A composition having biological activity and its use in cardiovascular protection products

Abstract

The present application provides a kind of composition with biological activity and its application in cardiovascular protection product, belong to the technical field of nutritional composition.The composition of the present application is composed of 4-18 parts of hydroxytyrosol, 2-120 parts of salidroside and 180-2000 parts of EPA by weight.The composition of the present application can significantly inhibit vascular endothelial inflammation, reduce the secretion of inflammatory factor IL-6, down-regulate the expression of pro-adhesion molecule ICAM-1, up-regulate the expression of vascular protection factor eNOS, and effectively improve vascular endothelial dysfunction;It can also significantly reduce the total cholesterol and low-density lipoprotein cholesterol level of the body, regulate blood lipid metabolism and reduce vascular lipid deposition;It can also regulate blood pressure smoothly.The composition of the present application has the triple core functions of anti-inflammatory endothelial protection, blood lipid regulation and blood pressure stabilization, and can achieve comprehensive cardiovascular protection.The formula is simple, and the preparation is convenient, which has wide application prospect in the field of cardiovascular health intervention food, health products and drugs.

Description

Technical Field

[0001] This invention belongs to the field of nutritional composition technology, specifically relating to a bioactive composition and its application in cardiovascular protection products. Background Technology

[0002] Cardiovascular disease is the leading cause of death worldwide. With the increasing prevalence of high-fat diets, irregular lifestyles, and an aging population, the number of people in my country suffering from vascular sub-health, hypertension, and hyperlipidemia is rising annually, placing increasing pressure on the prevention and control of chronic cardiovascular diseases. Modern vascular pathology research indicates that endothelial dysfunction, lipid metabolism disorders, and spontaneous hypertension are the three core independent risk factors for cardiovascular disease, forming a vicious cycle: external inflammatory factors and high-pressure blood flow damage the integrity of the vascular endothelium, inducing chronic vascular inflammation, upregulating adhesion factor expression, and reducing vasodilatory capacity; damaged endothelium accelerates the deposition of low-density lipoprotein cholesterol and total cholesterol, inducing atherosclerosis; plaque accumulation and decreased vascular elasticity further increase peripheral vascular resistance, exacerbating hypertension symptoms, and ultimately leading to a series of cardiovascular diseases such as coronary heart disease, myocardial infarction, stroke, and arteriosclerosis. Therefore, developing multi-target active compositions that can simultaneously repair vascular endothelium, regulate blood lipids, and stabilize blood pressure is a core research focus in the field of cardiovascular health nutritional intervention.

[0003] The application of natural active ingredients in vascular protection has been extensively studied. Hydroxytyrosol, a polyphenol derived from olives, possesses the ability to scavenge oxygen free radicals and inhibit vascular inflammation. Rhodioloside, the core functional monomer of Rhodiola rosea, can dilate blood vessels, improve microcirculation, and regulate vascular endothelial function. EPA, a core component of Omega-3 unsaturated fatty acids, can regulate lipid metabolism and reduce lipid deposition in blood vessels. However, individual ingredients target only a single vascular indicator, failing to simultaneously address the triple effects of anti-inflammatory endothelial protection, lipid regulation, and blood pressure stabilization. Furthermore, combining any two ingredients rarely creates a synergistic effect, and an imbalance in the ratio can significantly weaken the activity, failing to achieve comprehensive cardiovascular protection.

[0004] Existing compound vascular protective nutritional compositions are mostly multi-ingredient compound systems, with complex components, high costs, and limited adaptability. Patent document: CN115039880A discloses a composition for improving metabolic aging and inflammatory aging, comprising the following components in parts by weight: 20-40 parts of dihydroquercetin, 8-15 parts of hydroxytyrosol, 20-40 parts of purple tea polyphenols, 1-5 parts of proanthocyanidins, 5-8 parts of eicosapentaenoic acid, 5-8 parts of docosahexaenoic acid, and 5-8 parts of concentrated traditional Chinese medicine powder; the raw materials of the concentrated traditional Chinese medicine powder are selected from at least one of Polygonum multiflorum, ginseng, Panax notoginseng, Cordyceps sinensis, Acanthopanax senticosus, Ganoderma lucidum, Lycium barbarum, Rhodiola rosea, Gynostemma pentaphyllum, winged fruit oil, and Astragalus membranaceus. This composition has many components and an extremely low EPA dosage. Its research and development targets metabolic aging and inflammatory aging. However, its formulation compatibility and target of action are not compatible with the needs of cardiovascular endothelial repair, blood lipid regulation, and blood pressure control. Therefore, it cannot specifically address the triple damage to blood vessels, thus limiting its application in cardiovascular protection.

