阅读说明：本技术 磷脂在调控血管生成方面的应用 (Use of phospholipids for regulating angiogenesis ) 是由 李晓彬 李昊楠 刘可春 张梦启 张姗姗 张轩铭 王利振 李宁 盛文龙 于 2021-01-12 设计创作，主要内容包括：本发明涉及磷脂在调控血管生成方面的应用,本发明通过将各类磷脂及其产品应用于斑马鱼模型,评价其对血管内皮细胞生长因子受体抑制剂(PTK787)诱导的斑马鱼体节间血管(ISVs)损伤和对斑马鱼肠下静脉(SIVs)生长情况的影响,本发明首次发现,不同来源和不同形式的磷脂可以逆转PTK787诱导的斑马鱼ISVs损伤,并对斑马鱼SIVs生长有显著的促进作用,能不同程度的增加生成的血管长度,且毒副作用小；本发明可为制备血管疾病药物提供候选,应用前景良好。(The invention relates to the application of phospholipid in regulating angiogenesis, the invention evaluates the influence of the phospholipid on injury of internode blood vessels (ISVs) of zebra fish bodies induced by a vascular endothelial cell growth factor receptor inhibitor (PTK787) and growth conditions of inferior intestinal veins (SIVs) of the zebra fish by applying various phospholipids and products thereof to a zebra fish model, and discovers for the first time that phospholipids with different sources and different forms can reverse injury of ISVs of the zebra fish induced by the PTK787, have obvious promotion effect on growth of the zebra fish SIVs, can increase the length of generated blood vessels to different degrees and have small toxic and side effects; the invention can provide candidates for preparing vascular disease medicaments and has good application prospect.)

1. Use of phospholipids for modulating angiogenesis.

2. The application of phospholipid as effective component in preparing medicine for treating angiogenesis related diseases.

3. The use of claim 2, wherein the angiogenesis-related disease is a disease caused by damaged blood vessels or inhibited angiogenesis.

4. The application of phospholipid as an effective component in preparing medicaments related to angiogenesis promotion;

preferably, the use of a phospholipid as an active ingredient in the manufacture of a medicament for increasing the length of a blood vessel.

5. The phospholipid is used as effective component in preparing health food and special medical food related to angiogenesis.

6. The use according to any one of claims 1 to 5, wherein the phospholipids are egg yolk phospholipid extracts having a total phospholipid purity of greater than 80%;

preferably, the phospholipid component in the yolk phospholipid extract comprises the following components in relative content: PC accounts for 51.78%, PE accounts for 37.31%, PS accounts for 5.61%, and SM accounts for 4.47%.

7. The use of any one of claims 1 to 5, wherein the phospholipid is a soybean phospholipid extract having a total phospholipid purity of greater than 45%;

preferably, the phospholipid component in the soybean phospholipid extract comprises the following components in relative content: PC accounts for 37.06%, PE accounts for 45.19%, PS accounts for 11.78%, and PI accounts for 2.17%.

8. The use according to any one of claims 1 to 5, wherein, according to the invention, the phospholipids are penaeus vannamei phospholipid extracts having a total phospholipid purity of more than 70%;

preferably, the phospholipid component in the penaeus vannamei phospholipid extract comprises the following components in relative content: PC accounts for 86.63% of the total phospholipids, PE accounts for 7.29% of the total phospholipids, LPC accounts for 5.37% of the total phospholipids, and LPE accounts for 0.62% of the total phospholipids.

9. The method for producing the phospholipid extract according to any one of claims 6 to 8, comprising the steps of:

crushing and homogenizing a sample, adding an ethanol solution with the concentration of more than 90% according to the mass-volume ratio g/mL of 1 (7-10), stirring and extracting to obtain an extracting solution, carrying out solid-liquid separation on the extracting solution, concentrating the separated liquid into crude phospholipid, dissolving the crude phospholipid by using the ethanol solution with the concentration of 50% -70%, standing for 12-24h, carrying out solid-liquid separation, retaining a solid part, preparing a solution with the mass concentration of 0.3-1.0g/mL by using n-hexane, and mixing the solution with acetone according to the volume ratio of 1: (5-15) adding acetone, standing for precipitation, then carrying out solid-liquid separation to obtain a final solid, and drying the final solid to obtain the phospholipid extract;

preferably, in the preparation method, the sample is egg yolk, soybean or shrimp head of penaeus vannamei

Preferably, in the preparation method, the mixture is uniformly stirred and extracted twice, each time for 6 hours to obtain an extracting solution, the extracting solutions are combined, and the extracting solution is subjected to vacuum filtration and solid-liquid separation;

preferably, in the above preparation method, the separated liquid is subjected to a reduced pressure concentration at 45 ℃ to obtain crude phospholipid;

preferably, in the preparation method, after the acetone is added, the mixture is kept stand for 24 hours at the temperature of 4 ℃;

preferably, the final solid is dried under vacuum to obtain the phospholipid extract.

