Kelp extract and its preparation method and application

Abstract

The present invention discloses a kelp extract and a preparation method and application thereof. The present invention soaks kelp with ethanol to obtain an extract; the extract is concentrated to obtain the kelp extract; the ratio of ethanol to kelp is 199-201 mL: 1 g, and the volume ratio of kelp extract to ethanol is 3: 99-101. The kelp extract of the present invention has an anti-angiogenic effect, effectively inhibits the proliferation, migration and tubule formation of vascular endothelial cells, effectively inhibits the angiogenesis of the intestinal vein of zebrafish, has anti-angiogenic activity, and its effect is better than that of monomeric fucoidan. The kelp extract of the present invention blocks the signal pathway mediated by vascular endothelial growth factor receptor, inhibits the expression level of vascular endothelial growth factor receptor 2 and its downstream PI3K and Akt, and is used to prepare drugs that inhibit angiogenesis, including drugs for treating and preventing diseases dependent on neovascularization and neovascularization-related diseases.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a kelp extract and a preparation method and application thereof. Background Art

[0002] Angiogenesis is a physiological phenomenon in the human body that plays an important role in embryonic development and tissue repair. Angiogenesis is a complex process involving multiple cells and multiple molecules, including endothelial cells secreting proteases to degrade the basement membrane, endothelial cell proliferation and migration, the formation of new capillary lumen structure, the recruitment of pericytes to stabilize the newly formed capillary network, and the formation of a mature vascular network through the remodeling and expansion of primitive blood vessels.

[0003] The dynamic balance between angiogenesis-inhibiting molecules and angiogenesis-promoting molecules is considered to be the "switch" that regulates angiogenesis. Under normal physiological conditions, the angiogenesis-inhibiting molecules and anti-angiogenesis molecules are in a relative balance, and the angiogenesis mechanism is in a "closed" state; it only occurs during normal physiological processes of the body such as embryonic development, reproduction, wound repair, and the physiological cycle of women. However, in solid tumors, angiogenesis will get out of control, resulting in malignant consequences: on the one hand, new blood vessels can bring abundant oxygen and nutrients to tumor cells and take away metabolites; on the other hand, tumor cells can metastasize to distant sites through new blood vessels. If there is no angiogenesis in the tumor, the growth of the tumor will be stagnant or even degenerate; if there is angiogenesis in the tumor, the volume of the tumor will grow exponentially. For example, the occurrence and development of tumors caused by various reasons, new blood vessel formation in the eye, arthritis, skin diseases, atherosclerosis and other diseases are all closely related to pathological angiogenesis. Therefore, anti-angiogenesis is an effective method for treating malignant tumors, and tumor anti-angiogenesis therapy is considered to be one of the strategies with great medical value for treating malignant tumors. Anti-angiogenesis is an extremely complex process, including intervention in angiogenesis regulatory factors, inhibition of vascular basement membrane and extracellular matrix degradation, inhibition of endothelial cell division, migration and proliferation, etc.

[0004] There are many methods for treating tumors, but the effects vary. The advantages of inhibiting tumor angiogenesis are: 1. It has few toxic side effects and less damage to normal cells; 2. It has a broad spectrum of effects; 3. It is not easy to develop drug resistance; 4. It can complement other treatment methods. Therefore, this treatment method has broad application prospects. At the same time, this treatment method is still a relatively new treatment method, and relevant research is urgently needed.

[0005] Kelp (Laminaria Japonica), also known as kelp and Jiangbaicai, belongs to the Phaeophyceae, Laminariaceae, and Laminariales. It has good nutrition and flavor and has become a health food. Kelp is rich in a variety of functional ingredients, such as polyphenols, polyunsaturated fatty acids, sodium alginate, polysaccharides, mannitol, dietary fiber, etc. Modern medical research further shows that kelp is rich in functional substances such as iodine and mannitol, as well as trace elements such as selenium, cobalt, and chromium required by the human body. It has the functions of lowering blood pressure, lowering blood sugar, regulating blood lipids, anticoagulation, antithrombosis, antitumor, immunomodulation, anti-fatigue and hypoxia tolerance, anti-oxidation, antibacterial and antiviral, and is a large algae with edible, medicinal, health, industrial, and agricultural values. Kelp is widely used, but there is no report that kelp inhibits neovascularization and is directly used as an angiogenesis inhibitor. Summary of the invention

[0006] The purpose of the present invention is to overcome the above-mentioned deficiencies in the prior art and to provide a kelp extract and a preparation method and application thereof.