[0005] Given the shortcomings of existing technologies, there is an urgent need to develop a bioactive composition with simplified components, scientific formulation, and synergistic effects on multiple targets, which can simultaneously improve vascular endothelial damage, regulate blood lipids, and maintain stable blood pressure, thus filling the gap in specialized cardiovascular care formulations. Summary of the Invention

[0006] To address the above shortcomings, the present invention provides a bioactive composition and its application in cardiovascular protection products.

[0007] the term: In this invention, the term hydroxytyrosol is used, with the English name Hydroxytyrosol and the chemical name 2-(3,4-dihydroxyphenyl)ethanol, and the molecular formula C8H12H2O. 10 O3, with a molecular weight of 154.16, is a pale yellow to light brown liquid or white crystalline powder. It is readily soluble in polar solvents such as water, ethanol, and propylene glycol, but sparingly soluble in non-polar solvents. It is one of the core active ingredients in the composition of this invention. This invention does not specifically limit the source of hydroxytyrosol. It can be extracted from natural plant materials such as olive leaves and olive pomace, or obtained through conventional preparation methods in the art, such as chemical synthesis and biotransformation. Regardless of the extraction raw material or preparation process, as long as its chemical structure is consistent with the above definition of hydroxytyrosol, it falls within the scope of hydroxytyrosol referred to in this invention.

[0008] In this invention, the term rhodioloside is used, with the English name salidroside and the molecular formula C. 14 H 20O7, with a molecular weight of 300.3044, is a white crystalline powder. It is readily soluble in water, methanol, and ethanol, slightly soluble in acetone, and sparingly soluble in weakly polar solvents such as petroleum ether and chloroform. It is one of the core active components of the composition of this invention. This invention does not specifically limit the source of rhodioloside. Natural sources include the dried roots and rhizomes of plants in the Rhodiola genus such as Rhodiola grandiflora, Rhodiola alpineensis, and Rhodiola angustifolia; it can also be obtained using conventional preparation processes in the art, such as total chemical synthesis, enzymatic biotransformation, and microbial fermentation synthesis. Regardless of the origin of the raw material, the plant part, or the artificial synthesis route, as long as the chemical structure is consistent with the above definition of rhodioloside, it falls within the scope of rhodioloside referred to in this invention.

[0009] In this invention, the term EPA stands for Eicosapentaenoic acid, and its molecular formula is C2. 20 H 30 O2, with a molecular weight of 302.45, is a pale yellow to orange-yellow oily liquid at room temperature. It is readily soluble in organic solvents such as n-hexane, ethyl acetate, and ethanol, but insoluble in water. It is one of the core active components of the composition of this invention. This invention does not impose any special limitations on the form or source of EPA. EPA can be in the form of free fatty acids, ethyl esters, triglycerides, reconstructed triglycerides, natural triglycerides, or phospholipids. Sources include natural sources (such as marine animals, algae, and marine bacteria) and non-natural sources (such as microbial fermentation and genetically engineered oilseed plants). Regardless of the molecular binding form of EPA, the extraction / fermentation raw materials, or the purification process, as long as the core functional active ingredient is eicosapentaenoic acid with the above-mentioned structure, it falls within the scope of EPA as referred to in this invention.

[0010] The technical solution of this invention is as follows: On one hand, the present invention provides a bioactive composition comprising, by weight, 4-18 parts of hydroxytyrosol, 2-120 parts of rhodioloside and 180-2000 parts of EPA.

[0011] Specifically, the composition contains 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17 or 17-18 parts of hydroxytyrosol by weight.

[0012] Preferably, the composition contains 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14 or 14-15 parts of hydroxytyrosol by weight.

[0013] More preferably, the composition contains 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14 or 14-15 parts of hydroxytyrosol by weight.