10. The use of any one of claims 1 to 5, wherein the phospholipid is pure phosphatidylcholine;

preferably, the phospholipid is a pure product of phosphatidylserine;

preferably, the phospholipid is lecithin complex iodine tablets;

preferably, the phospholipid is a polyene phosphatidylcholine capsule.

Technical Field

The invention relates to application of phospholipid in angiogenesis, and belongs to the technical field of biological medicines.

Background

Angiogenesis refers to a process of sprouting a new vascular system based on an original vascular plexus or existing blood vessels, and is an essential mechanism for maintaining the function of the circulatory system and ensuring energy supply. The process is regulated by the balance of angiogenesis inducing factors and inhibiting factors, and various diseases can be caused if the regulation is unbalanced. When angiogenesis is insufficient, diseases such as myocardial ischemia, cerebral ischemia, slow healing of wound and fracture, obliterative vasculitis, diabetic peripheral vasculopathy and the like can be caused. The search for therapeutic agents with angiogenesis promoting effects is an important strategy for treating these ischemic diseases, and is a hot spot in clinical research today.

The discovery of angiogenic drugs requires the assistance of angiogenesis models, common angiogenesis models including in vitro models (endothelial cell model and rat arterial ring model), in vivo models (corneal microcapsules, chick chorioallantoic membrane, disc angiogenesis model, sponge-matrigel model, etc.), and whole animal models (zebrafish model and xenopus tadpole model). The zebra fish has the characteristics of small size, multiple spawning, fast development, transparent embryos, simple and convenient feeding and the like, has high flux of cells and high connotation of whole animals, and has become an important link for connecting the cells and a mammal model in a drug screening and evaluating system internationally. The transgenic zebra fish marked with green vascular fluorescence enables the process of observing the sample in the living embryo to act on angiogenesis to be more visual and rapid. Therefore, the zebrafish model is a dominant model for the evaluation of angiogenic activity.

Phospholipids have a wide range of biological activities and health-care functions, such as reducing blood lipid, resisting aging, promoting nerve conduction, improving brain activity, preventing cardiovascular and cerebrovascular diseases, protecting liver, enhancing immunity, resisting fatigue, promoting blood circulation, enhancing cell membrane permeability, resisting cancer, improving oxidation stability, resisting inflammation, etc. Researchers have found that supplementation with long-chain polyunsaturated fatty acids can promote regression of choroidal neovessels in mouse models of neovascular macular degeneration, suggesting that increasing dietary levels of polyunsaturated fatty acids is beneficial for attenuation of pathological angiogenesis in vivo (Ryoji Yanai et al, Cytochrome P450-produced metabolic acids from omega-3 fatty acids occlusion neovascularisation, PNAS, 2014, 111, 9603-. However, no report is found at present for evaluating the biological activity of phospholipids by using angiogenesis as a target.

The Chinese literature, "proliferation of vascular smooth muscle cells by lysophosphatidylcholine and its signaling pathway" (Baugeng, university of inner Mongolia, Master thesis, 6.2010) teaches that lysophosphatidylcholine can stimulate proliferation of vascular smooth muscle cells. However, the mechanism of angiogenesis is complex and mainly involves the proliferation and migration of endothelial cells. The proliferation of vascular smooth muscle cells can thicken the vascular wall and increase the strength and toughness of the blood vessel. Vascular smooth muscle cell proliferation does not directly correspond to angiogenesis. In addition, the phospholipid related in the invention comprises various phospholipid standard products, phospholipid commercial medicines and health-care products, self-made phospholipid extracts and the like, the components are complex, and the main evaluation index is the angiogenesis length of the zebra fish model. And the phospholipid sources are different, even though the lengths of the fatty acid side chains and the number and the positions of unsaturated bonds of the phospholipids of the same type are different, the structural difference is larger.

Disclosure of Invention

The invention aims at the defects of the prior art and provides the application of phospholipid in regulating angiogenesis.

The technical scheme of the invention is as follows:

use of phospholipids for modulating angiogenesis.

The application of phospholipid as effective component in preparing medicine for treating angiogenesis related diseases.

Preferably, according to the present invention, the angiogenesis-related disease is a disease caused by damaged blood vessels or inhibited angiogenesis.

