Use of tyrosol-beta-d-galactoside in the prevention and treatment of hyperlipidemia and atherosclerotic diseases

Abstract

The present application relates to the use of tyrosol-beta-D-galactoside in the prevention and treatment of hyperlipidemia and atherosclerotic diseases. Specifically, the present application relates to the use of tyrosol-beta-D-galactoside or a solvate or hydrate thereof in the preparation of a medicament for the treatment and / or prevention of hyperlipidemia, or atherosclerosis. In another aspect, the present application relates to the use of tyrosol-beta-D-galactoside or a solvate or hydrate thereof in the preparation of a food composition for assisting in maintaining a healthy level of blood lipids.

Description

Technical Field

[0001] This invention belongs to the technical fields of medicine, food, and health products. Specifically, this invention relates to the use of tyrosol-β-D-galactoside in the treatment of hyperlipidemia and atherosclerosis. Background Technology

[0002] Hyperlipidemia, also known as dyslipidemia, refers to elevated levels of total cholesterol (TC) and low-density lipoprotein (LDL-C) in plasma. It also refers to elevated levels of various blood lipids or lipoproteins, including mixed hyperlipidemia. It is a common metabolic disorder. Hyperlipidemia is a key risk factor for obesity, atherosclerosis, and type II diabetes. It is characterized by the absence of obvious symptoms in the early stages, with cardiovascular disease (CVD) being the first symptom [Alloubani A et al., Relationship between Hyperlipidemia, Cardiovascular Disease and Stroke: A Systematic Review [J]. Current Cardiology Reviews, 2021, 17(6).e051121189015]. Atherosclerosis (AS) is an important branch of CVD and a very common chronic vascular disease characterized by the formation and attachment of atherosclerotic plaques on the inner wall of the aorta. The main components of these plaques include lipids, necrotic tissue, and fibrous tissue [NCD Risk Factor Collaboration (NCD-RisC), Repositioning of the global epicentre of non-optimal cholesterol [J]. Nature, 2020, 582(7810): 73–7]. The most serious complications resulting from AS plaque rupture are myocardial infarction, cerebral ischemia, and peripheral tissue ischemia.

[0003] Hyperlipidemia is a key contributing factor to atherosclerosis (AS). Early stages of AS manifest as dyslipidemia. Clinically, hyperlipidemia is classified into high total cholesterol (TC), high triglyceride (TG), mixed hyperlipidemia, and low high-density lipoprotein cholesterol (HDL-C). Low-density lipoprotein cholesterol (LDL-C) levels are a core indicator for assessing cardiovascular disease risk and achieving treatment goals, and are the primary target for intervention in atherosclerotic cardiovascular disease (ASCVD) [Gaggini M et al., Lipids in Atherosclerosis: Pathophysiology and the Role of Calculated Lipid Indices in Assessing Cardiovascular Risk in Patients with Hyperlipidemia [J]. International Journal of Molecular Sciences, 2022, 24(1)].

[75] ; Mixed hyperlipidemia is a condition in which serum TC (≥5.20 mmol / L) and TG (≥1.70 mmol / L) are elevated simultaneously. Mixed hyperlipidemia not only leads to the occurrence and development of atherosclerosis, but also poses a risk of pancreatitis.

[0004] The development of atherosclerosis (AS) is mainly divided into four stages, including LDL-C elevation, AS initiation, atherosclerotic plaque formation, and atherosclerotic plaque rupture. AS is initiated by LDL-C. Excess free LDL-C particles in the blood pass through endothelial cells and are oxidized into oxidized low-density lipoprotein (ox-LDL) [Jebari-Benslaiman S et al., Pathophysiology of Atherosclerosis [J]. International Journal of Molecular Sciences, 2022, 23(6): 3346]. The formation of atherosclerotic plaques in AS is caused by the accumulation of ox-LDL after macrophages and vascular smooth muscle cells absorb ox-LDL and undergo foaming. Endothelial damage caused by atherosclerosis (AS) is generally accompanied by oxidative stress and inflammation, which disrupts the integrity of the endothelium. This makes it easier for free lipids to pass through the gaps in the endothelium and enter the vascular wall. The lipids adhere to the vascular wall and lead to the formation of atherosclerotic plaques [Batty M et al., The Role of Oxidative Stress in Atherosclerosis [J]. Cells, 2022, 11(23): 3843]. Vascular stenosis can induce various cardiovascular diseases such as coronary heart disease, angina pectoris, and myocardial infarction, and may also lead to heart failure [Jebari-Benslaiman S et al., Pathophysiology of Atherosclerosis [J]. International Journal of Molecular Sciences, 2022, 23(6): 3346].