[0014] More preferably, the composition contains 13-14 or 14-15 parts by weight of hydroxytyrosol.

[0015] More preferably, the composition contains 15 parts by weight of hydroxytyrosol.

[0016] Specifically, the composition contains, by weight, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, 18-19, 19-20, 20-21, 21-22, 22-23, 23-24, 24-25, 25-26, 26-27, 27-28, 28-29, 29-30, 30-31, 31-32, 32-33, 3 3-34, 34-35, 35-36, 36-37, 37-38, 38-39, 39-40, 40-41, 41-42, 42-43, 43-44, 44-45, 45-46, 46-47, 47-48, 48-49, 49-50, 50-51, 51-52, 52-53, 53-54, 54-55, 55-56, 56-57, 57-58, 58-59, 59-60, 60-61, 61-62, 62-63, 63-64, 64-6 5, 65-66, 66-67, 67-68, 68-69, 69-70, 70-71, 71-72, 72-73, 73-74, 74-75, 75-76, 76-77, 77-78, 78-79, 79-80, 80-81, 81-82, 82-83, 83-84, 84-85, 85-86, 86-87, 87-88, 88-89, 89-90, 90-91, 91-92, 92-93, 93-94, 94-95, 95-96, 9 6-97, 97-98, 98-99, 99-100, 100-101, 101-102, 102-103, 103-104, 104-105, 105-106, 106-107, 107-108, 108-109, 109-110, 110-111, 111-112, 112-113, 113-114, 114-115, 115-116, 116-117, 117-118, 118-119 or 119-120 parts of rhodioloside.

[0017] Preferably, the composition contains, by weight, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, 18-19, 19-20, 20-21, 21-22, 22-23, 23-24, 24-25, 25-26, 26-27, 27-28, 28-29, 29-30, 30-31, 31-32, 32-33, 33-34, 34-35, 35-36, 36-37, 3 7-38, 38-39, 39-40, 40-41, 41-42, 42-43, 43-44, 44-45, 45-46, 46-47, 47-48, 48-49, 49-50, 50-51, 51-52, 52-53, 53-54, 54-55, 55-56, 56-57, 57-58, 58-59, 59-60, 60-61, 61-62, 62-63, 63-64, 64-65, 65-66, 66-67, 67 -68, 68-69, 69-70, 70-71, 71-72, 72-73, 73-74, 74-75, 75-76, 76-77, 77-78, 78-79, 79-80, 80-81, 81-82, 82-83, 83-84, 84-85, 85-86, 86-87, 87-88, 88-89, 89-90, 90-91, 91-92, 92-93, 93-94, 94-95, 95-96, 96-97, 97- 98, 98-99, 99-100, 100-101, 101-102, 102-103, 103-104, 104-105, 105-106, 106-107, 107-108, 108-109, 109-110, 110-111, 111-112, 112-113, 113-114, 114-115, 115-116, 116-117, 117-118, 118-119 or 119-120 parts of rhodioloside.

[0018] More preferably, the composition contains 110-111, 111-112, 112-113, 113-114, 114-115, 115-116, 116-117, 117-118, 118-119 or 119-120 parts of rhodioloside by weight.

[0019] More preferably, the composition contains 120 parts by weight of rhodioloside.

