TW202327568A - Use of fucoxanthin for preparing composition for heart disease prevention for preventing, treating and reducing heart diseases - Google Patents

Abstract

The present invention provides a use of fucoxanthin for preparing a composition for preventing, treating or reducing heart disease. The present invention proves that fucoxanthin may alleviate the growth inhibition of rat primary heart valve interstitial cells caused by H2O2, prevent the cell destruction and DNA damages of valve interstitial cells caused by oxidative stress, reduce reactive oxygen density, recover apoptosis caused by H2O2, and alleviate calcification of valve interstitial cells caused by H2O2. The present invention may further reduce the mitral valve leakage, tricuspid valve leakage, and compensatory cardiomegaly of dogs in animal experiment.

Description

Use of phycoxanthin for preparing compositions for preventing, treating or improving heart diseases

The present invention relates to an application of phycoxanthin, especially an application for preparing a pharmaceutical composition or a food composition for preventing, treating or improving heart diseases.

The pathogenesis of cardiovascular diseases, such as atherosclerosis and aortic valve sclerosis, involves inflammatory responses to various stimuli, including high levels of low-density lipoprotein (LDL) and reactive oxygen species ( reactive oxygen species, ROS). ROS are caused by increased oxidative stress, infection and chemical damage. In cardiovascular disease, increased cardiomyocyte load or insufficient energy supply can lead to an imbalance between energy supply and demand, resulting in increased energy metabolism and mitochondrial redox. This eventually leads to increased reactive oxygen species and damage to heart cells.

Heart valves, such as the aortic valve, mainly include two types of cells: valve endothelial cells (VEC) on the valve surface and valve interstitial cells (VIC) in the valve matrix. VEC is responsible for the regulation of message transmission and permeability, and also prevents the formation of blood clots. The VIC maintains the tissue structure of the valve. Normally, healthy valve cells are predominantly of the fibroblast type, but apoptosis is induced in the presence of increased reactive oxygen species, and oxidative stress can promote heart valve calcification and lead to valve damage.

Elevated reactive oxygen species also affect extracellular matrix (ECM) remodeling. In heart valves, extracellular matrix remodeling occurs in VIC fibroblast-rich tissues and may further lead to valve fibrosis. In addition, reactive oxygen species-induced chronic inflammation leads to an influx of macrophages with increased osteopromotive and angiogenic activity, which in turn increases proteolytic enzyme production and triggers extracellular matrix remodeling.

The progression of heart valve disease is associated with increasing age, increased heart volume, and regurgitation of blood flow. Valve disease is not unique to humans, and small dogs weighing less than 20 kg can have heart valve disease. Example: In Taiwan, Maltese has the highest incidence.

Therefore, finding an effective composition for treating heart diseases is an urgently needed problem.

To achieve the above purpose, the present invention provides a use of Fucoxanthin, which is used to prepare a composition for preventing, treating or improving heart disease. That is to say, the composition can achieve the effects of treating and preventing heart diseases.

Preferably, the heart disease is heart valve disease.

According to the present invention, the heart disease includes: mitral valve leakage, tricuspid valve leakage, compensatory heart enlargement.

Preferably, the subject of administration of the composition of the present invention may be a mammal, including but not limited to human, mouse, dog or cat.

According to the present invention, the composition is a pharmaceutical composition or a food composition.

Preferably, the composition is a pharmaceutical composition, and the pharmaceutical composition contains an effective dose of phycoxanthin and a pharmaceutically acceptable carrier.

The "pharmaceutically acceptable carrier" in the present invention includes, but not limited to reducing agent, solvent, emulsifier, suspending agent, decomposer, Binder (binding agent), excipient (excipient), stabilizer (stabilizing agent), chelating agent (chelating agent), diluent (diluent), gelling agent (gelling agent), preservative (preservative), lubricant (lubricant), surfactant (surfactant), and other carriers similar or applicable to the present invention.

Preferably, the reducing agent includes, but not limited to, ascorbic acid, citric acid, acetic acid, propionic acid, butyric acid, lactic acid ), hydroxysuccinic acid (malic acid), sulfonic acid (sulfonic acid), succinic acid (succinic acid) or combinations thereof.