[0005] Statins are currently the first-line drugs for treating hyperlipidemia and AS, thereby improving the progression of CVD such as hyperlipidemia and AS. From the discovery of mevastatin in 1976 to the successive launches of structurally optimized analogues such as pravastatin and lovastatin, and their widespread global application, it has become an important milestone in the prevention and treatment of cardiovascular diseases. However, the therapeutic dose and toxic dose range of statins are relatively narrow. For example, the lipid-lowering dose of atorvastatin is 10 mg / kg, while 30 mg / kg of atorvastatin can increase liver drug-metabolizing enzymes in mice by 3 times [Herron. CE et al., Toxicokinetics and toxicity of atorvastatin dogs [J]. Toxicology and Applied Pharmacology, 2015, 289(1): 117–23]. Statins have various adverse reactions when used long-term in clinical practice, such as gastrointestinal adverse reactions, hepatotoxicity, rhabdomyolysis, nephrotoxicity and other toxic side effects. In addition, statins have very limited efficacy in reducing serum high TG in the treatment of mixed hyperlipidemia, and may also increase the risk of new-onset diabetes [Zhao Shuiping. Expert consensus on safety evaluation of statins [J]. Chinese Journal of Cardiovascular Diseases, 2014, 42(11): 890–4].

[0006] The most important goal of treating hyperlipidemia or ankylosing spondylitis (AS) is to lower LDL-C. Currently, the LDL-C target for adults in my country is <3.37 mmol / L (130 mg / dL). However, patients with concurrent hypertension, coronary heart disease, diabetes, carotid or coronary artery plaques, or peripheral artery disease should aim to lower their LDL-C to 2.6 mmol / L or even below 1.8 mmol / L [Scott M. Grundy et al., Executive Summary of the Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) [J]. Journal of the American Medical Association, 2001, 285(19): 2486–97]. Currently, the dosage of commonly used statins is difficult to achieve or maintain LDL-C <2.6 mmol / L.

[0007] The development of new drugs for the treatment of cardiovascular diseases, especially for the effective control of blood lipids, has always been a research hotspot. For example, Rhodiola rosea injection can significantly improve the serum levels of TC, TG, LDL-C and HDL-C in patients with acute cerebral infarction [Chen Shu et al., Effects of Rhodiola rosea injection as adjuvant therapy on blood glucose, blood lipids and clinical efficacy in patients with acute cerebral infarction [J]. Chinese Journal of Biochemical Pharmaceuticals, 2015, 35(11): 158–60]. Rhodiola rosea L. is a herbaceous plant belonging to the genus Rhodiola in the family Crassulaceae. More than 140 chemical structures have been identified, some of which have significant pharmacological activities, such as salidroside (tyrosol-β-D-glucopyranoside), tyrosol, and sorbitol. Animal studies have shown that salidroside can reduce LDL-C and TG, and delay the progression of atherosclerosis [Xing SS, et al., Salidroside improves endothelial function and alleviates atherosclerosis by activating a mitochondria-related AMPK / PI3K / Akt / eNOS pathway [J]. Vascul Pharmacol, 2015, 72: 141–52]. However, it has the following shortcomings: ① Rhodioloside can significantly prolong the tail bleeding time in mice, and long-term use carries the risk of internal bleeding [Lin Xiaojian et al., Rhodioloside's antiplatelet activity and antithrombotic effect on mice [J]. Journal of Guizhou Medical University, 2019, 44(09): 1039–43]. As a treatment for chronic diseases, the risk of internal bleeding with rhodioloside is a serious drawback; ② Some scholars have pointed out that long-term use of glucosinolate drugs may alter some normal metabolic balance in the body [Melisi D et al., D-galactose as a vector for prodrug design [J]. Current Topics in Medicinal Chemistry, 2011, 11(18): 2288–98; ③ In addition, high-purity rhodioloside (over 99%) is expensive (about 12,000 yuan / gram), which greatly limits its drugability (www.medchemexpress.cn / Salidroside.html or http: / / www.winherb.cn / product / 19855.html); Therefore, there is an urgent need to develop a drug that is highly safe, can effectively lower lipids (especially LDL-C), and can effectively prevent and treat hyperlipidemia and AS, in order to effectively inhibit the occurrence of malignant cardiovascular and cerebrovascular events.