[0020] Specifically, the composition contains, by weight, 180-190, 190-200, 200-210, 210-220, 220-230, 230-240, 240-250, 250-260, 260-270, 270-280, 280-290, 290-300, 300-310, 310-320, 320-330, 330-340, 340-350, 350-360, 360-370, 370-380, 380-390, 390-400, 400-410, 410-420, 420-430, 430-440, 440-450, 450-460, and 460-470. 470-480, 480-490, 490-500, 500-510, 510-520, 520-530, 530-540, 540-550, 550-560, 560-570, 570-580, 580-590, 590-600, 600-610, 610-620, 620 -630, 630-640, 640-650, 650-660, 660-670, 670-680, 680-690, 690-700, 700-710, 710-720, 720-730, 730-740, 740-750, 750-760, 760-770, 770-780 780-790, 790-800, 800-810, 810-820, 820-830, 830-840, 840-850, 850-860, 860-870, 870-880, 880-890, 890-900, 900-910, 910-920, 920-930, 930- 940, 940-950, 950-960, 960-970, 970-980, 980-990, 990-1000, 1000-1010, 1010-1020, 1020-1030, 1030-1040, 1040-1050, 1050-1060, 1060-1070, 1 070-1080, 1080-1090, 1090-1100, 1100-1110, 1110-1120, 1120-1130, 1130-1140, 1140-1150, 1150-1160, 1160-1170, 1170-1180, 1180-1190, 1190- 1200, 1200-1210, 1210-1220, 1220-1230, 1230-1240, 1240-1250, 1250-1260, 1260-1270, 1270-1280, 1280-1290, 1290-1300, 1300-1310, 1310-13201320-1330, 1330-1340, 1340-1350, 1350-1360, 1360-1370, 1370-1380, 1380-1390, 1390-1400, 1400-1410, 1410-1420, 1420-1430, 1430-1440, 1440-1450, 1450-1460, 1460-1470, 1470-1480, 1480-1490, 1 490-1500, 1500-1510, 1510-1520, 1520-1530, 1530-1540, 1540-1550, 1550-1560, 1560-1570, 1570-1580, 1580-1590, 1590-1600, 1600-1610, 1610-1620, 1620-1630, 1630-1640, 1640-1650, 1650-1660, 16 60-1670, 1670-1680, 1680-1690, 1690-1700, 1700-1710, 1710-1720, 1720-1730, 1730-1740, 1740-1750, 1750-1760, 1760-1770, 1770-1780, 1780-1790, 1790-1800, 1800-1810, 1810-1820, 1820-1830, 183 0-1840, 1840-1850, 1850-1860, 1860-1870, 1870-1880, 1880-1890, 1890-1900, 1900-1910, 1910-1920, 1920-1930, 1930-1940, 1940-1950, 1950-1960, 1960-1970, 1970-1980, 1980-1990, or 1990-2000 EPA copies.

[0021] Preferably, the composition contains, by weight, 180-190, 190-200, 200-210, 210-220, 220-230, 230-240, 240-250, 250-260, 260-270, 270-280, 280-290, 290-300, 300-310, 310-320, 320-330, 330-340, 340-350, 350-360, 360-370. 370-380, 380-390, 390-400, 400-410, 410-420, 420-430, 430-440, 440-450, 450-460, 460-470, 470-480, 480-490, 490-500, 500-510, 510-520, 520-530, 530-540, 540-550, 550-560, 560-570, 570-580, 58 0-590, 590-600, 600-610, 610-620, 620-630, 630-640, 640-650, 650-660, 660-670, 670-680, 680-690, 690-700, 700-710, 710-720, 720-730, 730-740, 740-750, 750-760, 760-770, 770-780, 780-790, 790 -800, 800-810, 810-820, 820-830, 830-840, 840-850, 850-860, 860-870, 870-880, 880-890, 890-900, 900-910, 910-920, 920-930, 930-940, 940-950, 950-960, 960-970, 970-980, 980-990, or 990-1000 EPA copies.

[0022] More preferably, the composition contains, by weight, 180-190, 190-200, 200-210, 210-220, 220-230, 230-240, 240-250, 250-260, 260-270, 270-280, 280-290, 290-300, 300-310, 310-320, 320-330, 330-340, 340-350, 350-360, 360-370, 370-380, 380-390, 390-400, 400-410, 410-420, 420-430, 430-440, 440-450, 450-460, 460-470, 470-480, 480-490, or 490-500 EPAs.

[0023] More preferably, the composition contains 180 parts by weight of EPA.

[0024] On the other hand, the present invention provides a method for preparing the composition described in any of the above claims, the method comprising mixing hydroxytyrosol, rhodioloside and EPA uniformly in parts by weight.

[0025] In another aspect, the present invention provides a food or health product comprising any of the compositions described above.

[0026] Specifically, the food or health product also includes any one or more of the following: nutritional additives, food-medicine homologous ingredients, and excipients acceptable in the food or health product field.

[0027] Preferably, the nutritional additives include one or more of dietary fiber, prebiotics, protein, lipids, minerals, and vitamins.