[0007] The first object of the present invention is to provide a method for preparing a kelp extract.

[0008] The second object of the present invention is to provide a kelp extract.

[0009] The third object of the present invention is to provide the use of the kelp extract in preparing a drug for inhibiting angiogenesis.

[0010] The fourth object of the present invention is to provide a method for detecting fucoidan in the kelp extract.

[0011] In order to achieve the above object, the present invention is implemented by the following scheme:

[0012] A method for preparing a kelp extract comprises soaking the kelp in ethanol to obtain an extract; and concentrating the extract to obtain the kelp extract.

[0013] The ratio of ethanol to kelp is 199-201 mL:1 g, and the volume ratio of kelp extract to ethanol is 3:99-101.

[0014] More preferably, the ratio of ethanol to kelp is 200 mL:1 g, and the volume ratio of kelp extract to ethanol is 3:100.

[0015] More preferably, the volume concentration of the ethanol is 49% to 51%.

[0016] More preferably, the kelp is soaked in 50% ethanol by volume for 2 to 3 days, and the solid and liquid are separated to obtain an extract; the extract is vacuum concentrated at 55 to 60° C. to obtain the kelp extract.

[0017] More preferably, the kelp is soaked in 50% ethanol by volume for 3 days, and the solid and liquid are separated to obtain an extract; the extract is concentrated in vacuum at 60°C to obtain the kelp extract.

[0018] A kelp extract is prepared by the preparation method.

[0019] The invention relates to an application of the kelp extract in preparing a drug for inhibiting angiogenesis.

[0020] Preferably, the drug is a drug that inhibits the proliferation of vascular endothelial cells.

[0021] Preferably, the drug is a drug that inhibits the migration of vascular endothelial cells.

[0022] Preferably, the drug is a drug that inhibits the formation of vascular endothelial cell tubules.

[0023] Preferably, the drug is a drug that blocks the signal pathway mediated by vascular endothelial growth factor receptor.

[0024] More preferably, the drug is a drug that inhibits the expression of vascular endothelial growth factor receptor.

[0025] More preferably, the drug is a drug that inhibits the expression of vascular endothelial growth factor receptor 2.

[0026] Preferably, the drug is a drug for treating and / or preventing one or more of tumors, arthritis, psoriasis, ophthalmic diseases and atherosclerosis.

[0027] Preferably, the dosage form of the drug is one or more of capsules, tablets, microcapsules, injections, suppositories, sprays and oral solutions.

[0028] The method for detecting fucoidan in kelp extract is characterized by comprising the following steps:

[0029] S1: Pretreatment: dissolving the kelp extract in methanol, performing ultrasonication and centrifugal filtration, wherein the ratio of the kelp extract to methanol is 24.9-25.1 mg:1 mL;

[0030] S2: HPLC detection: mobile phase A is acetonitrile, mobile phase B is a mixture of formic acid and aqueous solution, gradient elution, elution program is:

[0031] From 0 to 10 min, the volume ratio of mobile phase A to mobile phase B changed from 2:98 to 15:85;

[0032] From 10 to 25 min, the volume ratio of mobile phase A to mobile phase B changed from 15:85 to 45:55;

[0033] From 25 to 45 min, the volume ratio of mobile phase A to mobile phase B changed from 45:55 to 50:50;

[0034] From 45 to 75 min, the volume ratio of mobile phase A to mobile phase B changed from 50:50 to 80:20;

[0035] From 75 to 96 min, the volume ratio of mobile phase A to mobile phase B changed from 80:20 to 100:0.

