Application of pelargonidin-3-glucoside in preparation of product for improving vascular endothelial dysfunction

Abstract

The invention provides application of pelargonidin-3-glucoside in preparation of a product for improving vascular endothelial dysfunction, and belongs to the technical field of medical drugs. The invention provides an application of pelargonidin-3-glucoside in preparation of a product for improving vascular endothelial dysfunction. The pelargonidin-3-glucoside can effectively inhibit the active oxygen level of endothelial cells, reduce the expression of adhesion factors and recover the content of nitric oxide, and has an obvious improvement effect on the vasodilation function. According to the application, the effect of pelargonidin-3-glucoside in improving the vascular endothelial dysfunction is determined, and new choices and guiding significance can be provided for products (such as health care products or medicines) for preventing and improving the vascular endothelial dysfunction.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical drugs, and particularly relates to the application of pelargonidin-3-glucoside in the preparation of products for improving vascular endothelial dysfunction. Background Art

[0002] In the obese state, adipose tissue not only has abnormal functions, but also releases a large number of inflammatory factors, inducing a systemic chronic inflammatory response. These inflammatory factors act on vascular endothelial cells through blood circulation, directly damaging the functions of vascular endothelial cells. At the same time, insulin resistance caused by obesity will lead to a decrease in the production of nitric oxide in vascular endothelial cells. As an important factor maintaining vascular dilation, the decrease in its production will directly lead to impaired vascular dilation function. In addition, the increase in blood glucose level and lipid metabolism disorder further exacerbate the oxidative stress response of vascular endothelial cells, resulting in vascular endothelial cell damage, reducing the bioavailability of nitric oxide, and weakening the endothelium-dependent vascular dilation function. Finally, these mechanisms interact with each other, leading to vascular endothelial dysfunction, and further increasing the risk of cardiovascular diseases.

[0003] The intervention effects of obesity-related vascular endothelial dysfunction vary depending on the methods and mechanisms. Although bariatric surgery can significantly improve vascular endothelial function in the short term, its long-term efficacy is limited. Although obesity treatment drugs (such as orlistat, phentermine-topiramate, and bupropion-naltrexone, etc.) have weight management effects, their improvement mechanisms on endothelial function have not been fully evaluated, and their clinical prescription rates are low, with insufficient patient compliance and persistence. Exercise intervention can effectively improve endothelial function by reducing body weight, enhancing nitric oxide synthase activity, and anti-inflammatory mechanisms. However, the effects are affected by factors such as individual differences, exercise intensity, and intervention time, making it difficult to ensure universality. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide the application of pelargonidin-3-glucoside in the preparation of products for improving vascular endothelial dysfunction.

[0005] The present invention provides the application of pelargonidin-3-glucoside in the preparation of products for improving vascular endothelial dysfunction.

[0006] Preferably, the vascular endothelial dysfunction is obesity-related vascular endothelial dysfunction.

[0007] Preferably, the vascular endothelial dysfunction includes vascular endothelial dilation dysfunction.

[0008] Preferably, the vascular endothelial dysfunction includes thoracic aortic vascular endothelial dilation dysfunction.

[0009] Preferably, the vascular endothelial dysfunction is induced by palmitic acid.

[0010] Preferably, the improvement of vascular endothelial dysfunction is manifested in at least one of the following aspects: restoring the diastolic function of the thoracic aorta, restoring the nitric oxide content, restoring the activity of endothelial nitric oxide synthase, and inhibiting the generation of reactive oxygen species.

[0011] Preferably, the product includes drugs and / or health products.

[0012] Preferably, the product further includes excipients;

[0013] The excipients include diluents, binders, disintegrants, lubricants, absorbents, colorants, and coating materials.

[0014] Preferably, the dosage form of the product includes at least one of the following: tablets, powders, capsules, oral liquids, and granules.

[0015] Preferably, the mass percentage content of pelargonidin-3-glucoside in the drug is 10% - 90%.