The "pharmaceutical composition" of the present invention can exist in various forms, such forms include, but not limited to, liquid, semi-solid and solid pharmaceutical forms, such as solution, emulsion, suspension, powder (powder), lozenge (tablet), pill (pill), buccal lozenge (lozenge), tablet (troche), chewing gum (chewing gum), capsule (capsule), liposome, suppository and other similar or applicable The dosage form of the present invention.

Preferably, the pharmaceutical composition is in an oral dosage form or a parenteral dosage form.

More preferably, the oral dosage forms are solutions, emulsions, suspensions, powders, lozenges, pills, lozenges, tablets, chewing gums or capsules.

Preferably, the administration is twice a day.

Preferably, the dosage for the dog is 50 mg/kg (mg/kg) to 200 mg/kg per day. Preferably, the dosage for dogs is 75 mg/kg to 175 mg/kg per day. More preferably, the dosage for dogs is 100 mg/kg to 150 mg/kg per day.

Preferably, the effective dose of the composition for humans is between 25 mg/kg/day and 100 mg/kg per day; more preferably, between 50 mg/kg and 75 mg/kg per day. The above doses are calculated according to the estimation method (Estimating the maximum safe starting dose in initial clinical trials for therapeutics in adult healthy volunteers) announced by the US Food and Drug Administration in 2005.

Preferably, the composition is a food composition, and the food composition is a health food, health food, functional food, nutritional supplement or special nutritional food.

In one embodiment, the molecular formula of phycoxanthin is C ₄₂ H ₅₈ O ₆ , its structure is similar to ß-carotene or vitamin A, it belongs to a kind of carotenoid, and it is a common pigment in the chloroplast of Phaeaceae. It can be obtained from commercial Or extracted from the above-mentioned brown algae, preferably the highly stable phycoxanthin sold by Ocean Biotech Co., Ltd.

According to the present invention, phycoxanthin can reduce the concentration of active oxygen.

The "effective dose" mentioned in the invention refers to the amount effective for achieving the desired prevention, treatment or improvement of heart disease in terms of dosage and for the required time period; according to the present invention, it refers to the amount of Fucoxanthin can alleviate the growth inhibition of rat primary heart valve interstitial cells caused by hydrogen peroxide (H ₂ O ₂ ); avoid VIC cell damage and DNA damage caused by oxidative stress; reduce the density of reactive oxygen species; Apoptosis-related protein expression, such as: reducing the cleavage of adenosine diphosphate-ribose polymerase [Poly(ADP-Ribose) Polymerase, PARP] protein expression, cleavage of caspase 3 protein expression, BCL-2 related X protein (BCL2 Associated X, Bax) / B cell lymphoma-2 (B-cell lymphoma 2, Bcl-2) ratio, in order to restore the apoptosis caused by H ₂ O ₂ ; reduce the expression of calcification-related proteins, Such as: reducing the protein expression of phosphorylated protein kinase Akt, the protein expression of extracellular regulated protein kinases (ERK), matrix metalloproteinase 2 (matrix metalloproteinase-2, MMP) which is a marker of extracellular matrix remodeling -2) Protein expression to reduce VIC cell calcification caused by H ₂ O ₂ ; and improve mitral valve leakage, tricuspid valve leakage, and compensatory heart enlargement in dogs in animal experiments.

The present invention will be further illustrated by the following examples, which do not limit the foregoing disclosure of the present invention. Those skilled in the art of the present invention can make some improvements and modifications without departing from the scope of the present invention.

