Triphenylphosphonium Hydroxytyrosol TPP-HT, Its Synthesis Method and Application in the Preparation of Drugs for Improving Aortic Endothelial Cell Function

Abstract

Triphenylphosphonium hydroxytyrosol TPP-HT, its synthesis method and application in the preparation of drugs for improving aortic endothelial cell function. TPP-HT can significantly increase the reduction of human aortic endothelial cell activity caused by saturated fatty acids, inhibit the inflammatory response and oxidative stress caused by saturated fatty acids, and at the same time increase the synthesis of ATP in vascular endothelial cells and the expression of mitochondrial complex II. By anti-inflammatory and protecting mitochondrial function, it can prevent the occurrence and development of atherosclerosis. Therefore, TPP-HT has good application prospects in preventing cardiovascular diseases such as atherosclerosis caused by high-fat-induced endothelial damage, opening up a new medical approach for preventing a series of problems of cardiovascular diseases such as atherosclerosis caused by endothelial damage due to unbalanced dietary structure, and providing a new basis for the development of new drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and particularly relates to triphenylphosphonium hydroxytyrosol TPP-HT, its synthesis method, and its application in the preparation of drugs for improving the function of aortic endothelial cells. Background Art

[0002] According to the global data on the prevention and control of cardiovascular diseases released by the World Health Organization in 2011, deaths caused by cardiovascular diseases accounted for 31% of the global death toll, second only to the total number of other non-communicable diseases. It is predicted that the mortality rate caused by cardiovascular diseases from 2008 to 2030 will be significantly higher than that of other diseases and will be in the leading position. Cardiovascular diseases mainly include cardiovascular and cerebrovascular diseases, such as myocardial infarction and cerebral infarction. Atherosclerosis is the pathological basis of cardiovascular diseases.

[0003] The pathogenesis of atherosclerosis is very complex. It occurs in the blood vessel wall and is a chronic inflammatory response, forming plaques, and accompanied by vascular endothelial cell damage. The main factors leading to atherosclerosis are unhealthy diets such as high salt, high fat, high energy, smoking, and metabolic risk factors, including diseases such as hypertension, hyperlipidemia, and obesity.

[0004] At present, there are treatment methods for atherosclerosis such as drugs and surgery. However, this type of disease has a high degree of concealment, high lethality and disability rates. Therefore, early prevention is particularly important. There are research reports that natural active ingredients such as chlorogenic acid, xiongshao, and lignans have anti-atherosclerotic functions, and functional foods based on these ingredients have successively emerged. It is of great significance and prospect to explore more natural substances with better efficacy for preventing cardiovascular diseases such as atherosclerosis. Summary of the Invention

[0005] In order to overcome the defects of the above-mentioned prior art, the purpose of the present invention is to provide triphenylphosphonium hydroxytyrosol TPP-HT, its synthesis method, and its application in the preparation of drugs for improving the function of aortic endothelial cells. The obtained product TPP-HT can significantly increase the reduction of cell viability induced by saturated fatty acids, inhibit the inflammation and oxidative stress response of human aortic endothelial cells caused by saturated fatty acids, increase the generation of ATP in vascular endothelial cells and mitochondrial complex II, and prevent the occurrence and development of atherosclerosis by anti-inflammatory, antioxidant stress, and protecting mitochondrial function.

[0006] In order to achieve the above purpose, the present invention is realized through the following technical solutions:

[0007] Triphenylphosphonium hydroxytyrosol TPP-HT, its structural formula is:

[0008]

[0009] Synthesis method of triphenylphosphonium hydroxytyrosol TPP-HT, the reaction process is as follows:

[0010]

[0011] Using 3,4-dihydroxybenzaldehyde (1) as a raw material, a dibenzyloxy compound (2) is obtained through a benzyl bromide protection reaction; the dibenzyloxy compound (2) is reduced to benzyl alcohol (3) by NaBH4, and the benzyl alcohol (3) is chlorinated to a chloride (4); the chloride (4) reacts with potassium cyanide to obtain a nitrile compound (5); the nitrile compound (5) is hydrolyzed by sodium hydride to obtain a carboxylic acid compound (6); the carboxylic acid compound (6) is reduced by LiAlH4 to obtain dibenzyloxy hydroxytyrosol (7); dibenzyloxy hydroxytyrosol (7) reacts with 1,6-dibromohexane to obtain a bromide (8); taking the bromide (8) as a raw material, an intermediate bromide (9) is obtained through an Et3SiH reduction deprotection reaction; the bromide (9) reacts with triphenylphosphonium to obtain the target triphenylphosphonium-hydroxytyrosol triphenylphosphonium hydroxytyrosol TPP-HT.

[0012] Application of TPP-HT in the preparation of drugs for improving aortic endothelial cell function, which has a protective effect on the reduction of aortic endothelial cell activity caused by saturated fatty acids, an inhibitory effect on the inflammatory response of aortic endothelial cells caused by saturated fatty acids, and reduces the mRNA level contents of interleukin-6 (IL-6) and matrix metalloproteinase-1 (MMP-1) in aortic endothelial cells; it has a protective effect on the mitochondria damaged by the inflammatory response of aortic endothelial cells caused by saturated fatty acids.