[0036] Preferably, the high performance liquid chromatograph is an Agilent liquid chromatograph with an autosampler and a binary pump.

[0037] Preferably, the chromatographic column of the high performance liquid chromatograph is an Agilent Grace VisionHT C18 column, with specifications of 4.6×250 mm, 5 μm.

[0038] Preferably, the gradient elution flow rate is 0.9-1.1 mL / min.

[0039] More preferably, the gradient elution flow rate is 1 mL / min.

[0040] Preferably, the injection volume is 10 μL and the detection wavelength is 280 nm.

[0041] Preferably, the formic acid aqueous solution is a formic acid aqueous solution with a volume concentration of 0.19 to 0.21%.

[0042] More preferably, the formic acid aqueous solution is a formic acid aqueous solution with a volume concentration of 0.2%.

[0043] Preferably, the volume concentration of the methanol is 49% to 51%.

[0044] More preferably, the volume concentration of methanol is 50%.

[0045] More preferably, the ratio of the kelp extract to methanol is 25 mg:1 mL.

[0046] More preferably, the ultrasonic time is 14 to 16 minutes, and the centrifugation is performed at 900 to 1100×g for 4 to 6 minutes.

[0047] Preferably, the ultrasonic time is 15 min, and the centrifugation is performed at 1000×g for 5 min.

[0048] More preferably, the filter membrane specification of the filtration is 0.45 μm.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] The kelp extract of the present invention has an anti-angiogenic effect, effectively inhibits the proliferation, migration and tubule formation of vascular endothelial cells, effectively inhibits the formation of blood vessels in the intestinal vein of zebrafish, has anti-angiogenic activity, and its effect is better than that of monomeric fucoidan. The kelp extract of the present invention blocks the signal pathway mediated by the vascular endothelial growth factor receptor, inhibits the expression level of vascular endothelial growth factor receptor 2 and its downstream PI3K and Akt, and is used to prepare drugs for inhibiting angiogenesis, including drugs for treating and / or preventing tumors, arthritis, psoriasis, ophthalmic diseases, atherosclerosis and other diseases that are dependent on and related to neovascularization. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is the HPLC fingerprint of kelp extract.

[0052] Figure 2 This is a block diagram of the experiment on the inhibition of proliferation of human umbilical vein endothelial cells (HUVEC) by Laminaria japonica extract.

[0053] Figure 3 The experimental results of kelp extract inhibiting HUVEC scratch migration, where A is the experimental photo and B is the experimental quantitative graph.

[0054] Figure 4 The experimental results of kelp extract inhibiting the vertical migration of HUVEC in Transwell chamber. A is the experimental photo and B is the experimental quantitative graph.

[0055] Figure 5 The experimental results of kelp extract inhibiting HUVEC tubule formation. A is the experimental photo and B is the experimental quantitative graph.

[0056] Figure 6 The experimental results of kelp extract inhibiting angiogenesis of zebrafish intestinal vein. A is the experimental photo and B is the experimental quantitative graph.

[0057] Figure 7 The experimental results show that kelp extract can bind to VEGF and specifically block the signaling pathway mediated by vascular endothelial growth factor receptor 2 (VEGFR2) to exert its anti-angiogenic effect. A is the experimental photo and B is the experimental quantitative graph.

[0058] Figure 8 The experimental results show that kelp extract can bind to VEGF and specifically block the downstream signaling pathway (PI3K-AKT) mediated by vascular endothelial growth factor receptor 2 (VEGFR2) to exert its anti-angiogenic effect. A is the experimental photo and B is the experimental quantitative graph.

[0059] Fig. 9This is a block diagram comparing the effects of kelp extract and the fucoidan it contains on the inhibition of human umbilical vein endothelial cell (HUVEC) proliferation.