[0008] Although tyrosol β-D-galactoside (Tgal) has a similar structure to rhodioloside, it has not been reported in the identified components of Rhodiola rosea [Du Baoyu et al., Research progress on chemical constituents and pharmacological effects of Rhodiola rosea [J]. Pharmaceutical Research, 2025, 44(9): 905–12]. Tgal is an active molecule of non-natural origin, and there are also significant differences in pharmacological properties between the two. For example, ① Tgal did not show any risk of internal bleeding in tail hemorrhage and other tests. ② Tgal exhibits a unique dual regulation in hyperlipidemic mice, inhibiting cholesterol synthesis while promoting cholesterol excretion (via feces), and this dual regulation effect is the first discovery. ③ The test drug Tgal of this invention is synthesized by enzymatic method in our laboratory [He Bingfang et al., A high-efficiency galactosidase resistant to organic solvents and its application, CN108148824A [P / OL]], which is easy to prepare Tgal with a purity of over 99% and the cost is controllable.

[0009] Tyrosol-β-D-galactoside (Tgal) has tyrosol as its parent compound and is widely found in fermented foods such as beer, black tea, and cheese, rather than as a deliberately added functional additive. Hydroxytyrosol (3,4-dihydroxyphenylethanol) and tyrosol (4-hydroxyphenylethanol) are both natural phenolic compounds found in olive oil. Hydroxytyrosol has been officially approved for use as a food additive in China and is classified as an antioxidant food additive.

[0010] Currently reported pharmacological and pharmacodynamic studies on tyrosol-β-D-galactoside (Tgal) only utilize chemically synthesized products, specifically involving: Tgal significantly prolonging the time to exhaustion and effectively improving oxygen utilization in anti-hypoxia and fatigue resistance experiments [Sun Xiaoli et al., Application of tyrosol galactoside in the preparation of anti-hypoxia and anti-fatigue drugs, CN101653445A[P / OL]]; Tgal exerts in vitro and in vivo neuroprotective effects [SHI TY et al., Neuroprotective Effects of Salidroside and its Analogue Tyrosol Galactoside Against Focal Cerebral Ischemia In Vivo and H2O2-Induced Neurotoxicity In Vitro [J]. Neurotoxicity Research, 2011, 21(4): 358–67]; and protective effects against ischemia / reperfusion of the heart and brain. Tgal showed significant protective effects in myocardial and cerebral ischemia models [Shi Tianyao et al., Study on the synthesis of tyrosol galactoside and its protective effect on ischemic / reperfusion heart and brain [J]. Journal of Cardiology, 2010, 22(4): 524–8]. No applications of Tgal in the prevention and treatment of hyperlipidemia and atherosclerosis have been reported. Summary of the Invention

[0011] The inventors unexpectedly discovered that Tgal exhibits highly effective lowering of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C), demonstrating significantly superior lipid-lowering capabilities compared to clinical drugs that inhibit cholesterol synthesis in vivo, namely the statins rosuvastatin (RSV) and lovastatin (LOV). Furthermore, Tgal's inhibition of fatty acid (TG) synthesis further broadens its efficacy against mixed hyperlipidemia. Tgal significantly improves inflammatory markers in hyperlipidemia and atherosclerosis, inhibiting the formation of lipid plaques and fibrosis in the aortic intima. Tgal's effect on improving atherosclerotic plaques is also significantly superior to that of the statin rosuvastatin (RSV). Moreover, Tgal shows no risk of internal bleeding. Therefore, Tgal has great potential for development into a drug for the prevention and treatment of hyperlipidemia and atherosclerosis.