[0013] It has a protective effect on the oxidative stress of aortic endothelial cells caused by saturated fatty acids.

[0014] Reduces the level content of reactive oxygen species (ROS) in aortic endothelial cells.

[0015] The drug described above is a drug that has an improving effect on the reduction of energy in aortic endothelial cells caused by saturated fatty acids.

[0016] The drug described above increases ATP synthesis in aortic endothelial cells.

[0017] The drug described above is a drug that increases the expression of mitochondrial complex II protein. [

[0018] Application of TPP-HT in the preparation of drugs for preventing and treating cardiovascular diseases.

[0019] The drug described above is a drug for treating atherosclerosis.

[0020] Application of TPP-HT in the preparation of drugs for preventing the occurrence of atherosclerosis.

[0021] Compared with the prior art, the present invention has the following beneficial technical effects:

[0022] TPP-HT has an increasing effect on the activity of human aortic endothelial cells. The present invention first publicly points out that during the occurrence of vascular diseases such as atherosclerosis with vascular endothelial injury and inflammatory response, TPP-HT can significantly inhibit the decrease in the activity of human aortic endothelial cells caused by saturated fatty acids, inflammatory response, oxidative stress response, and increase the mitochondrial ATP generation and the expression of mitochondrial complex II protein in vascular endothelial cells, and prevent the occurrence and development of atherosclerosis by anti-inflammatory and protecting mitochondrial function.

[0023] TPP-HT can effectively increase the decrease in endothelial cell activity caused by saturated fatty acids; reduce the inflammatory response of human aortic endothelium caused by saturated fatty acids, such as the mRNA level contents of interleukin-6 (IL-6) and matrix metalloproteinase-1 (MMP-1); TPP-HT can effectively protect against oxidative stress and mitochondrial damage caused by saturated fatty acids to human aortic endothelium, such as reducing the generation of excessive reactive oxygen species induced by saturated fatty acids and increasing the production of mitochondrial ATP and the expression of mitochondrial complex II protein, etc.; it has good application prospects in preventing the occurrence and progression of vascular diseases. Brief Description of the Drawings

[0024] Figure 1 is the synthesis process of TPP-HT of the present invention.

[0025] Figures 2A to 2C is the protective effect of TPP-HT on the decrease in the viability of human aortic endothelial cells induced by saturated fatty acids. Among them: 2A to 2B respectively use the CCK-8 and MTT detection methods. The abscissa is the action concentration of TPP-HT, and the ordinate is cell activity; 2C is a cell microscope photograph.

[0026] Figures 3A to 3B is the inhibitory effect of TPP-HT on the inflammatory response of human aortic endothelial cells induced by palmitic acid. Among them: the abscissa is the action concentration of TPP-HT, and the ordinates are the mRNA level contents of IL-6 and MMP-1 respectively.

[0027] Figures 4A to 4B is that TPP-HT can inhibit the excessive production of ROS in human aortic endothelial cells induced by palmitic acid. Among them: Figure 4A is the result diagram by fluorescence microscopy method; Figure 4B is the result diagram by microplate reader method.

[0028] Figure 5 is that TPP-HT can up-regulate the decrease in mitochondrial ATP generation in human aortic endothelial cells induced by palmitic acid. Among them: the abscissa is different treatment groups, and the ordinate is the relative ATP level.

[0029] Figures 6A to 6B TPP-HT can increase the expression of mitochondrial complex II in human aortic endothelial cells, where: Figure 6A is the Western blot result diagram; Figure 6B is the Western blot statistical chart, with the protein name on the abscissa and the relative protein expression level content on the ordinate. Specific implementation manners

[0030] The following further elaborates on the present invention in conjunction with specific embodiments, which is an explanation rather than a limitation of the present invention.

[0031] 1. Synthesis process of TPP-HT

[0032] The synthesis process of TPP-HT is as Figure 1 shown. We used 3,4-dihydroxybenzaldehyde (1) as the raw material, and obtained dibenzyloxy compound (2) through a benzyl bromide protection reaction. Compound (2) was reduced to benzyl alcohol (3) by NaBH4, and benzyl alcohol was further chlorinated to chloride (4). Chloride (4) reacted with potassium cyanide to obtain nitrile compound (5), and was further hydrolyzed by base to obtain carboxylic acid compound (6). Carboxylic acid compound (6) was reduced by LiAlH4 to obtain dibenzyloxyhydroxytyrosol (7), which reacted with 1,6-dibromohexane to obtain bromide (8). Using bromide (8) as the raw material, intermediate bromide (9) was obtained through an Et3SiH reduction deprotection reaction, and bromide (9) reacted with triphenylphosphine to obtain the target triphenylphosphonium-hydroxytyrosol. The total yield of this synthesis process is about 23%. The reaction process is as follows:

[0033] The reaction reagents and conditions are as follows:

[0034] The TPP-HT synthesis process is as Figure 1 shown. Using 3,4-dihydroxybenzaldehyde (1) as the raw material, TPP-HT was synthesized in 9 steps. The total yield of the synthesis steps is about 23%.

