins or disappears, and endothelial cells activate, proliferate, and migrate. A new basement membrane forms, covering the endothelial cells and vascular smooth muscle cells, ultimately forming new blood vessels.

[0107] Angiogenesis is highly correlated with many major human diseases, such as malignant tumors, atherosclerosis, arthritis, skin and eye diseases (wet age-related macular degeneration, diabetic retinopathy, vascular glaucoma, neonatal retinal vascular disease), vascular dementia, endometriosis, etc.

[0108] Based on the research of the present invention, a preferred angiogenesis is angiogenesis that is not related to tumors. Representative examples include (but are not limited to): atherosclerosis, arthritis, skin and eye diseases (wet age-related macular degeneration, diabetic retinopathy, vascular glaucoma, neonatal retinal vasculopathy), vascular dementia, endometriosis, or a combination thereof.

[0109] Compositions and methods of administration

[0110] As used herein, the term "composition" includes anti-angiogenic compositions. In addition, the composition includes pharmaceutical compositions, food compositions, or health product compositions.

[0111] As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity on humans and / or animals and can be accepted by humans and / or animals.

[0112] As used herein, the term "pharmaceutically acceptable" refers to a substance that is suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent, including various excipients and diluents.

[0113] The pharmaceutical compositions of the present invention contain a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to) saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, pharmaceutical formulations should be compatible with the mode of administration. The pharmaceutical compositions of the present invention are available in the form of injections, oral preparations (tablets, capsules, oral liquids), transdermal formulations, and sustained-release formulations. For example, they can be prepared using conventional methods using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions are preferably manufactured under sterile conditions.

[0114] The effective amount of the active ingredient of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc. Generally, when the active ingredient of the present invention is administered at a dose of about 0.0001 mg-250 mg / kg animal body weight (preferably 0.001 mg-100 mg / kg animal body weight) per day, satisfactory results can be obtained. For example, depending on the urgency of the treatment condition, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0115] The pharmaceutically acceptable carriers of the present invention include, but are not limited to, water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be compatible with the mode of administration, as is well known to those skilled in the art.

[0116] Abbreviations

[0117] Hpf (hours post fertilization): hours after fertilization.

[0118] dpf (days post fertilization): days after fertilization.

[0119] DLAV (dorsal longitudinal anastomotic vessel): dorsal longitudinal anastomotic vessel.

[0120] ISV (intersegmental vessel): intersegmental vessel.

[0121] DA (dorsal aorta): dorsal aorta.

[0122] PCV (posterior cardinal vein): posterior cardinal vein.

[0123] Extraction method of Coptis chinensis

[0124] The extract for preparing anti-angiogenesis drugs of the present invention is an extract of Coptis chinensis, especially a solvent extract.

[0125] In one embodiment of the present invention, the method for extracting the Coptis chinensis plant extract may include the following steps:

[0126] a) Providing Coptis chinensis medicinal material powder;

[0127] b) extracting the Coptis chinensis medicinal material powder to obtain the Coptis chinensis plant extract.

[0128] In another preferred embodiment, the Coptis chinensis plant extract is obtained by extraction with a solvent selected from the group consisting of alcohol, water, or a mixed solvent of alcohol and water.

[0129] In another preferred embodiment, the alcohol is a C1-C3 lower alkanol.

[0130] In another preferred embodiment, the alcohol is methanol or ethanol.

[0131] In another preferred embodiment, the alcohol is methanol.

[0132] In another preferred embodiment, the alcohol content in the alcohol-water mixed solvent is 5wt% to 100wt%, preferably 30wt% to 100wt%, and more preferably 50wt% to 95wt%.

[0133] In another preferred embodiment, the medicinal material is ground into coarse powder using a grinder, the powder is added to a solvent, soaked at a certain temperature (such as room temperature), and filtered with filter paper;

[0134] The obtained filtrate was concentrated to dryness under reduced pressure using a rotary evaporator to obtain a concentrate;

[0135] The concentrate is dried using a freeze dryer to obtain an extract, which is then frozen and stored for future use.

[0136] In another preferred embodiment, the soaking time is 12-48 hours, preferably 16-32 hours.

[0137] In another preferred embodiment, the freezing condition is -25°C to -18°C, preferably about -20°C.

[0138] In another preferred embodiment, the method further comprises:

[0139] The obtained filtrate was concentrated to dryness under reduced pressure using a rotary evaporator to obtain a concentrate.