[0028] Preferably, the medicinal and edible ingredients include cloves, star anise, sword bean, fennel, thistle, yam, hawthorn, purslane, black-striped snake, dried plum, papaya, hemp seed, bitter orange flower, Solomon's seal, licorice, angelica, ginkgo, white hyacinth bean, white hyacinth bean flower, longan pulp, cassia seed, lily, nutmeg, cinnamon, amla, bergamot, almond, sea buckthorn, oyster, fox nut, Sichuan pepper, red adzuki bean, donkey-hide gelatin, chicken gizzard lining, malt, kelp, jujube, monk fruit, apricot kernel, honeysuckle, green plum, houttuynia cordata, ginger, Japanese raisin tree fruit, wolfberry, gardenia, amomum villosum, sterculia lychnophora, poria cocos, citron, elsholtzia ciliata, peach kernel, mulberry. Leaves, mulberries, tangerine peel, platycodon root, alpinia oxyphylla, lotus leaves, radish seeds, lotus seeds, galangal, bamboo leaves, fermented soybean, chrysanthemum, chicory, yellow mustard seeds, polygonatum, perilla, perilla seeds, kudzu root, black sesame, black pepper, sophora japonica buds, sophora japonica flowers, dandelion, honey, torreya nuts, jujube seeds, fresh imperata root, fresh reed root, viper, tangerine peel, mint, coix seed, allium macrostemon, raspberry, patchouli, angelica, galangal, saffron, cardamom, turmeric, long pepper, codonopsis, cistanche, dendrobium officinale, American ginseng, astragalus, ganoderma, cornus officinalis, gastrodia elata, eucommia ulmoides leaves, rehmannia glutinosa, ophiopogon japonicus, asparagus cochinchinensis, and tangerine peel.

[0029] Preferably, the excipients include any one or more of fillers, binders, disintegrants, lubricants, emulsifiers, antioxidants, antibacterial agents, isotonic regulators, suspending agents, solubilizers, cosolvents, preservatives, and flavoring agents.

[0030] Specifically, the food includes any one or more of finished food products, semi-finished food products, food additives, and food supplements.

[0031] Preferably, the dosage form of the food or health product includes any one or more of solid dosage forms, semi-solid dosage forms, and liquid dosage forms.

[0032] More preferably, the food includes, but is not limited to, any one or more of the following: candy, soy milk, yogurt, milk, fruit juice, canned food, biscuits, chocolate, pastries, cream, cheese, dairy products, milk powder, formula milk powder, ice cream, jam, fruit puree, dried fruit, bread, egg rolls, protein drinks, solid beverages, lactic acid bacteria drinks, carbonated beverages, coffee, jelly, protein bars, energy bars, energy gels, and puffed foods.

[0033] In another aspect, the present invention provides the use of the composition described in any one of the above claims in the preparation of cardiovascular protection products, wherein the cardiovascular protection products include any one or more of the following: (1) Foods, health products, or medicines that help maintain healthy blood pressure levels; (2) Foods, health products, or medicines that help maintain healthy blood lipid levels; (3) Drugs that help improve vascular endothelial dysfunction.

[0034] In another aspect, the present invention provides a medicine comprising the composition described in any of the preceding claims.

[0035] Specifically, the medicine also includes pharmaceutically acceptable excipients.

[0036] Preferably, the pharmaceutically acceptable excipients are selected from any one or more of the following: fillers, binders, disintegrants, lubricants, emulsifiers, antioxidants, antibacterial agents, isotonic modifiers, suspending agents, solubilizers, cosolvents, preservatives, and flavoring agents.

[0037] Specifically, the dosage form of the drug includes gastrointestinal dosage forms or non-gastrointestinal dosage forms.

[0038] Preferably, the gastrointestinal dosage forms include, but are not limited to, tablets, powders, granules, solutions, capsules, emulsions, suspensions, and oils.

[0039] Preferably, the gastrointestinal dosage forms include, but are not limited to, injectable dosage forms, respiratory dosage forms, skin dosage forms, mucosal dosage forms, and cavity dosage forms.

[0040] In another aspect, the present invention provides a method for protecting the cardiovascular system, the method comprising using any of the compositions, foods, health products or pharmaceuticals described above.

[0041] Specifically, the cardiovascular protection includes any one or more of the following methods: maintaining healthy blood pressure levels, maintaining healthy blood lipid levels, and improving vascular endothelial dysfunction.