[0060] Fig.10 The experimental results of kelp extract and its fucoidan inhibiting angiogenesis of zebrafish intestinal vein. A is the experimental photo and B is the experimental quantitative graph. DETAILED DESCRIPTION

[0061] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0062] Example 1 Preparation of kelp extract and HPLC fingerprint of kelp extract

[0063] 1. Experimental Methods

[0064] The kelp medicinal material is crushed, 50 g is weighed, and put into a volumetric flask, and soaked and extracted with 1000 mL of 50% volume concentration ethanol for three days. After filtering, a 50% ethanol extract is obtained. The 50% ethanol extract is vacuum concentrated to 30 mL at 60°C and then vacuum freeze-dried to obtain a kelp 50% ethanol extract freeze-dried powder, which is stored in a vacuum. The kelp 50% ethanol extract freeze-dried powder is the kelp extract.

[0065] Weigh 100 mg of kelp 50% ethanol extract freeze-dried powder, place it in a 15 mL centrifuge tube, add 4 mL of 50% volume concentration methanol, ultrasonicate for 15 minutes, centrifuge at 1000×g for 5 minutes to obtain the supernatant. Before injecting the supernatant, filter it with a 0.45 μm microporous filter membrane, take the filtrate, and inject it for analysis.

[0066] The analytical instrument used was an Agilent liquid phase with an autosampler and a binary pump, and the chromatographic column was an Agilent Grace VisionHT C18 column (4.6×250 mm, 5 μm). The mobile phase was acetonitrile (solvent A) and a 0.2% formic acid aqueous solution (solvent B), the flow rate was 1 mL / min, and the column temperature was 30° C. The mobile phase example for gradient elution is as follows:

[0067] From 0 to 10 min, the volume ratio of mobile phase A to mobile phase B changed from 2:98 to 15:85;

[0068] From 10 to 25 min, the volume ratio of mobile phase A to mobile phase B changed from 15:85 to 45:55;

[0069] From 25 to 45 min, the volume ratio of mobile phase A to mobile phase B changed from 45:55 to 50:50;

[0070] From 45 to 75 min, the volume ratio of mobile phase A to mobile phase B changed from 50:50 to 80:20;

[0071] From 75 to 96 min, the volume ratio of mobile phase A to mobile phase B changed from 80:20 to 100:0.

[0072] The injection volume was 10 μL, and the detection wavelength was 280 nm. Fucoidan was used as the index component of the kelp extract.

[0073] 2. Experimental Results

[0074] like Figure 1 As shown, under the analytical conditions of Example 1, the chemical components in the 50% ethanol extract of Laminaria japonica are separated, which can be used as a quality control method for Laminaria japonica medicinal materials. The content of fucoidan in the 50% ethanol extract of Laminaria japonica in Example 1 is 16.8 mg / g.

[0075] Example 2 Experiment on inhibition of proliferation of human umbilical vein endothelial cells (HUVEC) by Laminaria japonica extract

[0076] The MTT colorimetric method was used to observe the inhibitory effect of the Laminaria japonica extract of Example 1 at different administration concentrations on the proliferation of HUVEC.

[0077] 1. Experimental Methods

[0078] The test drugs were 10 ng / mL vascular endothelial growth factor (VEGF) diluted with Lonza medium for culturing HUVEC cells and the kelp extract of Example 1 at different concentrations (10, 30 and 100 μg / mL).

[0079] HUVEC cells were grown at 5.0×10 3 The cells were inoculated at a density of 100 μL / mL in a 96-well culture plate and cultured, with 100 μL per well. After the cells grew to 80% fusion, 10 ng / mL VEGF was added to each well, with 100 μL per well. Then, different concentrations of the kelp extract of Example 1 were added to the wells, with 100 μL per well. After 48 h of culture, 10 μL of MTT solution was added to each well. The culture was terminated after continuing to culture at 37°C for 4 h. The culture supernatant was discarded, and 150 μL of DMSO was added to each well. The DMSO was fully dissolved by shaking for 10 min. The wavelength of 490 nm was selected, and the absorbance (OD) value of each well was measured on an ELISA instrument. The wells without VEGF and the kelp extract of Example 1 were used as controls, and the cell viability was calculated according to the following formula:

[0080] Cell viability (%) = (OD 样品 -OD对照品 ) / OD 对照品 ×100%

[0081] 2. Experimental Results

[0082] like Figure 2 As shown, the inhibitory effect of the kelp extract of Example 1 on the proliferation of HUVEC is dose-dependent.