[0140] The main advantages of the present invention include:

[0141] (1) The present invention provides a new use of an ethanol extract of Coptis chinensis. In vivo pharmacodynamic experiments using a zebrafish angiogenesis model confirmed that the ethanol extract of Coptis chinensis can significantly inhibit angiogenesis in zebrafish, indicating that the ethanol extract of Coptis chinensis has anti-angiogenic activity and can be used as an angiogenesis inhibitor for the treatment of neovascularization-dependent and neovascularization-related diseases such as tumors, arthritis, skin and ophthalmological diseases, atherosclerosis, vascular dementia, and endometriosis.

[0142] (2) The alcohol extract of Coptis chinensis in the present invention is simple to prepare, inexpensive, and has a wide range of raw material sources. It can be made into various oral, injectable, and external preparations by conventional preparation technology with the addition of pharmaceutically acceptable excipients, and has good development prospects.

[0143] (3) The anti-angiogenic drug prepared by the research results of the present invention has the advantages of price advantage, safety, effectiveness, and low drug resistance, and has a broad development and application prospect.

[0144] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which no specific conditions are specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0145] Experimental Materials:

[0146] Zebrafish: The zebrafish used in Example 3 are vascular transgenic zebrafish (a gene specifically expressed by endothelial cells is used as a driver to drive the specific expression of green fluorescent protein in zebrafish vascular endothelial cells), and their breeding and use are strictly in accordance with the requirements of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

[0147] Fish water: Preparation method: Add 0.3g sea salt to 1L reverse osmosis water, and control the pH value between 6.9 and 7.2.

[0148] Methanol (for the preparation of Coptis chinensis ethanol extract): purchased from Sinopharm Group (National Medicine Code 10014118, CAS No. 67-56-1, specification AR (Shanghai test).

[0149] Dimethyl sulfoxide (DMSO, solvent control): purchased from Sigma (catalog number #D2650). 0.1% DMSO solution preparation: Prepare a 0.1% working solution with aquaculture water before use.

[0150] PTK787 (vatalanib) (positive control): The API was purchased from SelleckChem (Cat. #S1101). The desired concentration was prepared using 0.1% DMSO. The positive control used in this experiment was 10 μg / mL. PTK787 was developed by Novartis Pharmaceuticals, Switzerland, and serves as a standard anti-angiogenic reference drug.

[0151] Preparation of Coptis chinensis ethanol extract and Coptis chinensis water extract working solutions: 0.1% DMSO solution was used to prepare Coptis chinensis ethanol extract and Coptis chinensis water extract solutions of different concentrations, with the working concentrations being 25 μg / mL, 50 μg / mL, and 100 μg / mL, respectively.

[0152] Example 1 Preparation of Coptis chinensis aqueous extract

[0153] (1) Take the medicinal material of Coptis chinensis, grind it into coarse powder with a grinder, accurately weigh 100g of the coarse powder, add 500mL of deionized water, soak it at room temperature for 24h, and filter it with filter paper.

[0154] (2) The filtrate obtained in the first step is concentrated to dryness under reduced pressure using a rotary evaporator to obtain a concentrate.

[0155] (3) The concentrate was dried using a freeze dryer to obtain a water extract of Coptis chinensis, which was frozen and stored at -20°C for later use.

[0156] Example 2 Preparation of Coptis chinensis ethanol extract

[0157] (1) Take the medicinal material of Coptis chinensis and grind it into coarse powder with a grinder. Accurately weigh 100 g of the coarse powder, add 500 mL of methanol (anhydrous methanol), soak it at room temperature for 24 hours, and filter it with filter paper.

[0158] (2) The filtrate obtained in the first step is concentrated to dryness under reduced pressure using a rotary evaporator to obtain a concentrate.

[0159] (3) The concentrate was dried using a freeze dryer to obtain the Coptis chinensis alcohol extract, which was frozen and stored at -20°C for later use.

[0160] Example 3 Evaluation of the inhibitory effect of the aqueous and ethanol extracts of Coptis chinensis on the zebrafish intersegmental angiogenesis (ISV) model

[0161] In this example, the anti-angiogenic efficacy of the water and alcohol extracts prepared in Examples 1 and 2 was evaluated using the zebrafish intersegmental vessel (ISV) model. This in vivo model can be used to evaluate the pharmacodynamics of anti-angiogenic drugs.

[0162] Zebrafish intersegmental angiogenesis is a complete dynamic process. The sprouting of intersegmental vascular endothelial cells begins at 20hpf, and the main intersegmental vascular network, such as the dorsal long axis vessels (DLAVs) and intersegmental vessels (ISVs), is formed around 30-31hpf. The complete intersegmental vascular network is basically formed at 48hpf, at which time the complete intersegmental vessels (ISVs) are clearly visible.