[0012] In a first aspect, the present invention provides the use of tyrosol-β-D-galactoside or its solvate or hydrate in the preparation of medicaments for treating and / or preventing hyperlipidemia or atherosclerosis, wherein said tyrosol-β-D-galactoside has the structure shown in Formula I: Formula (I) Tyrosol galactoside, abbreviated as Tgal, chemical name: 1-(4-hydroxy)-phenylethyl-β-D-pyranoside, is an active molecule of non-natural origin.

[0013] In one aspect, the present invention provides the use of tyrosol-β-D-galactoside or its solvates or hydrates in the preparation of medicaments for the treatment and / or prevention of hyperlipidemia.

[0014] Specifically, the hyperlipidemia includes, but is not limited to, hypercholesterolemia, hypertriglyceridemia, and mixed hyperlipidemia.

[0015] Specifically, the hypercholesterolemia includes elevated serum total cholesterol and / or elevated serum low-density lipoprotein cholesterol.

[0016] As used in this article, mixed hyperlipidemia refers to elevated serum total cholesterol and / or low-density lipoprotein cholesterol, as well as elevated serum triglycerides.

[0017] Tyrosol-β-D-galactoside can lower serum total cholesterol, low-density lipoprotein cholesterol, and total triglycerides, and increase high-density lipoprotein cholesterol by inhibiting cholesterol and fatty acid synthesis and promoting cholesterol excretion in feces. In a further aspect, the present invention provides the use of tyrosol-β-D-galactoside or its solvate or hydrate in the preparation of medicaments for the treatment and / or prevention of atherosclerosis.

[0018] In this embodiment, tyrosol-β-D-galactoside can inhibit the formation of lipid plaques and fibrosis in the aortic intima and improve vascular inflammation markers in atherosclerosis. In this embodiment, the safety of tyrosol-β-D-galactoside, including risk of internal bleeding and acute toxicity, was evaluated. Experiments on the effect of Tgal on physiological hemostasis showed that it did not affect wound healing time, tail bleeding time, or bleeding volume. High-dose (4500 mg / kg) acute toxicity tests of Tgal verified that it had no effect on liver and kidney function, blood lipids, or organs such as the heart, liver, spleen, lungs, and kidneys in mice. In a second aspect, this invention provides the use of tyrosol-β-D-galactoside or its solvates or hydrates in the preparation of food compositions that help maintain the body's antioxidant levels, help maintain healthy blood lipids, and / or help improve hyperlipidemia and / or atherosclerosis, wherein the tyrosol-β-D-galactoside has the structure shown in Formula I: In a specific implementation scheme, the food composition of formula (I) is a health food, functional food, special dietary food, solid beverage, compressed candy, capsule, granule, oral liquid, powder or granule.

[0019] In a third aspect, the present invention provides a composition for lowering blood lipids and / or improving atherosclerosis, comprising tyrosol-β-D-galactoside or its solvate or hydrate and one or more pharmaceutically or food-acceptable carriers, excipients or excipients.

[0020] In embodiments of the composition, the tyrosol-β-D-galactoside or its solvate or hydrate is the main active ingredient, and its content is from 1% to 100%.

[0021] In embodiments of the composition, the composition is a pharmaceutical composition or a food composition, preferably, the dosage form of the composition is a tablet, pill, powder, capsule, injection, ointment, spray or pill.

[0022] In a fourth aspect, the present invention provides a method for treating and / or preventing hyperlipidemia or atherosclerosis, the method comprising administering to a subject in need a therapeutically effective amount of tyrosol-β-D-galactoside or its solvate or hydrate, or a pharmaceutical composition comprising thereto.

[0023] As used herein, the term "pharmaceutically acceptable carrier" refers to an inert or substantially inert substance used in the preparation of a pharmaceutical composition that, when used in conjunction with the active ingredient within an effective therapeutic dose range, does not produce significant toxicity, immunogenicity, or adverse reactions, and ensures the stability, bioavailability, and suitability for administration of the active ingredient.

[0024] The pharmaceutically acceptable carrier may be in solid, liquid or semi-solid form, including but not limited to excipients, diluents, solvents, solubilizers, stabilizers, disintegrants, lubricants, glidants, emulsifiers, buffers and preservatives.