[0016] The present invention provides the application of pelargonidin-3-glucoside in the preparation of a product for improving vascular endothelial dysfunction. Experiments at the animal level and cell level in this application demonstrate that pelargonidin-3-glucoside can inhibit the generation of reactive oxygen species in endothelial cells induced by obesity, restore the activity of endothelial nitric oxide synthase and the generation of nitric oxide in vascular endothelium, and has a significant improvement effect on the diastolic function of the thoracic aorta. This application determines the improvement effect of pelargonidin-3-glucoside on vascular endothelial dysfunction, providing new options and guiding significance for the prevention and treatment of vascular endothelial dysfunction. Description of the Drawings

[0017] Figure 1 Results of the effect of pelargonidin-3-glucoside on the diastolic function of the thoracic aorta of mice treated with a high-fat diet;

[0018] Figure 2 Results of the effect of pelargonidin-3-glucoside on the cellular reactive oxygen species of HUVECs treated with PA;

[0019] Figure 3 Results of the effect of pelargonidin-3-glucoside on the cellular endothelial nitric oxide synthase activity of HUVECs treated with PA;

[0020] Figure 4 Results of the effect of pelargonidin-3-glucoside on the cellular nitric oxide content of HUVECs treated with PA. Detailed Embodiments

[0021] The present invention provides the application of pelargonidin-3-glucoside in the preparation of a product for improving vascular endothelial dysfunction.

[0022] In the present invention, pelargonidin-3-glucoside is a kind of anthocyanin, with the molecular formula C 21 H 21 ClO 10 , with a molecular weight of 468.8384, and its structural formula is shown in Formula I; in the embodiments of the present invention, the pelargonidin-3-glucoside is purchased from Sigma-Aldrich.

[0023]

[0024] In the present invention, the vascular endothelial dysfunction is preferably obesity-related vascular endothelial dysfunction. The vascular endothelial dysfunction is preferably induced by palmitic acid. The vascular endothelial dysfunction preferably includes vascular endothelial relaxation dysfunction. The vascular endothelial dysfunction preferably includes thoracic aortic vascular endothelial relaxation dysfunction. The improvement of vascular endothelial dysfunction preferably shows in at least one of the following aspects: restoring the thoracic aortic relaxation function, restoring the nitric oxide content, restoring the endothelial nitric oxide synthase activity, and inhibiting the generation of reactive oxygen species. It can be seen that the pelargonidin-3-glucoside improves vascular endothelial dysfunction by anti-vascular endothelial cell oxidative stress and inflammatory effects, and the pelargonidin-3-glucoside provides a new pharmaceutical approach for the treatment of a series of cardiovascular-related diseases caused by vascular endothelial dysfunction.

[0025] In the embodiments of the present invention, the effects of pelargonidin-3-glucoside on improving vascular endothelial dysfunction are verified at the animal level and the cell level respectively. In the verification experiment at the animal level, a mouse obesity model was constructed by feeding a high-fat diet. After detection, the relaxation response of the thoracic aorta of the model group mice to acetylcholine was significantly lower than that of the control group, indicating that the model group mice had vascular endothelial relaxation dysfunction. After administration of pelargonidin-3-glucoside (150 mg / kg), the vascular relaxation response of the mice was significantly higher than that of the model group, which indicates that pelargonidin-3-glucoside can improve the vascular relaxation function of the thoracic aorta of obese mice. In the cell level experiment verification, using PA-induced human umbilical vein endothelial cells as the object, the pharmacodynamic effects of different concentrations of pelargonidin-3-glucoside were verified respectively. The results showed that PA treatment significantly increased the fluorescence intensity of reactive oxygen species in the cells, while pelargonidin-3-glucoside treatment significantly decreased the fluorescence intensity of reactive oxygen species in the cells, and showed a dose-dependent manner. In addition, compared with the control group, PA treatment significantly inhibited the activity of endothelial nitric oxide synthase, and the treatment with 50 μM and 100 μM of pelargonidin-3-glucoside significantly restored the activity of endothelial nitric oxide synthase. Moreover, PA treatment significantly decreased the relative content of NO in the cells, while 20 μM, 50 μM and 100 μM of pelargonidin-3-glucoside were able to restore the content of NO.

[0026] In the present invention, the product preferably includes drugs and / or health products. The product preferably further includes excipients. The excipients preferably include diluents, binders, disintegrants, lubricants, absorbents, colorants, and coating materials. The dosage form of the product preferably includes at least one of the following: tablets, powders, capsules, oral liquids, and granules. The excipients of the drug are selected from at least one of the following: mannitol, sorbitol, sodium metabisulfite, sodium bisulfite, sodium thiosulfate, cysteine hydrochloride, mercaptoacetic acid, methionine, vitamin C, disodium EDTA, calcium disodium EDTA, carbonates, acetates, phosphates of monovalent alkali metals or their aqueous solutions, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acids, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivatives, cellulose and its derivatives, alginates, gelatin, polyvinylpyrrolidone, glycerol, polysorbate 80, agar, calcium carbonate, calcium bicarbonate, surfactants, polyethylene glycol, cyclodextrin, β-cyclodextrin, phospholipid materials, kaolin, talc, calcium stearate, magnesium stearate, etc. The mass percentage content of pelargonidin-3-glucoside in the drug is preferably 10% to 90%, can be 20% to 80%, can also be 40% to 70%, and can further be 50% to 60%.