[0163] The complete intersegmental vessels mainly refer to the vessels connecting the dorsal aorta (DA) and the dorsal long axis vessels (DLAV). Figure 1 (2dpf vascular transgenic zebrafish intersegmental angiogenesis model). Zebrafish 2 days after fertilization (2dpf) have a total of 28 pairs of complete intersegmental vessels (ISVs).

[0164] If inhibitors are added before complete intersegmental vascularization has formed, the formation of the intersegmental vascular network can be inhibited. Therefore, the zebrafish intersegmental angiogenesis model can be used to conveniently and intuitively evaluate the effects of drugs on the inhibition of intersegmental angiogenesis.

[0165] 3.1 Experimental methods:

[0166] (1) Experimental grouping and embryo processing: 240 well-developed zebrafish embryos were randomly divided into 8 groups at the embryonic development stage of 8 hpf after fertilization (see the table below):

[0167] Table 1 Screening and processing conditions of water extract and alcohol extract of Geranium

[0168]

[0169] (1) Each group of zebrafish embryos consisted of 30. During the operation, the embryos were evenly distributed into a 6-well cell culture plate (BD Falcon), with 30 embryos per well. 30 embryos were treated with each drug concentration, and 4 mL of water was used for each embryo rearing well.

[0170] (2) Drug treatment: Use a pipette (range 100-1000 μl, Eppendorf, Germany) to quickly add the pre-prepared drug solution to the corresponding wells of a 6-well cell culture plate, 4 mL per well. Before adding the drug solution, use a pipette and pipette to remove as much water as possible from the 6-well plate used to incubate the embryos. Then wrap the 6-well plate with tin foil, mark the experiment, and quickly place it in a constant temperature and light incubator for further culture for 24 hours (the incubator temperature is controlled at 28.5±0.5℃).

[0171] (3) Phenotypic Observation and Statistics: Embryos treated with each drug concentration were observed and photographed under a stereo fluorescence microscope (Nikon SMZ-18 stereo fluorescence microscope) at 52 hpf to analyze the effects of each drug concentration on intersegmental vessel (ISV) formation in zebrafish. Ten embryos were randomly selected from each experimental group for quantitative analysis. The statistical indicators are as follows:

[0172] a) Number of intact intersegmental vessels (ISVs): vessels connecting the dorsal aorta (DA) and the dorsal long axis vessels (DLAV)

[0173]

[0174] GraphPad Prism 5 software was used for statistical drawing, and one-way ANOVA was used for statistical analysis. The inhibition rate of geranium extract at various concentrations on zebrafish intersegmental angiogenesis (SV) was calculated.

[0175] 3.2 Experimental Results

[0176] The experimental results are as follows Figure 2-Figure 4 shown.

[0177] The inhibitory effect of Coptis chinensis ethanol extract on the formation of intersegmental vessels (ISVs) in zebrafish increased gradually with increasing concentration. Compared with the solvent control (0.1% DMSO), Coptis chinensis ethanol extract can significantly inhibit the formation of intersegmental vessels (ISVs) in zebrafish, as manifested by the thinning and narrowing of intersegmental vessels (ISVs), the loss and rupture of dorsal long axis vessels (DLAVs), and only a few sporadic vascular endothelial cell sprouts (Sprouts) in the dorsal aorta (DA) (Figures 2a-2j). As the concentration increases, the inhibitory effect of Coptis chinensis ethanol extract on the formation of intersegmental vessels in zebrafish gradually increases, showing dose dependence; when the concentration of Coptis chinensis ethanol extract is 25μg / mL, the formation of intersegmental vessels in zebrafish is significantly inhibited ( Figure 2 k).

[0178] The inhibitory rate of the alcohol extract of Coptis chinensis on the formation of intersegmental vessels (ISV) in zebrafish increased with the increase of concentration. The inhibitory rates of the alcohol extract of Coptis chinensis on the formation of intersegmental vessels (ISV) in zebrafish at different concentrations were: 25μg / mL (11.96%), 50μg / mL (20.29%), 100μg / mL (65.58%) ( Figure 3 ).

[0179] The aqueous extract of Coptis chinensis also has a certain inhibitory effect on the zebrafish intersegmental blood vessel (ISV) generation model, and the inhibitory effect also shows a gradient increase with the increase of concentration. The proportion of embryos with intersegmental blood vessel (ISV) defects caused by different concentrations of Coptis chinensis aqueous extract are: 25μg / mL (6.67%), 50μg / mL (13.33%), 100μg / mL (26.67%) ( Figure 4 ).

[0180] The above experiments show that both the water extract and the alcohol extract of Coptis chinensis have significant inhibitory effects on angiogenesis, especially the alcohol extract of Coptis chinensis has a better effect.