[0083] Example 3 Kelp extract inhibits human umbilical vein endothelial cell (HUVEC) scratch migration experiment

[0084] 1. Experimental Methods

[0085] The test drugs were 10 ng / mL human vascular endothelial growth factor (VEGF) diluted with Lonza medium, 200 μg / mL of Carbendazim, and different concentrations (10, 30 and 100 μg / mL) of the kelp extract of Example 1.

[0086] HUVEC cells were grown at 20×10 4 Cells were inoculated at a density of 100 μg / well in a 12-well plate and cultured overnight (24 h) to allow the cells to adhere to the wall. After the cells grew to 80% fusion, a 200 μL pipette tip was used to scratch a horizontal wound in the middle of each well. The culture medium was discarded and the cells were washed once with PBS. 10 ng / mL of VEGF was added to each well, 200 μL per well, and then 200 μg / mL of Carbendazim and different concentrations of the kelp extract of Example 1 were added, 200 μL per well. A well without VEGF, Carbendazim and the kelp extract of Example 1 was set as a blank experiment. The changes in the coverage of the cell layer in each well were photographed at 0 and 8 h after 50 times magnification using a microscope equipped with a camera. The recovery growth rate was digitized using TScratch software, and the cell recovery growth rate was calculated according to the following formula:

[0087] Recovery rate % = (At0-At8) / At0×100%,

[0088] Wherein, At0: scratch area measured 0 h after drug administration; At8: scratch area measured 8 h after drug administration.

[0089] 2. Experimental Results

[0090] like Figure 3 As shown in A, the pictures represent scratch pictures of HUVEC cells before and after drug administration; Figure 3 As shown in B, the bar graph represents the quantification of the scratch area of ​​HUVEC cells in each group. Figure 3 This indicates that the Laminaria japonica extract of Example 1 has a dose-dependent inhibitory effect on the scratch migration of HUVEC.

[0091] Example 4 Experiment on inhibition of vertical migration of human umbilical vein endothelial cells (HUVEC) in Transwell chamber by Laminaria japonica extract

[0092] Transwell method is used to detect HUVEC migration. Transwell is an experimental technique. The main material of this technique is the Transwell chamber. The bottom layer of the chamber has a permeable membrane (usually a polycarbonate membrane) with micropores of 0.1 to 12.0 μm. In the experiment, cells are placed on the chamber. Under the action of chemokines or growth factors contained under the chamber, the cells pass through the polycarbonate membrane, thereby conducting co-culture, cell chemotaxis, cell migration, cell invasion and other research.

[0093] 1. Experimental Methods

[0094] The test drugs were 10 ng / mL human vascular endothelial growth factor (VEGF) diluted with HUVEC cell suspension, 200 μg / mL of Carbendazim and different concentrations (10, 30 and 100 ng / mL) of Laminaria japonica extract.

[0095] 500 μL HUVEC culture medium was added to the lower chamber, and 10 ng / mL human vascular endothelial growth factor (VEGF) was added to the upper chamber, and 10 ng / mL VEGF, 200 μg / mL of Acetin, and HUVEC cell suspensions (20×10 4 Cells / well), 200 μL per well, placed in a 37°C incubator for 24 hours, with the group without VEGF, carboplatin and kelp extract as the control. After 24 hours, the culture plate was taken out of the incubator, the culture medium in the wells was discarded, 500 μL of 100% methanol solution was added to each well and fixed at room temperature for 20 minutes, the unmigrated cells in the upper chamber were gently wiped with a cotton swab, and then 200 μL of 0.01% crystal violet was added for staining, and finally the excess crystal violet dye was washed away with PBS solution, and the cells were observed and photographed under an inverted microscope.