Preparation Example 1 Separation and Purification of Primary Rat Heart Valve Interstitial Cells (Valve Interstitial Cells, VIC)

According to Lin C., Zhu D., Markby G., Corcoran B.M., Farquharson C., Macrae V.E. Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification. J. Vis. Exp. 2017:56126 .Isolation and purification of rat heart valve interstitial cells, in short, after collecting all leaflets, centrifugation and co-action with collagenase II (collagenase II) for 2 hours to obtain heart valve interstitial fragments ( debris). The rat heart valve interstitial cells obtained from recipients were treated with 10% fetal bovine serum (Fetal Bovine Serum, FBS; CORNING, Manassas, VA, USA), 100 units/mL penicillin, 100 μg/mL streptomycin, 2.438 G/L sodium bicarbonate Dulbecco's Modified Eagle Medium (DMEM)/F12 medium (CASSION, Taichung, Taiwan) was cultured at 37°C in a humidified 5% carbon dioxide environment for 24 Hour.

Example 1 Cell Viability Test of VIC Cells

The VIC cells of Preparation Example 1 were planted on a 96-well plate at a cell density of 3000 cells per well. After waiting for 24 hours for the cells to attach, the following groups were treated respectively: (1) with concentrations of 0.1 mM and 0.2 mM , 0.5 mM, 1 mM, 10 mM H ₂ O ₂ for 15 minutes, 1 hour, 4 hours; (2) 0.01 mg/mL (mg/mL), 0.1 mg/mL, 0.5 mg/mL, 1 mg /mL, 5 mg/mL phycoxanthin aqueous solution for 24 hours, 48 hours, 72 hours; (3) 0.01 mg/mL (mg/mL), 0.1 mg/mL, 0.5 mg/mL, 1 mg/mL After the brownin aqueous solution was treated for 24 hours, it was treated with 0.5 mM H ₂ O ₂ for 15 minutes. Next, culture medium containing 1 mg/mL MTT reagent in serum-free medium for 3 hours and then remove the medium. Then formazan crystals were dissolved in 100 μL of dimethyl sulfoxide (DMSO) per well and read at 570 nm with a microplate analyzer (VERSA Max Molecular device, San Jose, CA, USA). (nm) and absorbance at 630 nm.

The results are shown in Figures 1A to 1C. It can be seen from Figure 1A that the treatment of H ₂ O ₂ in group (1) for 15 minutes, 1 hour and 4 hours will cause oxidative stress to the VIC cells, and the VIC will be affected at 15 minutes. The cells obviously produced oxidative stress, which inhibited the cell survival rate, so the group treated with 0.5 mM H ₂ O ₂ for 15 minutes (about 80% cell survival rate) was selected for subsequent phycoxanthin treatment experiments. According to Fig. 1B, in the treatment of group (2), except that phycoxanthin may be toxic to cells at 5 mg/mL, other concentrations will not significantly affect the cell survival rate of VIC cells. While Fig. 1C shows the results of group (3) treatment, phycoxanthin can alleviate the growth inhibition of VIC cells caused by H ₂ O ₂ treatment.

Example 2 Cell count

The VIC cells of Preparation Example 1 were planted on a 96-well plate at a cell density of 3000 cells per well, and waited for 24 hours for the cells to attach. Treat with 0.01 mg/mL, 0.1 mg/mL, 0.5 mg/mL, 1 mg/mL phycoxanthin aqueous solution for 24 hours and then treat with 0.5 mM hydrogen peroxide for 15 minutes. Next, cells were detached with trypsin, and the cell suspension was mixed with 0.4% trypan blue solution. Cell numbers were counted using a hemocytometer (Hausser scientific company, Horsham, PA, USA) and an inverted phase-contrast microscope at 200X magnification.

The results are shown in Figure 2, H ₂ O ₂ treatment can cause cell death, and phycoxanthin can alleviate the growth inhibition of VIC cells caused by H ₂ O ₂ treatment.

Example 3 Propidium iodide (PI) cell staining

The VIC cells of Preparation Example 1 were planted on a 6-well plate at a cell density of 3×10 ⁵ cells per well, and waited 24 hours for the cells to attach. Firstly, 0, 0.01 mg/mL, 0.1 mg/mL, 0.5 mg/mL, 1 mg/mL phycoxanthin aqueous solution was pretreated for 24 hours and then treated with 0.5 mM hydrogen peroxide for 15 minutes. and H ₂ O ₂ treatment group as the control group. The samples were stained with PI solution (1 μg/mL) dissolved in sterile water for 1 hour, and the DNA damage was observed with a phase-contrast inverted microscope at a magnification of 200 magnifications, and the results of PI staining were quantified with the image processing software Image J.