[0035] Step a: 3,4-bis(benzyloxy)benzaldehyde (2)

[0036] To a solution of compound 1 (15.18 g, 110 mmol) in acetone (250 mL) was added benzyl bromide (34.54 g, 220 mmol) and potassium carbonate (45.6 g, 330 mmol), and the mixture was stirred at 60 °C overnight. After completion of the reaction, the volatile components were evaporated in vacuo, and the residue was extracted with CH2Cl2 (3 x 200 mL). The combined organic layers were dried over anhydrous Na2SO4 and evaporated to dryness. Flash chromatography on silica gel using a mixture of cyclohexane / ethyl acetate: 4 / 1 as eluent afforded 32.5 g (93%) of 3,4-bis(benzyloxy)benzaldehyde (2). 1 H NMR (600 MHz, CDCl3) δ 9.82 (s, 1H), 7.50 (d, J = 1.8 Hz, 1H), 7.46 (t, J = 7.6 Hz, 2H), 7.45 (d, J = 8.2 Hz, 2H), 7.42 (dd, J = 8.2, 1.9 Hz, 1H), 7.38 (d, J = 8.2 Hz, 2H), 7.37 (t, J = 7.6 Hz, 2H), 7.33 (t, J = 7.6 Hz, 2H), 7.32 (t, J = 7.6 Hz, 2H), 7.03 (d, J = 8.2 Hz, 1H), 5.26 (s, 2H), 5.22 (s, 2H). 13 C NMR (151 MHz, CDCl3) δ 190.9, 154.5, 149.4, 136.7, 136.4, 130.5, 128.8, 128.71, 128.6, 128.2, 128.1, 127.5, 127.2, 126.7, 113.4, 112.8, 71.2, 71.1.

[0037] Step b: (3,4-Bis(benzyloxy)phenyl)methanol (3)

[0038] To a solution of compound 2 (18 g, 56.7 mmol) in methanol (150 mL) was added NaBH4 (3.21 g, 84.9 mmol) portionwise at 0 °C, and the resulting mixture was stirred at room temperature for 2 h. After completion of the reaction, saturated NaHCO3 (400 mL) and ethyl acetate (800 mL) were added, and the mixture was stirred at room temperature for an additional 20 min. The organic layer was removed, and the aqueous layer was washed with ethyl acetate (3 x 200 mL). The combined washed organic layers were dried over anhydrous Na2SO4 and evaporated to dryness. Flash chromatography on silica gel using a mixture of cyclohexane / ethyl acetate: 6 / 1 as eluent afforded 16.15 g (89%) of (3,4-bis(benzyloxy)phenyl)methanol (3).

[0039] Step c: ((4-(Chloromethyl)-1,2-phenylene)bis(oxy))bis(methylene))dibenzene (4)

[0040] To a solution of compound 3 (16 g, 50 mmol) in benzene (150 mL) at 0 °C was added dropwise thionyl chloride (7.3 mL, 100 mmol), and the resulting mixture was stirred at room temperature for 6 h. After completion of the reaction, the mixture was carefully poured into ice water (150 mL) and stirred for 5 min. The organic layer was removed, and the aqueous layer was washed with ethyl acetate (3 × 100 mL). The washed organic layers were combined, dried over anhydrous Na2SO4, and evaporated to dryness. Flash chromatography on silica gel using a mixture of cyclohexane / ethyl acetate: 10 / 1 as the eluent afforded 14.2 g (84%) of ((4-(chloromethyl)-1,2-phenylene)bis(oxy))bis(methylene))dibenzene (4). 1 1H NMR (600 MHz, CDCl3) δ 7.45 (t, J = 7.6 Hz, 4H), 7.37 (t, J = 7.6 Hz, 5H), 7.29–7.32 (m, 3H), 6.93 (d, J = 1.8 Hz, 1H), 6.91 (d, J = 8.2 Hz, 1H), 6.81 (dd, J = 8.2, 1.8 Hz, 1H), 5.15 (d, J = 8.2 Hz, 4H), 3.55 (s, 2H). 13 13C NMR (151 MHz, CDCl3) δ 177.7, 149.2, 148.6, 137.5, 137.3, 128.6, 127.9, 127.6, 127.4, 126.7, 122.6, 116.6, 115.4, 71.6.

[0041] Step d: 2-(3,4-bis(benzyloxy)phenyl)acetonitrile (5)

[0042] To a solution of compound 4 (14 g, 42.5 mmol) in DMSO (30 mL) was added KCN (11 g, 170 mmol), and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was poured into ice water (150 mL), and then washed with ethyl acetate (3 × 100 mL). The washed organic layers were combined, dried over anhydrous Na2SO4, and evaporated to dryness. Flash chromatography on silica gel using a mixture of cyclohexane / ethyl acetate: 8 / 1 as the eluent afforded 9.9 g (71%) of 2-(3,4-bis(benzyloxy)phenyl)acetonitrile (5). 11H NMR (600 MHz, CDCl3) δ 7.48 (t, J = 7.6 Hz, 2H), 7.47 (d, J = 8.2 Hz, 2H), 7.39 (t, J = 7.6 Hz, 2H), 7.38 (d, J = 8.2 Hz, 2H), 7.35 (t, J = 7.6 Hz, 1H), 7.34 (t, J = 7.6 Hz, 1H), 6.94 (d, J = 8.2 Hz, 1H), 6.93 (s, 1H), 6.83 (dd, J = 8.2, 1.9 Hz, 1H), 5.17 (d, J = 8.2 Hz, 4H), 3.61 (s, 2H). 13 13C NMR (151 MHz, CDCl3) δ 149.5, 148.8, 137.1, 136.9, 128.6, 128.0, 127.9, 127.5, 127.4, 123.1, 121.07, 118.1, 115.5, 114.9, 71.5, 71.42423.1.