The application of phospholipid as effective component in preparing medicine for promoting angiogenesis is provided.

According to a preferred embodiment of the present invention, the phospholipid is used as an active ingredient in the preparation of a medicament associated with increasing the length of a blood vessel.

The phospholipid is used as effective component in preparing health food and special medical food related to angiogenesis.

Preferably according to the invention, the phospholipids are egg yolk phospholipid extracts with a total phospholipid purity of more than 80%;

further preferably, the phospholipid component in the yolk phospholipid extract comprises the following components in relative content: PC accounts for 51.78%, PE accounts for 37.31%, PS accounts for 5.61%, and SM accounts for 4.47%.

Preferably, according to the invention, the phospholipid is a soybean phospholipid extract with a total phospholipid purity of more than 45%;

further preferably, the phospholipid component in the soybean phospholipid extract comprises the following components in relative content: PC accounts for 37.06%, PE accounts for 45.19%, PS accounts for 11.78%, and PI accounts for 2.17%.

According to the invention, the phospholipid is a penaeus vannamei phospholipid extract with the total phospholipid purity of more than 70%;

further preferably, the phospholipid component in the penaeus vannamei phospholipid extract comprises the following components in relative content: PC accounts for 86.63% of the total phospholipids, PE accounts for 7.29% of the total phospholipids, LPC accounts for 5.37% of the total phospholipids, and LPE accounts for 0.62% of the total phospholipids.

The preparation method of the phospholipid extract comprises the following steps:

crushing and homogenizing a sample, adding an ethanol solution with the concentration of more than 90% according to the mass-volume ratio g/mL of 1 (7-10), stirring and extracting to obtain an extracting solution, carrying out solid-liquid separation on the extracting solution, concentrating the separated liquid into crude phospholipid, dissolving the crude phospholipid by using the ethanol solution with the concentration of 50% -70%, standing for 12-24h, carrying out solid-liquid separation, retaining a solid part, preparing a solution with the mass concentration of 0.3-1.0g/mL by using n-hexane, and mixing the solution with acetone according to the volume ratio of 1: (5-15) adding acetone, standing for precipitation, then carrying out solid-liquid separation to obtain a final solid, and drying the final solid to obtain the phospholipid extract.

According to a preferred embodiment of the present invention, in the above preparation method, the sample is egg yolk, soybean or shrimp heads of penaeus vannamei.

According to the invention, in the preferable preparation method, the extract is obtained by uniformly stirring and extracting twice for 6 hours each time, the extract is combined, and the extract is subjected to vacuum filtration and solid-liquid separation.

According to a preferred embodiment of the present invention, in the above-mentioned production method, the separated liquid is subjected to a reduced pressure concentration at 45 ℃ to obtain a crude phospholipid.

According to the invention, in the preparation method, acetone is added and then the mixture is kept stand for 24 hours at the temperature of 4 ℃.

Further preferably, the final solid is dried under vacuum to obtain the phospholipid extract.

According to the invention, the phospholipid is pure PC (phosphatidyl choline).

According to the present invention, the phospholipid is preferably a pure PS (phosphatidylserine).

Preferably according to the invention, the phospholipid is a lecithin complexed iodine tablet.

Preferably according to the invention, the phospholipid is a polyene phosphatidylcholine capsule.

The technical scheme of the invention has the beneficial effects

The invention evaluates the influence of phospholipid on zebra fish internodal blood vessel (ISV) injury induced by a vascular endothelial cell growth factor receptor inhibitor (PTK787) and zebra fish enteron vein (SIV) growth condition by applying the phospholipid to a zebra fish model. After detection, different phospholipids can regulate and control PTK 787-induced damage of the ISVs of the zebra fish and have different degrees of promotion or inhibition effects on growth of the SIVs of the zebra fish for the first time. The invention can provide candidates for vascular disease drugs and has wide market prospect.

Drawings

FIG. 1 is a liquid chromatography base peak diagram of egg yolk phospholipids.

FIG. 2 is a diagram showing a liquid mass analysis base peak of soybean phospholipid.

FIG. 3 is a liquid mass analysis base peak diagram of phospholipid of Penaeus vannamei Boone.