The results are shown in Figures 3A and 3B. The treatment with H ₂ O ₂ can damage the cells, allowing PI to enter the cells to stain nucleic acids, while the group pretreated with phycoxanthin can avoid DNA damage caused by oxidative stress. damage.

Example 4 Active oxygen density analysis

The VIC cells of Preparation Example 1 were planted on a 6-well plate at a cell density of 3×10 ⁵ cells per well, and waited 24 hours for the cells to attach. Treat with 0.01 mg/mL, 0.1 mg/mL, 0.5 mg/mL, 1 mg/mL phycoxanthin aqueous solution for 24 hours and then treat with 0.5 mM hydrogen peroxide for 15 minutes. Next, cells were treated with 25 μM of 2 ,7 -dichlorofluorescin diacetate (DCFDA; Cayman) for 30 minutes. Fluorescence was captured using a fluorescence microscope at 200X magnification and the ROS density within single cells was quantified using Image J.

The results are shown in Figure 4A, 4B, H ₂ O ₂ treatment will lead to high reactive oxygen species, which may lead to valve structural damage and calcification. However, pretreatment with phycoxanthin can reduce the amount of fluorescence, that is, the density of active oxygen.

Example 5 Protein extraction and western blot method

The VIC cells of Preparation Example 1 were planted on a 6-well plate at a cell density of 3×10 ⁵ cells per well, and waited 24 hours for the cells to attach. Treat with 0.01 mg/mL, 0.1 mg/mL, 0.5 mg/mL, 1 mg/mL phycoxanthin aqueous solution for 24 hours and then treat with 0.5 mM H ₂ O ₂ for 15 minutes. Next, the cells were detached with trypsin. Cells in each group were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer, protease and phosphatase inhibitors. After quantification with bicinchoninic acid (BCA), carry out sodium dodecyl sulfate polyacrylamide gel electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) and transfer to polyvinylidene fluoride (polyvinylidene fluoride, PVDF) membrane. 5% bovine serum albumin (Bovine Serum Albumin, BSA) solution was used to block for 1 hour. Next, the PVDF membrane was mixed with primary antibodies: PARP (1:1000; Cell Signaling, Boston, MA, USA), glyceraldehyde 3-phosphate dehydrogenase (Glyceraldehyde 3-phosphate dehydrogenase, GAPDH) (1:10,000; Proteintech, Rosemont, IL, USA), p-Akt (1:1000; Cell Signaling), Akt (1:1000; Cell Signaling), p44/42 MAPK (Erk1/2) (1:1000; Cell Signaling), phospho-p44 /42 MAPK (Erk1/2) (1:1000; Cell signaling), MMP-2 (1:1000; Abcam), Bax (1:1000; cell signaling) and Bcl-2 (1:500; santacruz, Santa Cruz , CA, USA) at 4°C overnight, washed three times, and then reacted with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000 to 10,000) for 2 hours. The electronic tablet imager eBlot Touch Imager ^tm (eBlot Photoelectric Technology, Shanghai, China) was used to receive the signal, and the band concentration was determined using Image-J software version 1.52t (NIH, Bethesda, MD, USA). The expression of the target protein was analyzed in three different experiments.

The results are shown in Figure 5A, under the treatment of H ₂ O ₂ , the expression level of cleaved PARP protein was significantly increased. However, if pretreated with phycoxanthin, the protein expression level of cleavage PARP can be restored. As shown in Figure 5B, the upper thicker band is the intact Caspase 3, while the lower band is the cleaved Caspase 3, H ₂ O ₂ treatment will promote cell apoptosis, that is, the expression of the protein that promotes the cleaved caspase 3 will increase , Increase the ratio of Bax/Bcl-2, and this phenomenon can be restored if pretreated with phycoxanthin. The above data of PARP protein and caspase 3 protein show that the treatment of phycoxanthin has anti-apoptotic effect on VIC cells.