[0043] Step e: 2-(3,4-Bis(benzyloxy)phenyl)acetic acid (6)

[0044] To a solution of compound 5 (7 g, 20.11 mmol) in ethanol (30 mL) was added 40 ml of 40% NaOH solution, and the resulting mixture was stirred at 75 °C for 14 h. After completion of the reaction, the mixture was poured into ice water (200 ml), acidified with 9% HCl, and washed with CH2Cl2 (3 x 100 ml). The combined washed organic layers were dried over anhydrous Na2SO4 and evaporated to dryness. Flash chromatography on silica gel using a mixture of cyclohexane / ethyl acetate: 2 / 1 as the eluent gave 5.39 g (77%) of compound 2-(3,4-bis(benzyloxy)phenyl)acetic acid (6). 1 1H NMR (600 MHz, CDCl3) δ 7.44 (t, J = 7.6 Hz, 4H), 7.35 (t, J = 7.6 Hz, 4H), 7.31 (d, J = 4.8, 1H), 7.29 (d, J = 4.8, 1H), 6.92 (d, J = 1.9 Hz, 1H), 6.90 (d, J = 8.2 Hz, 1H), 6.80 (d, J = 8.2, 4H)., 3.55 (s, 2H). 13 13C NMR (151 MHz, CDCl3) δ 177.2, 149.2, 148.6, 137.5, 137.3, 128.6, 127.9, 127.6, 127.4, 126.7, 122.6, 116.6, 115.4, 71.6, 40.6.

[0045] Step f: 2-(3,4-Bis(benzyloxy)phenyl)ethan-1-ol (7)

[0046] Compound 6 (672 mg, 4 mmol) was added to a solution of lithium aluminum hydride (331 μl, 8 mmol) in tetrahydrofuran, and the mixture was stirred at room temperature for 2 h. The reaction mixture was filtered and evaporated in vacuo to give 2-(3,4-bis(benzyloxy)phenyl)ethan-1-ol (7). 1 H NMR (600 MHz, CDCl3) δ 7.45 (d, J = 8.2 Hz, 4H), 7.36 (t, J = 7.6 Hz 4H), 7.30 (d, J = 8.2, 1H), 7.29 (d, J = 8.2, 1H), 6.89 (d, J = 8.2, 1H), 6.83 (d, J = 1.9 Hz, 1H), 6.74 (dd, J = 8.2, 1.9 Hz, 1H), 5.14 (t, J = 7.1 Hz, 4H), 3.78 (t, J = 7.1 Hz, 2H), 2.76 (t, J = 7.1 Hz, 2H). 13 C NMR (151 MHz, CDCl3) δ 149.2, 148.0, 137.6, 137.5, 132.1, 128.6, 127.9, 127.9, 127.6, 127.5, 122.1, 116.5, 115.8, 71.7, 71.6, 63.8, 38.8.

[0047] Step g: ((4-(2-((6-bromohexyl)oxy)ethyl)-1,2-phenylene)bis(oxy))bis(methylene))dibenzene (8)

[0048] Compound 7 (666 mg, 2 mmol), NaH (85 mg, 3 mmol) and 1,6-dibromohexane (461 μl, 3 mmol) were added to dimethylformamide, and the suspension was stirred at room temperature for 24 h. After completion of the reaction, the mixture was evaporated in vacuo and the residue was purified by column chromatography (silica gel 60 mm) using cyclohexane / ethyl acetate: 6 / 1 as eluent to give ((4-(2-((6-bromohexyl)oxy)ethyl)-1,2-phenylene)bis(oxy))bis(methylene))dibenzene (8). 11H NMR (600 MHz, CDCl3) δ 7.45 (t, J = 7.3 Hz, 4H), 7.36 (q, J = 6.7 Hz, 4H), 7.31 (q, J = 6.8, 6.0 Hz, 2), 6.90–6.84 (m, 2H), 6.75 (d, J = 8.0 Hz, 1H), 5.14 (d, J = 9.3 Hz, 4H), 3.57 (t, J = 7.2 Hz, 2H), 3.43–3.38 (m, 41H), 2.79 (t, J = 7.2 Hz, 2H), 2.07 (q, J = 7.2 Hz, 1H), 1.84–1.88 (m, 1H), 1.55–1.61 (m, 2H), 1.42–1.49 (m, 2H), 1.34–1.39 (m, 21H). 13 13C NMR (151 MHz, CDCl3) δ 149.1, 147.7, 147.7, 138.9, 137.7, 137.7, 137.61, 132.9, 132.9, 128.6, 127.9, 127.9, 127.8, 127.5, 127.5, 121.9, 121.9, 116.5, 116.44, 115.62, 115.6, 114.6, 77.4, 77.2, 76.9, 72.0, 71.8, 71.7, 71.6, 71.6, 71.1, 70.9, 70.9, 36.0, 33.9, 33.7, 32.8, 32.6, 29.6, 29.7, 29.3, 28.1, 27.4, 26.2, 25.7, 25.5.