[0096] 2. Experimental Results

[0097] The migrated cells appear purple-red after being stained with crystal violet. Figure 4 As shown in A, the picture represents the number of HUVEC cells passing through the chamber before and after drug administration; Figure 4 As shown in B, the bar graph represents the quantification of the number of HUVEC cells that passed through the chamber in each group. Figure 4 As shown, the inhibitory effect of Laminaria japonica extract on the vertical migration of HUVEC in Transwell chamber was dose-dependent.

[0098] Example 5 Experiment on inhibition of tubulogenesis of human umbilical vein endothelial cells (HUVEC) by Laminaria japonica extract

[0099] Matrigel was used to detect the tubule formation of HUVEC. Matrigel is a basement membrane component extracted from mouse EHS sarcoma. It can spontaneously form a gel-like artificial basement membrane at 37°C and has the biological effects of a natural basement membrane. Human umbilical vein endothelial cells can adhere to Matrigel to form tubes, which can be used to study the effects of drugs on endothelial cell tubule formation.

[0100] 1. Experimental Methods

[0101] The test drugs were 10 ng / mL human vascular endothelial growth factor (VEGF) diluted with HUVEC cell suspension, 200 μg / mL of Carbendazim, and different concentrations (10, 30 and 100 μg / mL) of the kelp extract of Example 1.

[0102] 200 μL of Matrigel gel solution was added to a pre-cooled 12-well culture plate, and then placed in a 37°C incubator for 1 h to solidify the gel. 10 ng / mL VEGF, 200 μg / mL of Carbothionin and different concentrations of the kelp extract of Example 1 were added to HUVEC (20×10 4 The cells / well) were added to each well of a 12-well plate covered with Matrigel, and cultured at 37°C for 8 h. The group without VEGF, carbendazim and kelp extract was used as the control. After 8 h, the angiogenesis was observed under an inverted optical microscope.

[0103] 2. Experimental Results

[0104] Human umbilical vein endothelial cells (HUVEC) extend and grow in tubular form on Matrigel and connect with each other to form a three-dimensional network structure. Figure 5 As shown in A, the picture represents the number of tubules formed by HUVEC cells before and after drug administration; Figure 5 As shown in B, the bar graph represents the quantification of the number of tubules formed by HUVEC cells in each group. Figure 5 As shown, the inhibitory effect of Laminaria japonica extract on HUVEC tubule formation was dose-dependent.

[0105] Example 6 Experiment on inhibition of zebrafish subintestinal vein (SIV) formation by kelp extract

[0106] The subintestinal vessel (SIV) of zebrafish grows on both sides of the yolk sac and is shaped like a basket. The length of the SIV extending downward from the ventral side of the body segment is about 50 to 100 μm.

[0107] 1. Experimental Methods

[0108] The test drugs were 10 ng / mL human vascular endothelial growth factor (VEGF) diluted with Lonza medium, 200 μg / mL of Carbendazim, and different concentrations (10, 30 and 100 μg / mL) of the kelp extract of Example 1.

[0109] Experimental grouping and embryo processing: 70 well-developed zebrafish embryos were randomly divided into 7 groups, with 10 embryos in each group. During the operation, the embryos were evenly distributed into 12-well cell culture plates, with 10 embryos in each well and 1 mL of water for embryo rearing in each well.

[0110] Drug treatment: Use a pipette (range 100-1000 μL, Eppendorf) to quickly add the pre-prepared 10 ng / mL VEGF to each well of the 12-well cell culture plate, 1 mL per well, and then add 200 μg / mL of carbothion and different concentrations of the kelp extract of Example 1, 1 mL per well. The wells without VEGF, carbothion and kelp extract are used as blank experimental groups. Before adding the drug solution, use a pipette (range 100-1000 μL, Eppendorf) to remove the feeding water for incubating embryos in the 12-well cell culture plate as much as possible. This operation needs to be completed in advance within a short time to prevent the embryos from drying. The experimental environment temperature is controlled at about 28.5°C and the relative humidity is 40-70%. Wrap the 12-well plate with the number, mark the experiment, and quickly place it in the zebrafish incubator to continue culturing for 48 hours (the incubator temperature is controlled at 28.5°C). After 48 hours, zebrafish embryos were fixed with 4% paraformaldehyde at 4°C for 20 hours, and then washed with PBST (0.1% Tween), 50% methanol and 100% methanol for 5 minutes each time. The zebrafish embryos were then immersed in methanol and dehydrated at -20°C for 2 hours. After dehydration, excess methanol was washed away with PBST (0.1% Tween), and zebrafish embryos were stained with nitro-blue tetrazolium / 5-bromo-4-chloro-3-indol-yl-phosphate (NBT / BCIP), and angiogenesis was observed under an inverted optical microscope.