[0042] Specifically, the method includes administering an effective amount of the composition, food, health product, or medicine to the subject.

[0043] Preferably, the subject is a mammal.

[0044] More preferably, the subject is a human being.

[0045] The beneficial effects of this invention are as follows: The composition of this invention can simultaneously achieve three effects: the formulation of this invention can significantly reduce the secretion of the endothelial inflammatory factor IL-6, downregulate the expression of the adhesion-promoting molecule ICAM-1, upregulate the expression of eNOS, effectively repair TNF-α-induced vascular endothelial damage, inhibit chronic vascular inflammation, and restore vascular dilation function; at the same time, the composition of this invention can significantly reduce serum total cholesterol and low-density lipoprotein cholesterol levels, improve hyperlipidemia, reduce vascular lipid deposition, and prevent atherosclerosis; blood pressure intervention experiments show that the composition of this invention can significantly reduce systolic and diastolic blood pressure, stably regulate blood pressure health, and fundamentally block the vicious cycle of cardiovascular disease.

[0046] The composition of this invention can be prepared simply by uniform mixing, with a simple process, no complex steps, and low production cost, making it suitable for large-scale industrial production. Furthermore, this composition can be widely used in the food, health product, and pharmaceutical industries. It can be combined with various nutritional additives, food-medicine homologous ingredients, and conventional pharmaceutical and edible excipients. It can be processed into various dosage forms such as solids, semi-solids, and liquids, adapting to diverse market product forms and having a wide range of applications. Attached Figure Description

[0047] Figure 1 The results show the IL-6 secretion levels; in the figure: compared with the control group, There was no significant difference in ns. p<0.05, p<0.001, p<0.0001; Compared with the damage model group, there was no significant difference in #ns, #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001.

[0048] Figure 2 The results show the expression levels of ICAM-1 and eNOS; in the figure: compared with the control group, There was no significant difference in ns. p<0.05, p<0.01, p<0.001, p<0.0001; Compared with the damage model group, there was no significant difference in #ns, ##p<0.01, ###p<0.001, ####p<0.0001.

[0049] Figure 3 The results show the concentrations of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) in serum; in the figure: compared with the control group, There was no significant difference in ns. p<0.01, p<0.0001; compared with the model control group, p<0.0001.

[0050] Figure 4 The results are blood pressure measurements; in the figure: compared with the control group, There was no significant difference in ns. p<0.01, p<0.001, p<0.0001; compared with the model control group, p<0.0001. Detailed Implementation

[0051] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0052] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0053] Examples 1-4 The compositions of Examples 1-4 consist of the components listed in Table 1, and the amounts of each component are shown in Table 1.

[0054] Table 1

[0055] The composition is prepared by mixing the components evenly according to their weight proportions to obtain the composition.

[0056] Comparative Examples 1-5 The compositions of Comparative Examples 1-5 consist of the components listed in Table 2, and the amounts of each component in the compositions are shown in Table 2.

[0057] Table 2

[0058] The composition is prepared by mixing the components evenly according to their weight proportions to obtain the composition.

[0059] Experimental Example 1: The composition helps improve vascular endothelial dysfunction. Using a TNF-α-induced human umbilical vein endothelial cell (HUVEC) injury model as the research subject, this study investigated the protective effect of the composition on vascular endothelial cell injury, inflammatory response, and functional disorder.

[0060] 1. Construction of TNF-α injury model and administration of each raw material (1) Human umbilical vein endothelial cells (HUVECs) were purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number SCSP-5535. They were cultured in complete culture medium for HUVECs (containing 90% F-12K basal medium, 10% fetal bovine serum, 0.1 mg / mL heparin, and 30 μg / mL endothelial cell growth supplement) at 5% CO2 and 37℃. They were passaged at a ratio of 1:2 and passaged once every 3-4 days.

[0061] (2) HUVEC cells were grown at a concentration of 1.0 × 10⁻⁶. 4 Seeds were seeded at a density of 10 cells / well in 96-well plates, and 0.1 mL of complete culture medium was added to each well. Three replicates were set up for each group. The plates were incubated overnight at 37°C with 5% CO2.