[0027] In the present invention, the administration method of the drug is oral administration. The dosage is administered once a day according to the dosage of pelargonidin-3-glucoside at 12.16 mg / kg.

[0028] The following combines examples to elaborate in detail on the application of pelargonidin-3-glucoside provided by the present invention in the preparation of products for improving vascular endothelial dysfunction, but they cannot be construed as limiting the protection scope of the present invention.

[0029] Example 1

[0030] Pharmacodynamic verification of pelargonidin-3-glucoside in improving vascular endothelial dysfunction

[0031] 1. Experimental content

[0032] In vivo, C57BL / 6J mice were fed a high-fat diet to establish a mouse obesity model, and the effect of pelargonidin-3-glucoside on improving the diastolic function of the thoracic aorta was explored by detecting the diastolic amplitude of the thoracic aorta; human umbilical vein endothelial cells (HUVECs) were cultured in vitro, and a cell model of vascular endothelial dysfunction was established using palmitic acid (PA). After adding pelargonidin-3-glucoside, the content of reactive oxygen species in endothelial cells was detected by fluorescence microscopy, the activity of endothelial nitric oxide synthase was detected by the DAF-FMDA probe method, and the content of nitric oxide was detected by the Griess reagent method to explore the effect of pelargonidin-3-glucoside (Pg3G) on improving vascular endothelial dysfunction.

[0033] 2. Experimental methods

[0034] 2.1 In vivo experiments

[0035] 2.1.1 Establishment of animal model: Six-week-old male C57BL / 6 mice were selected. After 1 week of adaptive feeding, they were fed with 60% high-fat diet for 12 weeks to induce vascular endothelial dysfunction. After establishing the obesity model, the mice were randomly divided into a control group, a high-fat group, a low-dose Pg3G group (50 mg / kg), and a high-dose Pg3G group (150 mg / kg), with 10 mice in each group. Two days before the intervention, the first Pg3G gavage was performed, and then the corresponding dose of Pg3G was given by gavage every morning. The intervention period lasted for 8 weeks.

[0036] 2.1.2 Vascular function detection: After sacrificing the mice, the thoracic aorta of the mice was isolated. The removed aorta was placed in pre-cooled PSS solution, and the fat and connective tissue on the blood vessel surface were removed with dissecting forceps. Subsequently, the tissue was cut into 2-mm lengths. Then, 5 mL of room-temperature PSS was filled in each chamber of the DMT 620M Myograph. A 40-μm wire was passed through the blood vessel and fixed to the chamber. The chamber was placed on the interface, ventilation was turned on, and it was heated to 37 °C and balanced for 20 min. After stretching the blood vessel to a certain initial tension, the tension was further adjusted to reach the tension when the blood vessel in vivo withstood 100 mmHg. After replacing with 5 mL of room-temperature PSS, it was balanced for 20 min, and this operation was repeated 3 times. Subsequently, 5 mL of 60 mM KPSS was added and stimulated for 5 min. Then, it was eluted with PSS to the baseline to restore the vascular tension to the initial tension. Then, acetylcholine (ACh) with gradient concentrations of 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 M, 10 -5 M, 10 -4 M was added to relax for 2 min.

[0037] 2.2 In vitro experiments

[0038] 2.2.1 Construction and grouping of human umbilical vein endothelial cell model: Human umbilical vein endothelial cells were divided into a control group, a model group, and a model drug administration group. The cells in the control group were not treated with anything. The cells in the model group were added with 250 μM of palmitic acid. The cells in the model drug administration group were first incubated with 250 μM of PA for 24 h and then added with 20 μM, 50 μM, and 100 μM of pelargonidin-3-glucoside respectively. Then, the above groups were cultured with DMEM complete medium containing 10% fetal bovine serum by volume and placed in an incubator at 37 °C and 5% CO 2 constant temperature incubator.

[0039] 2.2.2 Detection of the content of reactive oxygen species in endothelial cells by fluorescence microscopy: After the cells were treated accordingly, they were washed three times with PBS. Subsequently, the DHE probe was diluted with DMEM colorless medium at a ratio of 1000:1. An appropriate amount of DHE-containing medium was added to each well, and the cells were incubated at 37 °C in the dark for 30 min. After the incubation, the cells were washed three times with PBS to remove the residual probe. An appropriate amount of colorless DMEM medium was added, and under the fluorescence microscope, the cells were excited with red light at an excitation wavelength of 485 nm and an emission wavelength of 610 nm, and the cells were observed and photographed.