The Akt/ERK pathway is activated due to oxidative stress that promotes valve calcification. As shown in Figures 6A and 6B, H ₂ O ₂ treatment significantly increased the protein expression of phosphorylated Akt (p-Akt) and ERK, while pretreatment with phycoxanthin decreased the activation of the aforementioned proteins. And as shown in FIG. 6C , the pretreatment with phycoxanthin can reduce the increase in the expression level of the extracellular matrix remodeling marker MMP-2 protein in VIC cells caused by H ₂ O ₂ treatment. Therefore, fucoxanthin treatment can prevent or reduce heart valve calcification.

Example 6 Alizarin red staining

Calcification progression was assessed by Alizarin Red staining. The VIC cells of Preparation Example 1 were planted on a 6-well plate at a cell density of 3×10 ⁵ cells per well, and waited 24 hours for the cells to attach. Treat with 0.01 mg/mL, 0.1 mg/mL, 0.5 mg/mL, 1 mg/mL phycoxanthin aqueous solution for 24 hours and then treat with 0.5 mM hydrogen peroxide for 15 minutes. Cells were fixed with 4% paraformaldehyde at room temperature for 10 minutes and stained with Alizarin Red (Sciencell, Carlsbad, CA, USA) for 30 minutes. Finally, the stained image was captured by a fluorescent microscope, and the absorbance value at 405 nm was read with a microwell plate analyzer (VERSA Max Molecular device, San Jose, CA, USA).

The experimental results are shown in Figures 7A and 7B. The degree of calcification of VIC cells increased after treatment with H ₂ O ₂ , while the degree of calcification could be reduced under pretreatment with phycoxanthin.

Example 7 Animal experiments

For 26 dogs diagnosed with heart disease by veterinarians, the age of the dogs was 7-15 years old, and the weight was between 1.85 and 8.3 kg. The heart problem of the dogs was degenerative heart valve disease, and the degenerative changes of the valves were pathological. It is scientifically called myxomatous degeneration (myxomatous degeneration, MXD), which is related to genes and aging. During the period, a large number of endocardial and valvular cells age and die, causing glycosaminoglycan (GAG) infiltration and increased valve thickness during the course of the disease. , The number of interstitial cells increases, the collagen bundles are destroyed and the elastic fibers split, and the chordae tendineae are extended or even broken. Due to valve deformation and degeneration, resulting in incomplete closure (mitral regurgitation), systolic blood backflow to the atrium. In the way of two times a day, give Zhaxinpei (Zhonghua Ocean, Taipei, Taiwan) containing phycoxanthin. The composition of Zaoxinpei (300 mg) is: 125 mg fucoxanthin, 125 mg small molecule fucose, 10 mg coenzyme Q10, 10 mg astaxanthin, 24.5 mg microcrystalline cellulose, 5.5 mg stearic acid magnesium. Among them, phycoxanthin was administered at a dose of 60 mg/kg, combined with medical treatment for 0.5 to 2 years, and the medical treatment was based on ACVIM 2019 consensus guidelines for the diagnosis and treatment of myxomatous mitral valve disease in dogs. Triple drug therapy (pimobendan, ACEI, furosemide, etc.) is the main therapy, and the types and doses of drugs are selected according to the clinical manifestations and needs of the cases. Cardiac ultrasound combined with Doppler (Doppler) ultrasound is used to detect and track the function of heart valves, which is equipped with an array of ultrasound probes (PA-122, frequency 3-8 megahertz (MHz)) Diagnostic system (Esaote's MyLab™ClassC ^® , Italy) for examination. Left atrial to aortic ratio (LA/AO) was measured in B-mode short-axis five-chamber view of the right sternum wall.