[0062] (3) After overnight culture, discard the original culture medium in the wells, wash the cells twice with PBS, and add 10 ng / ml TNF-α (prepared with complete culture medium) to each group except the blank control group. At the same time, add 100 μL of the test sample solution (prepared with complete culture medium) to each well and incubate at 5% CO2 and 37℃ for 24 h. The group settings and treatment methods are shown in Table 3: Table 3

[0063] 2. IL-6 measurement After 24 hours of culture, the supernatant from each well was collected, and the IL-6 content in the cell culture supernatant was detected using a Human IL-6 ELISA kit. The supernatant was centrifuged at 300g for 10 min to remove precipitate, then transferred to an ELISA plate (100 μL per well). Two-fold serially diluted standards were added to the standard wells; 100 μL of cell culture medium was added to the blank wells; 50 μL of detection antibody was added to each test well; the plate was sealed with a sealing membrane, vortexed at 150 rpm, and incubated at room temperature for 2 hours. After incubation, the liquid was discarded, and the plate was washed 6 times with 300 μL of washing buffer per well. 100 μL of HRP working solution was added to each well, the plate was sealed with a sealing membrane, vortexed at 150 rpm, and incubated at room temperature for 45 minutes. After incubation, the liquid was discarded, and the plate was washed 6 times with 300 μL of washing buffer per well. 100 μL of chromogenic substrate was added to each well, and the plate was incubated in the dark for 10 minutes. 100 μL of stop solution was added to each well, and the OD value was measured at wavelengths of 450 / 630 nm. A standard curve was established using different concentrations of standards and their OD values. The actual IL-6 content of each group was calculated using the standard curve. The measurement results are shown in Table 4 and... Figure 1 As shown.

[0064] Table 4

[0065] 3. Expression level determination of ICAM-1 and eNOS After 24 hours of culture, the original culture medium in the wells was discarded, and the cells were washed twice with PBS. Gene CT values ​​were measured using a cell lysis, reverse transcription, and qPCR kit. 125 μL of washing buffer was added to each well, and the liquid was discarded. 50 μL of the prepared cell processing solution was added to each well and incubated at room temperature for 5 minutes. The cell processing solution in each well was repeatedly pipetted and transferred to a microcentrifuge tube and incubated at 75°C for 5 minutes. The resulting cell lysate was used as a template for reverse transcription. The reaction solution was prepared according to the recommended components, and finally, the RNA sample was added. The mixture was incubated at 37°C for 15 minutes, 85°C for 5 seconds, and cooled to 4°C. The resulting reaction solution was then used for real-time PCR. The PCR reaction solution was prepared according to the recommended components, and a two-step PCR reaction was performed: pre-denaturation at 95°C for 30 seconds, followed by PCR at 95°C for 3 seconds and 60°C for 30 seconds, for 40 cycles. The system's default melting curve program was then used. (Formula 2) -δδCT Gene expression levels were calculated. The results are shown in Table 5 and... Figure 2 As shown.

[0066] Table 5

[0067] Compared with the control group, the TNF-α-induced injury model group showed significantly increased IL-6 secretion in cell supernatant, significantly upregulated expression of the adhesion-promoting molecule ICAM-1 gene, and significantly downregulated expression of the vascular protective factor eNOS gene. This indicates that TNF-α successfully induced inflammatory damage and endothelial dysfunction in HUVECs, and the modeling system is stable and reliable, which can be used to evaluate the vascular endothelial protective activity of each group of samples.

[0068] After intervention with the compositions in Examples 1-4, the release of IL-6 in cells was significantly reduced; simultaneously, it significantly inhibited damage-induced ICAM-1 overexpression and upregulated eNOS gene expression to near the level of blank normal cells. This indicates that the combination of hydroxytyrosol, rhodioloside, and EPA within the ratio range of this invention can synergistically inhibit endothelial inflammation, reduce the expression of endothelial adhesion molecules, and increase nitric oxide synthase levels, thus protecting damaged vascular endothelium through a dual pathway of anti-inflammation and improved vasodilatory function.