FIG. 4 is a photograph of ISVs grown after treatment of zebrafish with different phospholipid extracts, drugs and health products;

in the figure: 20. 40 and 80. mu.g/mL represent the phospholipid sample treatment groups at the corresponding concentrations, respectively; sample 1 represents an egg yolk phospholipid extract, i.e. samples 1 at three concentrations were: 1-20, 1-40, 1-80; sample 2 represents the soy phospholipid extract, i.e. sample 2 at three concentrations is: 2-20, 2-40, 2-80; sample 3 represents a phospholipid extract of penaeus vannamei, i.e. three concentrations of sample 3 are: 3-20, 3-40, 3-80; sample 4 represents a lecithin complexed iodine tablet, i.e. sample 4 at three concentrations is: 4-20, 4-40, 4-80; sample 5 represents a polyene phosphatidyl choline capsule, i.e. samples 5 at three concentrations are: 5-20, 5-40, 5-80; sample 6 represents soya lecithin softgels, i.e. samples 6 at three concentrations are: 6-20, 6-40, 6-80; sample 7 represents a fish oil soft capsule, i.e. samples 7 at three concentrations are: 7-20, 7-40 and 7-80.

FIG. 5 is a bar graph of ISVs length statistics after treatment of zebrafish with different phospholipid extracts, drugs and health products;

in the figure: control represents the blank Control group; PTK787 represents a model group; SAAS (sodium danshensu) represents the positive control group; 20. 40 and 80. mu.g/mL represent the phospholipid sample treatment groups at the corresponding concentrations, respectively; each sample number represents the same meaning as figure 4; in comparison to the set of models,^**P＜0.01，^****P＜0.0001。

FIG. 6 is a photograph of growth of SIVs following treatment of zebrafish with different phospholipid extracts, drugs and health products;

in the figure: 20. 40 and 80. mu.g/mL represent the phospholipid sample treatment groups at the corresponding concentrations, respectively; each sample number is as defined in FIG. 4.

FIG. 7 is a bar graph of SIVs length statistics after treatment of zebrafish with different phospholipid extracts, drugs and health products;

in the figure: control represents the blank Control group; 20. 40, 80. mu.g/mL minRespectively representing the phospholipid sample treatment groups at corresponding concentrations; each sample number represents the same meaning as figure 4; in comparison to the set of models,^*P＜0.05，^**P＜0.01，^***P＜0.001。

FIG. 8 is a photograph of ISVs grown after treatment of zebrafish with different phospholipid standards;

in the figure, 20, 40 and 80. mu.g/mL represent the treatment groups of the phospholipid standard substance at the corresponding concentrations, respectively.

FIG. 9 is a bar graph of ISVs length statistics after treatment of zebrafish with different phospholipid standards;

in the figure: control represents the blank Control group; PTK787 represents a model group; SAAS (sodium danshensu) represents the positive control group; 20. 40 and 80 mu g/mL respectively represent the phospholipid standard substance treatment groups under corresponding concentrations; in comparison to the set of models,^*P＜0.05，^**P＜0.01，^****P＜0.0001。

FIG. 10 is a photograph of growth of SIVs following treatment of zebrafish with different phospholipid standards;

in the figure, 20, 40 and 80. mu.g/mL represent the treatment groups of the phospholipid standard substance at the corresponding concentrations, respectively.

FIG. 11 is a histogram of SIVs length statistics after treatment of zebrafish with different phospholipid standards;

in the figure: control represents the blank Control group; 20. 40 and 80 mu g/mL respectively represent the phospholipid standard substance treatment groups under corresponding concentrations; in comparison to the set of models,^*P＜0.05，^**P＜0.01，^****P＜0.0001。

Detailed Description

The technical scheme of the invention and the technical effects thereof are further explained below by combining a specific test method and a drawing. The following description is intended only to illustrate the invention and not to limit it in any way, and any alterations or substitutions made on the basis of the teachings of the present invention are within the scope of the invention.

The contents of the examples, which are not specified in specific conditions, were carried out under conventional conditions; the reagents or instruments used are not indicated by manufacturers, and are all common commercial products.

Sources of materials

The penaeus vannamei boone is purchased from a local seafood market and is a common commercial product; eggs and soybeans are purchased from local supermarkets and are common commercial products; the soybean lecithin soft capsules, the fish oil soft capsules, the lecithin complexed iodine tablets and the polyene phosphatidyl choline amide capsules are purchased from local drugstores and are common commercial products; the zebra fish is provided for a drug screening research laboratory of biological research institute of academy of sciences in Shandong province and is a common commercially available product; streptokinase E, PTK787(N- (4-chlorophenylyl) -4- (pyridine-4-ylmethyl) phthalazin-1-amine succinate), sodium danshensu, PE (phosphatidylethanolamine), PC (phosphatidylcholine), PI (phosphatidylinositol), PS (phosphatidylserine), SM (sphingomyelin) and the like were purchased from Sigma, and Danhong injection (10 mL/lot) was produced by Shandong steplength pharmaceutical Co., Ltd and was a product generally sold on the market.