[0111] 2. Experimental Results

[0112] like Figure 6 As shown in A, the picture represents the area of ​​the zebrafish intestinal vein formed before and after drug administration; Figure 6 As shown in B, the bar graph represents the quantification of the area of ​​the inferior intestinal vein of each group of zebrafish. Figure 6 As shown, the inhibitory effect of Laminaria japonica extract on the formation of blood vessels in the intestinal vein of zebrafish is dose-dependent. Example 7 Laminaria japonica extract specifically blocks the signaling pathway mediated by vascular endothelial growth factor receptor 2 (VEGFR2) to exert its anti-angiogenic effect

[0113] 1. Experimental Methods

[0114] The test drugs were 10 ng / mL human vascular endothelial growth factor (VEGF) diluted with HUVEC cell suspension, 200 μg / mL of Carbendazim, and different concentrations (10, 30 and 100 μg / mL) of the kelp extract of Example 1.

[0115] Western-blot experiments were used to evaluate the effect of kelp extract on VEGF-dependent angiogenesis. HUVEC cells were seeded in 12-well plates (20×10 4 Cells / well) were placed in a 37°C incubator and incubated for 24 hours. After starvation culture with serum-free medium for 1 hour, 10 ng / mL of VEGF was added to each well, and then 200 μg / mL of Anticancer and different concentrations of the kelp extract of Example 1 were added, 200 μL per well. The group without VEGF, Anticancer and kelp extract was used as a blank control. HUVEC cells were lysed using Sigma-Aldrich low-concentration lysis buffer, and the cell lysate was collected and loaded on 8% SDS-PAGE gel.

[0116] 2. Experimental Results

[0117] like Figure 7 As shown in A, the picture represents the expression of phosphorylated VEGFR2 protein in HUVEC cells before and after drug administration; Figure 7 As shown in B, the bar graph represents the quantification of the phosphorylated VEGFR2 protein expression in each group. Figure 7 As shown, the kelp extract inhibited the expression level of phosphorylated VEGFR2 in a dose-dependent manner.

[0118] like Figure 8 As shown in A, the picture represents the expression of phosphorylated PI3K and Akt proteins in HUVEC cells before and after administration; Figure 8 As shown in B, the bar graph represents the quantification of the phosphorylated PI3K and Akt protein expression levels in each group. Figure 8As shown, kelp extract inhibited the expression levels of PI3K and Akt downstream of phosphorylated VEGFR2 in a dose-dependent manner.

[0119] Example 8 Comparison of Laminaria japonica extract and Dunaliella polysaccharide in inhibiting the proliferation of human umbilical vein endothelial cells (HUVEC)

[0120] 1. Experimental Methods

[0121] The experimental method was the same as that of Example 2, wherein the test drugs were human vascular endothelial growth factor (VEGF), the kelp extract of Example 1, and fucoidan, and the concentrations were all 10 ng / mL.

[0122] 2. Experimental Results

[0123] like Fig. 9 As shown, both kelp extract and salt algae polysaccharide have an inhibitory effect on HUVEC proliferation, and the efficacy of kelp extract is better than that of fucoidan, with a significant difference.