[0049] Step h: 4-(2-(6-bromohexyloxy)ethyl)benzene-1,2-diol (9)

[0050] To a methanol solution of compound 8 (996 mg, 2 mmol) was added palladium carbon Pd / C (200 mg) and triethylsilane (4.64 g, 40 mmol). The mixture was stirred under argon at room temperature for 14 h. After vacuum concentration of the mixture, water (50 mL) was added and the mixture was washed with ethyl acetate (3 x 20 mL). The combined washed organic layers were dried over anhydrous Na2SO4, concentrated and purified by flash column chromatography (silica gel 60 mm), using cyclohexane / ethyl acetate: 3 / 1 as the eluent to give compound 4-(2-(6-bromohexyloxy)ethyl)benzene-1,2-diol (9). 11H NMR (600 MHz, CDCl3) δ 6.74 (s, J = 8.2 Hz, 1H), 6.70 (d, J = 1.9 Hz, 1H), 6.62 (m, 2H), 3.62 (t, J = 7.1 Hz, 2H), 3.46 (t, J = 7.1 Hz, 2H), 3.39 (t, J = 7.1 Hz, 2H), 2.76 (t, J = 7.1 Hz, 2H), 1. (m, 2H), 1.58 (m, 2H), 1.42 (m, 2H), 1.34 (m, 2H). 13 13C NMR (151 MHz, CDCl3) δ 1: 43.9, 142.3, 131.8, 121.2, 116.1, 115.4, 72.1, 71.4, 71.0, 68.2, 35.6, 33.9, 32.8, 31.8, 29.7, 29.5, 28.1, 25.9, 25.7, 25.4, 22.7, 14.1.

[0051] Step i: (6-(3,4-Dihydroxyphenethyloxy)hexyl)triphenylphosphonium bromide (10) (TPP-HT)

[0052] A mixture of compound 9 (670 mg, 234 mmol), triphenylphosphine (1.1 g, 4.2 mmol) and acetonitrile was stirred at 82 °C for 24 h. After vacuum concentration of the mixture, it was washed 3 times with ether and filtered to obtain the title compound (6-(3,4-dihydroxyphenethyloxy)hexyl)triphenylphosphonium bromide (10) (TPP-HT). 1 1H NMR (600 MHz, DMSO-d6) δ 8.62 (s, 1H), 7.89 (tt, J = 7.1, 1.8 Hz, 1H), 7.85–7.68 (m, 6H), 6.64–6.54 (m, 1H), 6.43 (dd, J = 7.9, 2.1 Hz, 0H), 3.61–3.49 (m, 1H), 3.44 (t, J = 7.1 Hz, 1H), 3.31 (t, J = 6.5 Hz, 3H), 2.58 (t, J = 7.1 Hz, 1H), 1.49–1.56 (m, J = 7.8 Hz, 2H), 1.49–1.37 (m, 2H), 1.27-1.31 (m, 2H). 1313C NMR (151 MHz, DMSO-d6) δ 144.9, 143.4, 134.8, 134.8, 133.5, 133.5, 130.2, 130.1, 129.7, 119.3, 118.8, 118.2, 116.2, 115.3, 71.3, 69.7, 59.7, 40.1, 39.9, 39.9, 39.8, 39.7, 39.5, 39.4, 39.2, 39.1, 34.9, 29.6, 29.5, 28.8, 24.8, 21.7, 21.7, 20.7, 20.4, 20.0, 14.0.

[0053] 2. Experimental materials

[0054] CCK-8 was purchased from Suzhou Youyilan Di Biotechnology Co., Ltd.; TRIzol reagent was purchased from Invitrogen; RNA reverse transcription kit and SYBR fluorescent dye were purchased from TaKaRa Biotechnology (Dalian) Co., Ltd. RNA primer sequences were ordered and synthesized from Xi'an Tsingke Jersey Biotechnology Co., Ltd.

[0055] 3. Experimental cell culture and model establishment

[0056] Human aortic endothelial cells (HAEC) were purchased from Shanghai Baili Biotechnology Co., Ltd., and palmitic acid was purchased from sigma. Cells were cultured in a constant temperature, humidified, sterile incubator at 37 °C with 95% air and 5% CO2. The experiment was divided into three groups: (1) control group; (2) TPP-HT protection group; (3) 500 μM palmitic acid (PA) treatment group; (4) TPP-HT + PA pre-protection group.

[0057] 4. Experimental methods

[0058] (1) CCK-8 assay

[0059] HAEC were treated with different concentrations of TPP-HT and cultured in a cell incubator for 24 h, then treated with 500 μM palmitic acid for 24 h. After that, the cells were washed once with PBS, and the culture medium containing CCK-8 reagent was added to each well. After incubation in a 37 °C incubator with 95% air and CO2 for 4 h, the cells were washed three times with PBS, and the absorbance was measured at a wavelength of 450 nm.