[0181] Example 4 Anti-angiogenic effects of various other Chinese herbal extracts in the screening of zebrafish angiogenesis model

[0182] In this example, the inventors studied four other traditional Chinese medicines that have been reported to have anti-tumor and other indications, and obtained water extracts and alcohol extracts, respectively, using the same extraction methods as in Examples 1 and 2. The research method is the same as in Example 3.

[0183] The inventors screened the anti-angiogenic effects of the water and alcohol extracts using a zebrafish angiogenesis model.

[0184] The results of this example and the results of Example 3 are summarized in Table 2.

[0185] Table 2 Anti-angiogenic effects of various Chinese herbal medicine extracts in the zebrafish angiogenesis model

[0186]

[0187] Note: The concentrations of the extracts used for anti-angiogenesis screening are: 25 μg / mL, 50 μg / mL, and 100 μg / mL. Ethanol extract: methanol extract.

[0188] discuss

[0189] The present inventors studied Coptis chinensis and other traditional Chinese herbal medicines, using their aqueous and methanol extracts to screen for anti-angiogenic effects in a zebrafish angiogenesis model. Unexpectedly, the present inventors discovered that the alcohol extract of Coptis chinensis exhibited significant anti-angiogenic effects against intersegmental blood vessel (ISV) formation in zebrafish, and that the aqueous extract of Coptis chinensis also exhibited some anti-angiogenic effects. However, aqueous and alcohol extracts of other traditional Chinese medicines failed to inhibit angiogenesis in vivo at the selected experimental concentrations.

[0190] Both the aqueous and alcoholic extracts of the present invention significantly inhibit angiogenesis, with the alcoholic extract of Coptis chinensis being particularly effective. This suggests that the Coptis chinensis plant, its Chinese medicinal materials, or Coptis chinensis plant extracts can be used to prepare anti-angiogenic drugs. Furthermore, given that angiogenesis inhibition is present in both aqueous and alcoholic extracts, this suggests that extracts prepared by conventional solvent extraction, supercritical carbon dioxide extraction, extraction, chromatography, or combinations thereof also exhibit similar anti-angiogenic properties.

[0191] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A use of a Coptis chinensis plant, a Chinese medicinal material of the Coptis chinensis plant, or a Coptis chinensis plant extract, characterized in that: Used for preparing anti-angiogenic drugs.

2. The use according to claim 1, characterized in that The Coptis chinensis plant extract includes extracts of roots, stems, branches, leaves, and / or flowers of the Coptis chinensis plant, or extracts of the whole plant.

3. The use according to claim 1, characterized in that The Coptis chinensis plant extract is an extract extracted using the following solvents: alcohol, water, or a mixed solvent of alcohol and water.

4. The use according to claim 3, characterized in that The alcohol content in the alcohol-water mixed solvent is 5wt% to 100wt%, preferably 30wt% to 100wt%, and more preferably 50wt% to 95wt%.

5. The use according to claim 1, characterized in that The anti-angiogenic drugs are used to treat one or more diseases selected from the following groups: (a) Inhibit angiogenesis in psoriatic lesion tissues; (b) inhibiting angiogenesis in Paget's disease; (c) inhibiting angiogenesis in benign vascular proliferative diseases; (d) inhibiting angiogenesis in arthritis lesions; (e) inhibiting angiogenesis in neovascular eye diseases; (f) inhibiting angiogenesis in atherosclerotic lesions; (g) Inhibit angiogenesis in endometriosis lesion tissue.

6. The use according to claim 1, wherein The anti-angiogenesis drugs are also used to inhibit vascular dementia.

7. The use according to claim 1, characterized in that The Coptis chinensis plant extract is prepared by an extraction method comprising the following steps: (i) providing Coptis chinensis medicinal material powder; (ii) extracting the Coptis chinensis medicinal material powder to obtain the Coptis chinensis plant extract.

8. A composition for preparing an anti-angiogenic drug, characterized in that: The composition comprises: (i) the Coptis chinensis plant, a Chinese medicinal material of the Coptis chinensis plant, or an extract of the Coptis chinensis plant; and (ii) pharmaceutically acceptable carriers or excipients; The Coptis chinensis plant extract is extracted using a solvent selected from the group consisting of alcohol, water, or a mixed solvent of alcohol and water.

9. A use of a composition, characterized in that: For preparing anti-angiogenesis drugs, the composition comprises: (a) Coptis chinensis plant, Chinese medicinal materials of Coptis chinensis plant or Coptis chinensis plant extract; and (b) carriers or excipients acceptable in pharmaceuticals, foods or health products.

10. A non-therapeutic in vitro method for inhibiting angiogenesis, characterized in that: The method comprises the steps of administering the Coptis chinensis plant extract to a subject in need.

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