[0069] Experimental Example 2: The composition helps maintain healthy blood lipid levels. This experiment used male, spontaneously atherosclerotic ApoE- / - mice, 8 weeks old, weighing 20-25g. After 2 weeks of acclimatization, the animals were randomly divided into a normal control group, a model control group, and Example 1-Example 4 groups, with 5 mice in each group. The model control group and Example 1-Example 4 groups were fed a high-fat diet (containing 21% fat and 0.15% cholesterol), while the normal control group was fed a normal diet.

[0070] ApoE- / - mice were fed a high-fat diet for 12 weeks, while the normal control group was fed a normal diet for 12 weeks. During this period, starting from week 5, groups 1-4 were administered the compositions prepared in Examples 1-4 by gavage at a dose of 50 mg / kg, while the normal control group and the model control group were administered physiological saline by gavage. The administration lasted for a total of 8 weeks.

[0071] Lipid level testing: After the experiment, blood was collected via the tail vein, serum was separated, and the concentrations of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) in the serum were measured. The results are shown in Table 6. Figure 3 As shown: Table 6

[0072] ApoE- / - mice are a recognized susceptibility model for atherosclerosis, and excessive accumulation of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) is a core contributing factor to vascular plaque formation and cardiovascular disease. The composition of this invention can significantly inhibit the abnormal increase of total cholesterol and low-density lipoprotein cholesterol under high-fat conditions, reducing the risk of lipid deposition in the vascular wall from the source and thus regulating blood lipid levels.

[0073] Experimental Example 3: Effect of the Composition on Blood Pressure Eighteen four-week-old male spontaneously hypertensive rats (SHR) were randomly divided into a model group, a positive control group, and three rats in each of the Example 1-Example 4 groups. Three male Wistar Kyoto rats (WKY) of the same age were used as a normal control group. They were fed a normal diet and had free access to food and water. The experimental period was eight weeks.

[0074] Rats in the model group and blank control group were administered physiological saline by gavage daily as controls; the positive control group was administered 10 mg / kg captopril by gavage daily; and the groups in Examples 1-4 were administered 50 mg / kg of the composition prepared in Examples 1-4 by gavage daily. Blood pressure was measured weekly in the tail artery using the BP-2000 non-invasive rat blood pressure monitoring system. The results are shown in Table 7 and... Figure 4 As shown: Table 7

[0075] Compared with the SHR model group, the positive control drug captopril significantly reduced systolic and diastolic blood pressure. After 8 weeks of gavage intervention with the compositions of Examples 1-4 of this invention, the systolic and diastolic blood pressure of rats decreased significantly, and the antihypertensive effect was better than that of the positive control group.

[0076] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A biologically active composition, characterized in that, The composition comprises, by weight, 4-18 parts hydroxytyrosol, 2-120 parts rhodioloside and 180-2000 parts EPA.

2. The composition according to claim 1, characterized in that, The composition comprises, by weight, 4-15 parts hydroxytyrosol, 10-120 parts rhodioloside and 180-2000 parts EPA.

3. The composition according to claim 2, characterized in that, The composition comprises, by weight, 5-15 parts hydroxytyrosol, 10-120 parts rhodioloside and 180-2000 parts EPA.

4. A method for preparing the composition according to any one of claims 1-3, characterized in that, The preparation method includes mixing hydroxytyrosol, rhodioloside, and EPA evenly in parts by weight.

5. A food or health product, characterized in that, The food or health product described herein comprises the composition according to any one of claims 1-3.

6. The food or health product according to claim 5, characterized in that, The food or health product also includes any one or more of the following: nutritional additives, food-medicine homologous ingredients, and excipients acceptable in the food or health product field.

7. The food or health product according to claim 5, characterized in that, The dosage forms of the food or health products mentioned include liquid dosage forms, semi-solid dosage forms, or solid dosage forms.

8. The use of the composition according to any one of claims 1-3 in the preparation of cardiovascular protection products, characterized in that, The cardiovascular protection products mentioned above include any one or more of the following: (1) Foods, health products, or medicines that help maintain healthy blood pressure levels; (2) Foods, health products, or medicines that help maintain healthy blood lipid levels; (3) Drugs that help improve vascular endothelial dysfunction.

9. A medicine, characterized in that, The medicine comprises the composition according to any one of claims 1-3.

10. The pharmaceutical product according to claim 9, characterized in that, The medicine also includes pharmaceutically acceptable excipients.

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