[0040] 2.2.3 Detection of the activity of endothelial nitric oxide synthase by DAF-FMDA probe method: The DAF-FMDA probe and the eNOS inhibitor (L-NAME) were used to calculate the activity of eNOS. First, an appropriate amount of 2×NOS buffer was taken and an equal volume of ultrapure water was added, and they were mixed well to prepare the NOS detection buffer. Then, the detection reaction solution was prepared, which included 50 μL of NOS detection buffer (2×), 39.8 μL of ultrapure water, 5 μL of arginine solution, 5 μL of 0.1 mM NADPH, and 0.2 μL of DAF-FMDA, with a total volume of 100 μL. Then, 100 μM of L-NAME was added to the detection reaction solution to inhibit the activity of eNOS. Subsequently, the medium was aspirated, and 100 μL of NOS detection buffer was added to each well. Next, the reaction solution with or without L-NAME was added to the wells with the same treatment, and the cells were incubated at 37 °C for 60 min. Finally, a fluorescence microplate reader with an excitation wavelength of 495 nm and an emission wavelength of 515 nm was used for detection.

[0041] 2.2.4 Detection of the content of nitric oxide by Griess reagent method: The supernatant of HUVEC cell culture was collected and reacted with the sample in the pre-prepared Griess reagent to form a pink complex. The absorbance of the reactant was measured by a fully automatic microplate reader at a wavelength of 540 nm, and then the content of NO in each sample was calculated through the standard curve.

[0042] 3. Experimental results

[0043] 1.1 Experimental results of animal models

[0044] The results are shown in Figure 1 . The relaxation response of the thoracic aorta of the mice in the model group to ACh was significantly lower than that of the control group, while after treatment with 150 mg / kg of pelargonidin-3-glucoside, the vascular relaxation response of the mice was significantly higher than that of the model group. The results indicate that pelargonidin-3-glucoside can improve the relaxation function of the thoracic aorta in obese mice.

[0045] 1.2 Experimental results of in vitro cells

[0046] The results of the relative fluorescence intensity of reactive oxygen species are shown in Figure 2 . Compared with the control group, PA treatment significantly increased the fluorescence intensity of reactive oxygen species in HUVECs, while pelargonidin-3-glucoside treatment significantly decreased the fluorescence intensity of reactive oxygen species in HUVECs, and showed a dose-dependent manner.

[0047] The results of the activity assay of nitric oxide synthase are shown in Figure 3 . Compared with the control group, PA treatment significantly inhibited the activity of endothelial nitric oxide synthase, and the treatment with 50 μM and 100 μM of pelargonidin-3-glucoside significantly restored the activity of endothelial nitric oxide synthase.

[0048] The results of the nitric oxide content assay are shown in Figure 4 . PA treatment significantly decreased the relative content of NO in HUVECs, while 20 μM, 50 μM and 100 μM of pelargonidin-3-glucoside could restore the content of NO. The results indicate that pelargonidin-3-glucoside can improve PA-induced vascular endothelial dysfunction.

[0049] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Application of pelargonidin-3-glucoside in the preparation of products for improving vascular endothelial dysfunction.

2. The application according to claim 1, characterized in that: The vascular endothelial dysfunction is obesity-related vascular endothelial dysfunction.

3. The application according to claim 1, characterized in that: The vascular endothelial dysfunction includes vascular endothelial relaxation dysfunction.

4. The use according to claim 1, characterized in that: The vascular endothelial dysfunction includes thoracic aorta endothelial vasodilation dysfunction.

5. The use according to claim 1, characterized in that: The endothelial dysfunction is induced by palmitate.

6. The use according to any one of claims 1 to 5, characterized in that: The improvement of vascular endothelial dysfunction is manifested in at least one of the following aspects: restoring the thoracic aorta diastolic function, restoring the nitric oxide content, restoring the activity of endothelial nitric oxide synthase and inhibiting the generation of reactive oxygen species.

7. The use according to claim 1, characterized in that: The products include medicines and / or health products.

8. The use according to claim 7, characterized in that: The product also includes excipients; The auxiliary materials include diluents, binders, disintegrants, lubricants, absorbents, colorants and coating materials.

9. The use according to claim 7, characterized in that: The dosage form of the product includes at least one of the following: tablets, powders, capsules, oral liquids and granules.

10. The use according to claim 7, characterized in that: The mass percentage of pelargonidin-3-glucoside in the medicine is 10% to 90%.

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