[0060] (2) MTT assay

[0061] HAECs were treated with different concentrations of TPP-HT and cultured in a cell incubator for 24 h. Then, they were treated with 500 μM palmitic acid for 24 h. After that, the cells were washed once with PBS, 0.5 mg / ml MTT was added, and they were incubated in a 37 °C incubator containing 95% air and CO2 for 4 h. Then, they were washed three times with PBS, DMSO was added to dissolve them, and the absorbance value was detected at a wavelength of 490 nm.

[0062] (3) Detection of mRNA contents of interleukin-6 and matrix metalloproteinase-1

[0063] The detection was carried out by reverse transcription RNA-real-time fluorescence quantitative PCR. The specific method is as follows:

[0064] 1) RNA extraction

[0065] In a 12-well cell culture plate, 500 μL of TRIzol reagent was added to each well. At room temperature, it was shaken on a shaker for 5 min. Then, 200 μl of chloroform (1 / 5 of the total volume) was added to extract proteins. It was vigorously mixed for 15 s and left at room temperature for 15 min. Then, it was centrifuged at 12,000 g at 4 °C for 10 minutes. The upper aqueous phase was transferred to another EP tube, an equal volume of isopropanol was added, and after mixing, it was left at -20 °C for 1 h. Then, it was centrifuged at 12,000 g at 4 °C for 10 min, and the supernatant was discarded. 1 mL of pre-cooled 75% ethanol was added, and it was inverted and mixed. Then, it was centrifuged at 12,000 g at 4 °C for 10 min. The supernatant was discarded, and it was placed in a laminar flow hood for 30 min to completely volatilize the ethanol and dissolved in 10 μl of DEPC water. Its concentration was measured using an ultraviolet spectrophotometer for reverse transcription.

[0066] 2) RNA reverse transcription

[0067] The transcription volume was 20 μl. 2 μg of RNA was taken out, and 0.5 μg of random primers was added. 4 μl of 5X Master Mix was added, and the volume was made up to 20 μl with DEPC water. It was incubated at 37 °C for 60 min and treated at 80 °C for 15 s. It was placed at -20 °C for standby.

[0068] 3) Real-time fluorescence quantitative PCR (Real-time PCR)

[0069] It was carried out by the SYBR Green method. The reaction system included 1 μl of cDNA, 5 μl Premix ExTaq TMII, 0.5 μl of the upstream and downstream primer mixture (10 μM), add sterile water to 10 μl. The reaction conditions follow the instructions: denaturation at 95°C for 10 min, followed by 40 cycles of PCR (each cycle includes 30 s at 95°C, 30 s at 55°C, and 20 s at 72°C), and finally observe the melting curve (15 s at 95°C, 15 s at 60°C, and 15 s at 95°C). β-actin is used as an internal reference, and the primer sequences used in the experiment are:

[0070] IL-6:

[0071] forward: 5’-TTTTGTACTCATCTGCACAGC-3’

[0072] reverse: 5’-GGATTCAATGAGGAGACTTGC-3’

[0073] MMP-1:

[0074] forward: 5’-ACGCCAGATTTGCCAAGAG-3’

[0075] reverse: 5’-TTGACCCTCAGAGACCTTGGT-3’

[0076] β-actin:

[0077] forward: 5'-ATCATGTTTGAGACCTTCAA-3'

[0078] reverse: 5'-AGATGGGCACAGTGTGGGT-3’

[0079] (4) Determination of ROS in cells

[0080] 1) Reagent preparation

[0081] (1) Dissolve H2DCF-DA in DMSO to prepare a stock solution with a concentration of 10 mM, and then store it at -20°C using a light-protected method.

[0082] (2) Prepare the lysis buffer used to detect ROS. The formula is shown in Table 1 below. Then adjust the pH to 7.5 and store it at 4°C.

[0083] Table 1 Preparation of ROS lysis buffer

[0084]

[0085] 2) Detection by microplate reader

[0086] (1) Seed HAEC cells in a 6-well plate. Treat the HAEC cells with TPP-HT and incubate them in a cell culture incubator for 24 h. Then, treat the cells with 500 μM palmitic acid for 24 h, and the cell treatment is completed.

[0087] (2) Dilute the mother liquor of H2DCF-DA 1000-fold with serum-free medium to a final concentration of 10 μM for the working solution, and mix it well by inverting the tube up and down.

[0088] (3) Use a vacuum pump to aspirate the existing medium in the 6-well plate. Add 1 mL of the H2DCF-DA working solution to each well of the 6-well plate, and incubate it in a 37 °C incubator with 5% CO2 for 30 min, paying attention to avoiding light.

[0089] (4) Discard the existing H2DCF-DA working solution, wash the cells three times with PBS. Then, add 300 μL of ROS lysis solution to each well, gently shake the 6-well plate to completely wet the bottom cells with the ROS lysis solution, and place it on ice for 10 min.