Experimental example 1

Determination of phospholipid content

The invention adopts spectrophotometry (molybdenum blue colorimetry) to determine the content of phospholipid. The standard curve equation for potassium dihydrogen phosphate was made as follows: y 0.8458x +0.0057 (R)²＝0.9998)。

Accurately measuring 0.3mL of phospholipid sample solution (taking dichloromethane as a solvent, dissolving the phospholipid sample in dichloromethane to obtain a phospholipid sample solution with the mass concentration of 0.5 mg/mL), placing in a 10mL graduated test tube (taking 0.3mL of dichloromethane as a blank control), volatilizing the solvent in a water bath, adding 4 drops of concentrated sulfuric acid and 3 drops of perchloric acid, digesting on an electric furnace until the solution is colorless and clear, cooling, adding water to 2mL, adding 1 drop of phenolphthalein indicator, neutralizing with 50% sodium hydroxide solution until the solution turns red, slowly adding dilute sulfuric acid (5/200, v/v) to eliminate the red, adding water to 5mL, shaking, sequentially adding 1.0mL of sulfuric acid for adjusting acidity, shaking, 1.0mL of ammonium molybdate solution, shaking, 0.6mL of ascorbic acid solution, adding water to 10mL, adding a plug, mixing uniformly, quickly placing in a 70 ℃ water bath for color development for 30min, taking out, placing in cold water for cooling for 10min, and (3) taking a blank control group as a reference, measuring a light absorption value at the wavelength of 820nm, substituting into a standard curve to obtain the content of the inorganic phosphorus, and multiplying by a coefficient of 26.3 to obtain the content of the total phospholipid.

Experimental example 2

The UPLC-Q-active Orbitrap/MS method is adopted to determine the composition and relative content of various phospholipids in the phospholipid sample, and the specific reference of the calculation method of the relative content is as follows: xiaobin Li, et al, lipid finger printing of differential material sources by UPLC-Q-reactive Orbitrap/MS approach and the z zebraphis-based activities company, Journal of Agricultural and Food Chemistry 2020,68,2007 company 2015.

Experimental example 3

Activity evaluation of phospholipid for promoting generation of ISVs in zebra fish

(1) Pretreatment of zebra fish

The experimental animals used in the invention are vascular fluorescence transgenic Tg (fli-1: EGFP) zebra fish, which are fed under the conditions of 28 ℃ and 10h darkness/14 h illumination period, and the giant shrimps are fed regularly and quantitatively every day. Taking healthy and mature zebra fish according to the ratio of male to female being 1:2, putting the zebra fish into a mating tank, placing a partition plate in the middle, and placing the zebra fish in a dark environment. And (3) before lighting the next day, removing the partition plate, lighting to stimulate the partition plate to ovulate, collecting fertilized eggs after 1h of ovulation, and washing the fertilized eggs for 3 times by using a new zebra fish embryo culture solution. In order to avoid the influence of pigmentation during the development process, 3 percent of 1-phenyl-2-thiourea (1-phenyl-2-thiourea, PTU, Sigma) is added into fish culture water of embryos, the fish culture water is put into a light incubator at the temperature of 28 ℃ for light control culture, 1/3 culture solution is changed every 24 hours in the middle, and dead embryos are sucked out in time.

(2) Zebra fish ISVs generation experiment

Zebrafish embryos developed to 24hpf (hours post fertilization) were stripped of the egg membrane with 1mg/mL solution of streptokinase E. Embryos were randomly divided into 6 groups, blank control, model, positive control and phospholipid sample (three doses), placed in 3 parallel wells of 24-well plates, with 10 embryos per well. 2mL of culture solution is added into a blank control group, PTK787 with the final concentration of 0.25 mu g/mL is added into 2mL of culture solution of a model group, PTK787 (with the final concentration of 0.25 mu g/mL) and danshensu sodium (with the final concentration of 100 mu g/mL) are simultaneously added into 2mL of culture solution of a positive control group, and PTK787 (with the final concentration of 0.25 mu g/mL) and phospholipid samples (with the final concentrations of 20, 40 and 80 mu g/mL) are simultaneously added into 2mL of culture solution of a phospholipid three-dose group respectively. The components are respectively put into a constant temperature incubator at 28 ℃ and cultured for 24 hours.

(4) Data processing

After 24h of administration, the generation of zebrafish ISVs was observed by a fluorescence microscope and images thereof were collected, and after measuring the length of ISVs using Image Pro Plus 5.0 software, data statistics was performed using GraphPad Prism 6.01 software.