[0124] Example 9 Experiment on inhibition of zebrafish subintestinal vein (SIV) formation by kelp extract

[0125] 1. Experimental Methods

[0126] Experimental grouping and embryo processing: 50 well-developed zebrafish embryos were randomly divided into 5 groups, with 10 embryos in each group. During the operation, the embryos were evenly distributed into 12-well cell culture plates, with 10 embryos in each well and 1 mL of water for embryo rearing in each well.

[0127] The drug treatment experiment was the same as that in Example 6, wherein the test drugs were 10 ng / mL of human vascular endothelial growth factor (VEGF), 200 μg / mL of Carbendazim, 100 μg / mL of kelp extract, and 100 μg / mL of fucoidan.

[0128] 2. Experimental Results

[0129] like Fig.10 As shown in A, the picture represents the area of ​​the zebrafish intestinal vein formed before and after drug administration; Fig.10 As shown in B, the bar graph represents the quantification of the area of ​​the inferior intestinal vein of each group of zebrafish. Fig.10 As shown, both kelp extract and fucoidan have an inhibitory effect on the angiogenesis of the inferior intestinal vein of zebrafish, and the pharmacological effect of kelp extract is better than that of fucoidan, with a significant difference.

[0130] Example 10 Preparation of Kelp Extract

[0131] 1. Experimental Methods

[0132] The kelp extract of Example 1 is mixed with suitable excipients and can be made into different dosage forms such as capsules, tablets, oral preparations, microcapsule preparations, injections, suppositories, sprays or ointments according to different processes, as a pharmaceutical preparation for the treatment and prevention of neovascularization-dependent and neovascularization-related diseases such as tumors, arthritis, psoriasis, ophthalmic diseases, and atherosclerosis. The preparation methods of each dosage form are commonly used in the prior art and will not be repeated here.

[0133] Comparative Example 1 Kelp Extracts Prepared by Other Extractants

[0134] 1. Prepare kelp extract using water as an extractant: Crush the kelp medicinal material, weigh 50 g, put it into a volumetric flask, soak it in 1000 mL of water overnight (24 h), ultrasonically extract it for 1 h, repeat the water soaking and ultrasonic extraction twice, combine the supernatants of the two extractions, take the supernatant and concentrate it to 30 mL at 60°C, and then vacuum freeze-dry it to obtain the kelp water extract freeze-dried powder, which is stored in a vacuum.

[0135] 2. Prepare kelp extract using 95% ethanol by volume as an extractant: Crush the kelp medicinal material, weigh 50 g, put it into a volumetric flask, soak it in 1000 mL of 95% ethanol by volume overnight, ultrasonically extract it for 1 hour, repeat the 95% ethanol soaking and ultrasonic extraction twice, combine the supernatants of the two extractions, vacuum concentrate to 30 mL at 60°C, and then vacuum freeze-dry to obtain the kelp alcohol extract freeze-dried powder, which is stored in a vacuum.

[0136] However, the Laminaria japonica extracts prepared by the above two methods are not as effective as that of Example 1 in inhibiting angiogenesis.

[0137] 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 the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. Use of kelp extract in the preparation of a medicament for treating arthritis, psoriasis or atherosclerosis by inhibiting angiogenesis, characterized in that: The preparation method of the kelp extract comprises: soaking the kelp in a 50% ethanol solution to obtain an extract; concentrating the extract to obtain the kelp extract; The ratio of the 50% ethanol solution to the kelp is 20 mL:1 g, and the volume ratio of the kelp extract to the ethanol is 3:100; The active ingredients of the kelp extract include fucoidan.

2. The use according to claim 1, characterized in that: The drug is a drug for inhibiting the proliferation of vascular endothelial cells.

3. The use according to claim 1, characterized in that: The drug is a drug for inhibiting the migration of vascular endothelial cells.

4. The use according to claim 1, characterized in that: The drug is a drug for inhibiting the formation of vascular endothelial cell tubules.

5. The use according to claim 1, characterized in that: The drug is a drug that blocks the signal pathway mediated by vascular endothelial growth factor receptor.

6. The use according to claim 5, characterized in that: The drug is a drug that inhibits the expression of vascular endothelial growth factor receptor.

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