[0090] (5) Scrape the cells with a cell scraper and collect them in a 1.5 mL EP tube. Centrifuge at 13000 g and 4 °C for 10 min, and transfer the supernatant to a new 1.5 mL EP tube for standby.

[0091] (6) Then, transfer the obtained supernatant to a 96-well plate, 200 μL per well. Use the ROS lysis solution as a blank control, and detect the fluorescence intensity under the conditions of an excitation wavelength of 485 nm and an emission wavelength of 538 nm.

[0092] (7) Detect the protein content in the supernatant by the BCA method, and compare the fluorescence value obtained in (5) with the protein concentration obtained in (6) to obtain the final ROS content.

[0093] 3) Detection by fluorescence microscopy

[0094] The steps of cell seeding and drug treatment are the same as those in steps (1)-(3) of the detection method using an enzyme-labeled instrument. After washing three times with PBS, add 1 mL of PBS to completely wet the cells, and then take pictures using a fluorescence microscope.

[0095] (5) Protein detection

[0096] 1) Protein extraction

[0097] Add 150 μL of IP lysis buffer to each well of a 6-well cell culture plate, remove cells with a cell scraper, oscillate for 15 seconds, and place on ice for 10 minutes. Repeat this process three times, ensuring that the ice bath time is at least 30 minutes. Centrifuge at 12,000 rcf at 4°C for 10 minutes, collect the supernatant, quantify the protein using the BCA method, adjust the level, add 5X loading buffer and mercaptoethanol, boil for 10 minutes to denature the protein, and store at -80°C until use.

[0098] 2) Western blot

[0099] 10% acrylamide gel was used for running, and 10 μg of protein was loaded. The sample was transferred to PVDF membrane, blocked, incubated with primary antibody at 4°C overnight, washed with primary antibody, incubated with secondary antibody at room temperature for 1 hour, washed with secondary antibody, and subjected to chemiluminescence.

[0100] (6) Statistical analysis

[0101] The results are expressed as Mean ± SEM. The data were analyzed using One Way-ANOVA analysis method. Statistically significant differences were set at *p < 0.05, **p < 0.01, and ***p < 0.001.

[0102] 4. TPP-HT has a protective effect on palmitic acid-induced decrease in human aortic endothelial cell activity

[0103] Human endothelial cells (HAEC) were treated with different concentrations of TPP-HT for 24 hours, and then treated with 500 μM palmitic acid for 24 hours. Cell activity was detected using CCK-8 and MTT, respectively. Figures 2A to 2C The results showed that the use of 5μM TPP-HT alone can increase the activity of human aortic endothelial cells, and the use of 5μM TPP-HT can prevent the decrease in human aortic endothelial cell activity induced by palmitic acid.

[0104] 5. Inhibitory effect of TPP-HT on palmitic acid-induced inflammatory response in human aortic endothelial cells

[0105] TPP-HT was first applied to human endothelial cells for 24 h and then 500 μM palmitic acid was added for 24 h. Figure 2A~B showed that TPP-HT could significantly inhibit the inflammatory response induced by palmitic acid in human vascular endothelial cells. Palmitic acid at a concentration of 500 μM induced an inflammatory response in human aortic endothelial cells, with significant increases in the levels of the pro-inflammatory cytokines interleukin IL-6 and human matrix metalloproteinase MMP-1 compared to the group without palmitic acid treatment. At the mRNA level, IL-6 increased by approximately 10-fold and MMP-1 increased by approximately 7-fold. The inflammatory level in the model group was significantly elevated. When the concentration of TPP-HT was 5 μM and 10 μM, an obvious inhibitory effect on the inflammatory response was observed, suggesting the anti-inflammatory and anti-atherosclerotic effects of TPP-HT.

[0106] 6. TPP-HT can inhibit the excessive production of reactive oxygen species induced by palmitic acid

[0107] Reactive oxygen species (ROS) are mainly produced during the oxidative phosphorylation process of the mitochondrial inner membrane respiratory chain. In endothelial cells, the occurrence of an inflammatory response may be triggered by oxidative stress. Therefore, we further detected the changes in reactive oxygen species. Figure 3A ~B showed that when measuring the levels of reactive oxygen species in cells using two different detection methods, it was found that the ROS level in the model group was significantly higher than that in the control group. Pretreatment of HAECs cells with 10 μM punicalagin could significantly inhibit the excessive production of reactive oxygen species caused by palmitic acid.

[0108] 7. TPP-HT can upregulate the decrease in mitochondrial ATP synthesis induced by palmitic acid in human aortic endothelial cells

[0109] ATP is an important "energy currency" of cells, an energy carrier and a signaling molecule, involved in regulating various life activities of cells. Mitochondria are one of the metabolic centers of cells, which can produce a large amount of ATP to provide energy for cells. Therefore, the ATP content level can reflect the mitochondrial function to a certain extent. Figure 5 It was shown that palmitic acid could reduce the production of mitochondrial ATP in human aortic endothelial cells, while TPP-HT could upregulate the production of ATP, suggesting that TPP-HT could improve mitochondrial function.