Experimental example 4

Evaluation of Activity of phospholipid for promoting Generation of SIVs in Zebra Fish

(1) The pretreatment of zebra fish was the same as in experimental example 3.

(2) Zebra fish SIVs generation experiment

Zebrafish embryos that developed to 72hpf were randomly divided into 4 groups, a placebo group and three dose groups of phospholipids, placed in 24-well plates with 3 parallel wells per group, with 10 embryos per well. 2mL of culture solution was added to the blank control group, and each phospholipid sample (final concentrations of 20, 40, and 80. mu.g/mL, respectively) was added to 2mL of culture solution to each of the three phospholipid dose groups. The components are respectively put into a constant temperature incubator at 28 ℃ and cultured for 24 hours.

(3) Data processing

After 24h of administration, generation of zebrafish SIVs was observed by a fluorescence microscope and images thereof were collected, and after measuring ISVs length using Image Pro Plus 5.0 software, data statistics was performed using GraphPad Prism 6.01 software.

Example 1

Effect of yolk phospholipid extract on angiogenesis of zebra fish

Separating egg white and yolk of fresh purchased eggs, weighing 100g of yolk, homogenizing, adding 800mL of 90% ethanol-water solution, uniformly stirring and extracting for 2 times (6 h each time), combining extracting solutions, carrying out vacuum filtration, and concentrating the filtrate at 45 ℃ under reduced pressure to obtain crude phospholipid. Dissolving crude phospholipid with 60% ethanol-water solution as solvent, standing for 24 hr, centrifuging for 15min, and retaining precipitate. Using n-hexane to prepare a solution of 0.5g/mL, adding 50mL of acetone, and standing in a refrigerator at 4 ℃ for precipitation for 24 hours. Centrifuging for 15min to remove supernatant, and vacuum drying insoluble substances to obtain yolk phospholipid with yield of 4.63% (based on the fed yolk) and total phospholipid purity of 83.79% by mass, and determining the composition of yolk phospholipid and the relative content of phospholipids according to the experimental method of experimental example 2, wherein the phospholipid components comprise: PC accounts for 51.78%, PE accounts for 37.31%, PS accounts for 5.61%, and SM accounts for 4.47%.

The liquid quality analysis and detection pattern of the yolk phospholipids is shown in figure 1.

The effect of egg yolk phospholipids on promoting the production of ISVs in zebrafish was determined according to the experimental method of experimental example 3, and the experimental results are shown in fig. 4 and 5. The experimental result shows that compared with a blank control group, the ISVs of the zebra fish in the model group are remarkably reduced in length (P is less than 0.0001), and the model drug remarkably inhibits angiogenesis of the zebra fish, so that the model is successfully molded. Compared with the model group, the length of the zebra fish ISVs in the egg yolk phospholipid group of each dosage is obviously increased, and the statistical difference proves that the egg yolk phospholipids can effectively promote the growth of the ISVs.

The effect of egg yolk phospholipids on the generation of zebrafish SIVs was measured according to the experimental method of experimental example 4, and the experimental results are shown in fig. 6 and 7. According to the experimental result, compared with a blank control group, the length of zebra fish SIVs in the yolk phospholipid group with medium concentration (40 mug/mL) is obviously increased, the significant difference is achieved (P is less than 0.01), and the activity of the rest concentration groups is not obvious.

Example 2

Effect of Soybean Phospholipids extract on Zebra Fish angiogenesis

Weighing 100g of soybean, crushing, homogenizing, adding 900mL of 90% ethanol-water solution, uniformly stirring and extracting for 2 times, 6h each time, combining the extracting solutions, carrying out vacuum filtration, and concentrating the filtrate at 45 ℃ under reduced pressure to obtain crude phospholipid. Dissolving crude phospholipid with 62% ethanol-water solution as solvent, standing for 24 hr, centrifuging for 15min, and collecting precipitate. Using n-hexane to prepare a solution of 0.5g/mL, adding 60mL of acetone, and standing in a refrigerator at 4 ℃ for precipitation for 24 hours. Centrifuging for 15min to remove supernatant, and vacuum drying the insoluble substance to obtain soybean phospholipid with yield of 0.15% (based on charged soybean) and total phospholipid purity of 47.32% by mass; the composition of soybean phospholipids and the relative contents of various types of phospholipids were determined according to the experimental method of experimental example 2, wherein the phospholipid components included, in terms of relative contents: PC accounts for 37.06%, PE accounts for 45.19%, PS accounts for 11.78%, and PI accounts for 2.17%.