In heart valve disease, there will be an increase in VHS value and LA/AO value. Veterinarians generally use cardiac ultrasound index: Tei index to evaluate the systolic and diastolic function of the heart and use the E/e' ratio to evaluate the mitral valve Inflow and analysis of mitral valve leakage and abnormal left ventricular diastolic pressure, while the E/e' ratio is generally <12 in dogs aged 6 to 10 years. The measurement results of relevant parameters are shown in Figures 8A to 8H, in which the cone heart score (vertebral heart size, VHS) is shown in Figure 8A and Figure 8B. Normalized to reflect compensated cardiomegaly. Long-term administration of phycoxanthin to dogs was shown to restore heart valve function and leakage in dogs as VHS decreased. Figure 8C and Figure 8D show the improved LA/AO value; Figure 8E shows the reduced Tei index after phycoxanthin treatment; Figure 8F shows the reduction of E/e' ratio and other improvements. Among them, Figures 8B, 8D, 8E, and 8F are respectively expressed by the improvement degree of VHS, LA/AO value, Tei index, and E/e' ratio.

Figure 8G and Figure 8H show that the connection between the mitral valve and the tricuspid valve is significantly reduced in the echocardiogram. Thus, phycoxanthin treatment may improve overall ventricular contraction and relaxation, valve function, and compensatory cardiomegaly.

Therefore, this example proves that phycoxanthin combined with traditional heart medication can improve the valve degeneration in dogs, restore the appearance of the valve to a smoother appearance, reduce regurgitation, and improve the overall heart function and deformation.

In summary, phycoxanthin has the potential to protect heart valve cells from damage caused by high oxidative stress. It can reduce the growth inhibition of VIC cells caused by H ₂ O ₂ , avoid the damage of VIC cells and DNA damage caused by oxidative stress; reduce the density of reactive oxygen species, restore the apoptosis caused by H ₂ O ₂ , and alleviate the damage caused by H ₂ O ₂ valve calcification; improve mitral valve leakage, tricuspid valve leakage, and compensatory heart enlargement in dogs in animal experiments.