[0110] 8. Houttuynia cordata extract can increase the expression of mitochondrial complex IV and mitochondrial antioxidant protein SOD2 in human aortic endothelial cells

[0111] Mitochondrial respiratory chain enzymes are called mitochondrial respiratory chain complexes and mitochondrial respiratory chain complex enzymes. The mitochondrial respiratory chain is located on the inner mitochondrial membrane and consists of 5 complex groups: NADH (called complex I), succinate oxidoreductase (called complex II), cytochrome C oxidoreductase (complex III), cytochrome C oxidase (called complex IV), and ATP synthase (called complex V). Mitochondrial complexes are closely related to electron transfer and energy production. An increase in the expression level of the complex indicates an improvement in the mitochondrial function of cells.Figure 4A Group B showed that TPP-HT could significantly increase the expression of mitochondrial complex II protein, thereby enhancing mitochondrial function.

[0112] The above experimental results demonstrated that the houttuynia cordata extract could effectively inhibit the reduction of the activity, inflammatory response, and mitochondrial damage of human aortic endothelial cells induced by high fat, thereby improving the function of human aortic endothelial cells. Since the impairment of endothelial cell function is the initial manifestation, basis, and cause of the occurrence and development of atherosclerosis, atherosclerosis is a chronic inflammatory response, and the occurrence of the inflammatory response is one of the important reasons for the disease. Moreover, mitochondrial damage can cause insufficient energy and decreased function of endothelial cells, which has also been reported to be possibly one of the important reasons for the occurrence of atherosclerosis. TPP-HT showed excellent characteristics of protecting endothelial cell inflammation and mitochondrial damage in the above endothelial cell injury experiments. Thus, it can be seen that TPP-HT has good application prospects in preventing cardiovascular diseases such as atherosclerosis caused by high-fat-induced endothelial damage, opening up a new medical approach for preventing a series of problems of cardiovascular diseases such as atherosclerosis caused by endothelial damage due to unbalanced dietary structure, and providing a new basis for the development of new preventive foods and drugs.

[0113] The above-given embodiments are preferred examples for implementing the present invention, and the present invention is not limited to the above embodiments. Any non-essential addition or replacement made by those skilled in the art based on the technical features of the technical solution of the present invention shall fall within the protection scope of the present invention.

Claims

1. Triphenylphosphonium hydroxytyrosol TPP-HT, characterized in that Its structural formula is:

2. The synthetic method of triphenylphosphonium hydroxytyrosol TPP-HT is characterized in that, The reaction process is as follows: Using 3,4-dihydroxybenzaldehyde (1) as a raw material, a benzyl bromide protection reaction is performed to obtain a dibenzyloxy compound (2); the dibenzyloxy compound (2) is reduced to benzyl alcohol (3) by NaBH4, and the benzyl alcohol (3) is chlorinated to a chloride (4); the chloride (4) reacts with potassium cyanide to obtain a nitrile compound (5); the nitrile compound (5) is hydrolyzed with sodium hydride to obtain a carboxylic acid compound (6); the carboxylic acid compound (6) is reduced with LiAlH4 to obtain dibenzyloxyhydroxytyrosol (7); dibenzyloxyhydroxytyrosol (7) reacts with 1,6-dibromohexane to obtain a bromide (8); using bromide (8) as a raw material, an Et3SiH reduction and deprotection reaction is performed to obtain an intermediate bromide (9); the bromide (9) reacts with triphenylphosphonium to obtain the target triphenylphosphonium-hydroxytyrosol triphenylphosphonium hydroxytyrosol TPP-HT.

3. The use of TPP-HT in the preparation of a drug for improving the function of aortic endothelial cells, characterized in that: It has a protective effect on the decreased activity of aortic endothelial cells caused by saturated fatty acids, inhibits the inflammatory response of aortic endothelial cells caused by saturated fatty acids, reduces the mRNA levels of interleukin-6 and matrix metalloproteinase-1 in aortic endothelial cells, and protects mitochondria damaged by the inflammatory response of aortic endothelial cells caused by saturated fatty acids. The structural formula of triphenylphosphonium hydroxytyrosol TPP-HT is:

4. The use of TPP-HT in the preparation of a medicament for improving aortic endothelial cell function according to claim 3, characterized in that: It has a protective effect on the oxidative stress of aortic endothelial cells caused by saturated fatty acids and reduces the level of reactive oxygen species in aortic endothelial cells.

5. The use of TPP-HT according to claim 3 in preparing a drug for improving aortic endothelial cell function, characterized in that: The medicine is a medicine that has an improving effect on the reduction of energy of aortic endothelial cells caused by saturated fatty acids.

6. The use of TPP-HT according to claim 3 in preparing a drug for improving aortic endothelial cell function, characterized in that: The drug increases ATP synthesis in aortic endothelial cells.

7. The use of TPP-HT in the preparation of a drug for preventing and treating cardiovascular diseases, characterized in that: The structural formula of triphenylphosphonium hydroxytyrosol TPP-HT is:

8. Use of -HT in the preparation of a drug for improving aortic endothelial cell function, characterized in that: The structural formula of triphenylphosphonium hydroxytyrosol TPP-HT is:

9. The use of -HT in the preparation of a drug for preventing atherosclerosis, characterized in that: The structural formula of triphenylphosphonium hydroxytyrosol TPP-HT is:

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