The liquid mass analysis detection spectrum of soybean phospholipid is shown in figure 2.

The effect of soybean phospholipids on promoting the production of ISVs in zebrafish was measured according to the experimental method of experimental example 3, and the experimental results are shown in fig. 4 and 5. According to experimental results, compared with a model group, the length of the ISVs of the zebra fish in each dose of soybean phospholipid group is obviously increased, statistical differences exist, and the soybean phospholipid can effectively promote the growth of the ISVs.

The effect of soybean phospholipids on the generation of zebrafish SIVs was measured according to the experimental method of experimental example 4, and the experimental results are shown in fig. 6 and 7. According to the experimental results, compared with a blank control group, the length of the zebra fish SIVs in each soybean phospholipid group is obviously increased, statistical differences exist, and the soybean phospholipids can effectively promote the growth of the SIVs.

Example 3

Effect of penaeus vannamei phospholipid extract on angiogenesis of zebra fish

Weighing 100g of heads of the penaeus vannamei boone which is frozen and preserved for 30 days, crushing, homogenizing, adding 850mL of ethanol-water solution with the concentration of 90%, stirring and extracting for 2 times at constant speed for 6h each time, combining extracting solutions, carrying out vacuum filtration, and carrying out vacuum concentration on filtrate at 45 ℃ to obtain crude phospholipid. Dissolving crude phospholipid with 62% ethanol-water solution as solvent, standing for 24 hr, centrifuging for 15min, and collecting precipitate. The solution was made up with n-hexane to 0.5g/mL, 56mL acetone was added, and the mixture was allowed to stand in a refrigerator at 4 ℃ for precipitation for 24 hours. Centrifuging for 15min to remove supernatant, and finally vacuum drying insoluble substances to obtain the penaeus vannamei phospholipid, wherein the yield of the penaeus vannamei phospholipid is 0.43% (calculated on the frozen penaeus vannamei heads fed with materials), the total phospholipid purity is 73.28% by mass percent, the composition of the penaeus vannamei phospholipid and the relative content of various phospholipids are determined according to the experimental method of experimental example 2, and the phospholipid components comprise the following components in relative content: PC accounts for 86.63%, PE accounts for 7.29%, LPC (lysophosphatidylcholine) accounts for 5.37%, and LPE (lysophosphatidylethanolamine) accounts for 0.62%.

The liquid mass analysis and detection map of the penaeus vannamei phospholipid is shown in figure 3.

The effect of penaeus vannamei phospholipid on promoting the generation of ISVs in zebrafish was determined according to the experimental method of experimental example 3, and the experimental results are shown in fig. 4 and 5. According to experimental results, compared with a model group, the length of the zebra fish ISVs in each dose of penaeus vannamei phospholipid group is obviously increased, statistical differences exist, and the penaeus vannamei phospholipid can effectively promote the growth of the ISVs, and the phospholipid angiogenesis promoting activity is enhanced along with the increase of the dose. In addition, the angiogenesis promoting activity of the penaeus vannamei phospholipid is obviously stronger than that of egg yolk phospholipid and soybean phospholipid.

The effect of penaeus vannamei phospholipid on promoting the generation of zebrafish SIVs was determined according to the experimental method of experimental example 4, and the experimental results are shown in fig. 6 and 7. According to the experimental results, compared with a blank control group, the length of the zebra fish SIVs in each penaeus vannamei phospholipid group is obviously increased, statistical differences exist, and the penaeus vannamei phospholipid can effectively promote the growth of the SIVs.

Example 4

Effect of lecithin-complexed iodine tablets on angiogenesis of zebra fish

The effect of lecithin complexed iodine tablets on the generation of ISVs by zebrafish was determined according to the experimental method of Experimental example 3. The experimental results are shown in fig. 4 and 5. The results show that compared with the model group, the lecithin complexed iodine tablets of each dosage can obviously promote the growth of the ISVs of the zebra fish, and have statistical difference.

The effect of lecithin-iodine complex tablets on the generation of zebrafish SIVs was measured according to the experimental method of experimental example 4, and the experimental results are shown in fig. 6 and 7. As can be seen from the experimental results, compared with the blank control group, only the lecithin-complexed iodine tablet of the medium concentration group (40 mug/mL) has an obvious promotion effect on the growth of the zebra fish SIVs, and has statistical difference.

Example 5

Effect of polyene phosphatidyl choline capsule on angiogenesis of zebra fish

The content of the polyene phosphatidyl choline capsule was taken out, and the effect of the polyene phosphatidyl choline capsule on the growth of ISVs in zebra fish was determined according to the experimental method of Experimental example 3. The experimental results are shown in fig. 4 and 5. The results show that compared with the model group, each dose of polyene phosphatidyl choline can obviously promote the growth of zebra fish ISVs, has statistical difference, and increases the phospholipid angiogenesis promoting activity along with the increase of the dose.