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Figure 1A is the cell survival rate of VIC cells treated with H ₂ O ₂ at a concentration of 0.1 mM to 10 mM for 15 minutes, 1 hour, and 4 hours, *** indicates that compared with the control group, p <0.001; Figure 1B is VIC Cell survival rates were treated with phycoxanthin at a concentration of 0.01 mg/mL to 5 mg/mL for 24 hours, 48 hours, and 72 hours. *** indicates that compared with the control group, p <0.001; Figure 1C is VIC Cell survival rate after pretreatment with phycoxanthin at a concentration of 0.01 mg/mL to 1 mg/mL for 24 hours, then treated with 0.5 mM H ₂ O ₂ for 15 minutes, *** indicates that compared with the control group, p <0.001;# means p < 0.05 compared with H ₂ O ₂ treatment group; ## means p < 0.01 compared with H ₂ O ₂ treatment group; After pretreatment with mg/mL phycoxanthin for 24 hours, the cell survival rate was treated with 0.5 mM H ₂ O ₂ for 15 minutes, *** indicates that compared with the control group, p <0.001;### indicates the difference with H ₂ Compared with the O ₂ treatment group, p <0.001; Figure 3A is the VIC cells pretreated with phycoxanthin at a concentration of 0.01 mg/mL to 1 mg/mL for 24 hours, and then treated with 0.5 mM H ₂ O ₂ for 15 minutes PI staining; Figure 3B is the quantitative result of PI staining of VIC cells pretreated with phycoxanthin at a concentration of 0.01 mg/mL to 1 mg/mL for 24 hours, then treated with 0.5 mM H ₂ O ₂ for 15 minutes,*** Indicates that compared with the control group, p <0.001;### indicates that compared with the H ₂ O ₂ treatment group, p <0.001; Figure 4A shows the VIC cells treated with phycoxanthin at a concentration of 0.01 mg/mL to 1 mg/mL After 24 hours of pretreatment, DCFDA presents the results of DCFDA treated with 0.5 mM H ₂ O ₂ for 15 minutes; Figure 4B shows VIC cells pretreated with phycoxanthin at a concentration of 0.01 mg/mL to 1 mg/mL for 24 hours After treatment with 0.5 mM H ₂ O ₂ for 15 minutes, the reactive oxygen species density, *** indicates that compared with the control group, p <0.005;### indicates that compared with the H ₂ O ₂ treated group, p <0.001; Figure 5A is the total PARP protein and cleaved PARP protein expression levels of VIC cells pretreated with phycoxanthin with a concentration of 0.01 mg/mL to 1 mg/mL for 24 hours, and treated with 0.5 mM H ₂ O ₂ for 15 minutes, *** indicates that compared with the control group, p <0.001;### indicates that compared with the H ₂ O ₂ treatment group, p <0.001; After 24 hours of phycoxanthin pretreatment, the expression levels of apoptosis-related proteins were treated with 0.5 mM H ₂ O ₂ for 15 minutes; After 24 hours of pretreatment, the protein expression of phosphorylated Akt and Akt treated with 0.5 mM H ₂ O ₂ for 15 minutes, *** indicates that compared with the control group, p <0.001;### indicates the difference with H ₂ O ₂ Compared with the treatment group, p < 0.001, ## indicates that compared with the H ₂ O ₂ treatment group, p <0.01; Figure 6B shows that VIC cells were pretreated with phycoxanthin at a concentration of 0.01 mg/mL to 1 mg/mL After 24 hours, the expression levels of phosphorylated ERK and ERK proteins were treated with 0.5 mM H ₂ O ₂ for 15 minutes, ** indicates that compared with the control group, p <0.01; After pretreatment with phycoxanthin from mL to 1 mg/mL for 24 hours, the expression of MMP-2 protein was treated with 0.5 mM H ₂ O ₂ for 15 minutes; /mL of phycoxanthin pretreated for 24 hours, then treated with 0.5 mM H ₂ O ₂ for 15 minutes to stain the calcification results; After 24 hours of pretreatment, the staining and quantification results of the degree of calcification treated with 0.5 mM H ₂ O ₂ for 15 minutes, *** indicates that compared with the control group, p <0.001;### indicates that compared with the H ₂ O ₂ treated group Ratio, p <0.001; Figure 8A is the VHS score measured by X-ray film after the long-term administration of phycoxanthin to the dogs in Example 7; VHS score, *** indicates p < 0.001 compared with the baseline (before the administration of phycoxanthin); Figure 8C is the LA/AO value obtained by cardiac ultrasound after long-term administration of phycoxanthin to the dog in Example 7 ; Figure 8D is the LA/AO value of the dogs in Example 7 after long-term administration of phycoxanthin; Figure 8E is the Tei index of the dogs in Example 7 after long-term administration of phycoxanthin, *** indicates the difference from the baseline ( before administration of phycoxanthin), p <0.001; Figure 8F is the E/e' ratio of the dogs in Example 7 after long-term administration of phycoxanthin, *** indicates the difference from the baseline (before the administration of phycoxanthin ) compared with p <0.001; Figure 8G is the connection of the mitral valve after long-term administration of phycoxanthin in the dog of Example 7; Figure 8H is the connection of the tricuspid valve after long-term administration of phycoxanthin in the dog of Example 7 link.

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Claims (10)

A use of phycoxanthin for preparing a composition for preventing, treating or improving heart diseases. According to the use described in claim 1, the heart disease is a heart valve disease. According to the use described in Claim 1, the heart disease includes: heart valve calcification, mitral valve leakage, tricuspid valve leakage and compensatory heart enlargement. The use according to claim 1, wherein the subject of administration of the composition is a mammal. The use according to claim 4, wherein the mammal is a human being. The use according to any one of claims 1 to 5, wherein the composition is a pharmaceutical composition or a food composition. The use as described in Claim 6, wherein the composition is a pharmaceutical composition, and the pharmaceutical composition contains an effective dose of phycoxanthin and a pharmaceutically acceptable carrier. The use as described in Claim 7, wherein the pharmaceutical composition is an oral dosage form. The use according to claim 8, wherein the subject of administration of the pharmaceutical composition is a human being, and the administration dose is 25 mg/kg to 100 mg/kg per day. The use according to claim 6, wherein the composition is a food composition, and the food composition is health food, health food or functional food.