The accelerating effect of the polyene phosphatidyl choline capsules on the formation of zebrafish SIVs was determined according to the experimental method of experimental example 4, and the experimental results are shown in fig. 6 and 7. As can be seen from the experimental results, the zebrafish SIVs in the medium concentration group (40. mu.g/mL) and the high concentration group (80. mu.g/mL) were significantly increased in length and were statistically different from the blank control group, and the low concentration group (20. mu.g/mL) had no activity of promoting the growth of SIVs.

Example 6

Influence of health product soybean phospholipid soft capsule on zebra fish angiogenesis

The contents of the soybean phospholipid soft capsules were taken out, and the effect of the contents of the soybean phospholipid soft capsules on the generation of ISVs in zebra fish was determined according to the experimental method of experimental example 3. The experimental results are shown in fig. 4 and 5. The results show that compared with the model group, the soybean phospholipid soft capsule has no obvious promotion effect on the generation of the zebra fish ISVs.

The effect of the contents of the soybean phospholipid soft capsules on the generation of zebrafish SIVs was measured according to the experimental method of experimental example 4. The experimental results are shown in fig. 6 and 7. The results showed that the medium concentration group (40. mu.g/mL) and the high concentration group (80. mu.g/mL) of the soybean lecithin soft capsules had a promoting effect on the generation of zebrafish SIVs, as compared with the model group.

Example 7

Influence of health-care product fish oil soft capsule on angiogenesis of zebra fish

The contents of the fish oil soft capsules were taken out, and the effect of the contents of the fish oil soft capsules on promoting the generation of ISVs in zebrafish was measured according to the experimental method of experimental example 3. The experimental results are shown in fig. 4 and 5. The result shows that compared with the model group, the fish oil soft capsule has no obvious promotion effect on the generation of the zebra fish ISVs.

The effect of the contents of the fish oil soft capsules on the generation of zebrafish SIVs was determined according to the experimental method of experimental example 4. The experimental results are shown in fig. 6 and 7. The results show that, compared with the model group, only the medium concentration group (40 mug/mL) of the fish oil soft capsule has a promoting effect on the generation of zebra fish SIVs, and the other two concentration groups are inactive.

Comparative example

Effect of phospholipid standards on Zebra Fish angiogenesis

The growth promoting effect of various phospholipid standards (PC, PE, PS, PI, SM) on the ISVs and SIVs of zebra fish was evaluated according to the experimental methods in Experimental example 3 and Experimental example 4, respectively. The experimental results are shown in FIGS. 8 to 11.

As is clear from fig. 8 and 9, each of the phospholipid standards showed different degrees of activity of promoting the growth of ISVs in zebrafish, except PI, compared to the model group. The activities of PC and PE are obvious, but the activity of PC shows obvious concentration dependence, and the activity of PE is reduced along with the increase of the concentration. The activity of the low, medium and high concentration groups of PS is not greatly different, and only the high concentration group (80 mug/mL) of SM shows obvious growth promotion activity on ISVs.

As can be seen from fig. 10 and 11, the class 5 phospholipid standards all exhibited different degrees of activity of promoting the growth of zebrafish SIVs as compared to the model group. Wherein, each concentration group of PI has the activity of promoting the growth of SIVs, the medium and high concentration groups of PC, PS and SM show obvious activity, and PE only has the activity of the high concentration group.

In conclusion, the phospholipid and phospholipid products are found for the first time to reverse PTK 787-induced damage to the ISVs of the zebra fish, have a remarkable promoting effect on growth of the SIVs of the zebra fish, can obviously increase the length of blood vessels, and have small toxic and side effects. In contrast, the activity of promoting ISVs of the penaeus vannamei phospholipid is obviously stronger than that of soybean phospholipid and yolk phospholipid, and the activity of the contents of soybean phospholipid soft capsules and fish oil soft capsules is relatively unobvious, possibly because the content of phospholipid in the contents of the soft capsules is relatively low. In combination with the vascular-promoting effects of each phospholipid standard on ISVs and SIVs, the PC and PS activities were relatively better. The invention can provide candidates for vascular disease drugs and has wide market prospect.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in other forms, and it will be apparent to those skilled in the art that several modifications and variations can be made in the above embodiment without departing from the spirit of the present invention, and these modifications and variations also fall into the protection scope of the present invention.