[0025] In some embodiments, the pharmaceutically acceptable carrier can be used to prepare different dosage forms, such as oral formulations, injectable formulations, topical formulations, sustained-release formulations, or controlled-release formulations, for example, tablets, capsules, granules, solutions, suspensions, emulsions, or lyophilized formulations.

[0026] In a fifth aspect, the present invention provides the use of tyrosol-β-D-galactoside or its solvation or hydrate in the preparation of a pharmaceutical composition for lowering blood lipids and / or improving atherosclerosis, wherein the pharmaceutical composition comprises tyrosol-β-D-galactoside or its solvation or hydrate and one or more pharmaceutically acceptable carriers, excipients or excipients.

[0027] In a specific implementation, the tyrosol-β-D-galactoside or its solvate or hydrate is the main active ingredient, with a content of 1 to 100%.

[0028] In specific implementation schemes, the dosage form of the pharmaceutical composition is tablets, pills, powders, capsules, injections, ointments, sprays, or pills.

[0029] Those skilled in the art should understand that any carrier that can be used in the preparation of pharmaceutical formulations and has safety, compatibility and stability under administration conditions, and does not affect the therapeutic effect of the active ingredient, falls within the scope of the "pharmaceutically acceptable carriers" described in this invention.

[0030] As used herein, the term "pharmaceutical composition" refers to a formulation system containing one or more active ingredients and one or more pharmaceutically acceptable carriers or excipients, which may be used to prevent, treat or improve a disease, symptom or pathological condition and may be suitable for different routes of administration.

[0031] The active ingredient in the pharmaceutical composition may be a single compound or a combination of multiple compounds, and may coexist with pharmaceutically acceptable carriers, excipients, diluents, solvents, stabilizers, solubilizers, disintegrants, lubricants or other excipients to form a dosage form suitable for clinical use.

[0032] In some embodiments, the pharmaceutical composition may be a solid dosage form, a liquid dosage form, or a semi-solid dosage form, such as tablets, capsules, granules, solutions, suspensions, emulsions, injections, or lyophilized preparations. In some preferred embodiments, the pharmaceutical composition is suitable for oral, injectable, topical administration, or other systemic or topical routes of administration.

[0033] As used in this article, “treatment” refers to the process of intervening in a disease, symptom or pathological state that has occurred or is at risk of occurring, with the aim of alleviating or eliminating disease signs and symptoms, delaying or halting disease progression, improving or restoring bodily functions, or reducing disease-related complications and the risk of death.

[0034] The term "treatment" includes not only the management of clinically diagnosed diseases, but also the regulation of disease-related pathological processes, such as the improvement of inflammatory responses, immune abnormalities, tissue damage, or fibrosis.

[0035] In some implementations, "treatment" may include varying degrees of therapeutic effects such as complete remission, partial remission, reduced disease activity, reduced relapse frequency, or improved quality of life.

[0036] The term "therapeutic effective dose" refers to the minimum dosage range required to achieve the desired therapeutic or improvement effect. Attached Figure Description

[0037] Figure 1 This diagram shows the enzymatic synthesis of tyrosol-β-D-galactoside Tgal.

[0038] Figure 2 The image shows a high-performance liquid chromatogram of the Tgal purified sample.

[0039] Figure 3 This diagram shows a high-cholesterol, high-fat diet-induced hyperlipidemia model (HCHFD) and its grouped treatment. Figure 4 The effects of Tgal on liver coefficient and liver function transaminase activity in hyperlipidemic mice are shown: (A) Liver tissue condition, with a scale bar of 1 cm; (B) Changes in mouse body weight during administration; (C) Mouse liver coefficient; (D) AST activity; (E) ALT activity.

[0040] Figure 5 The effects of Tgal on serum lipids in hyperlipidemic mice are shown as follows: (A) total cholesterol (TC), (B) total triglycerides (TG), (C) low-density lipoprotein (LDL-C), and (D) high-density lipoprotein (HDL-C).

[0041] Figure 6 The effects of Tgal on the transcriptional levels of key genes involved in cholesterol and fatty acid synthesis in the liver of hyperlipidemic mice were shown: (A) HMGCR (3-hydroxy-3-methylglutaryl-CoA reductase), (B) SREBF (sterol regulatory element-binding transcription factor), and (C) Acaca (acetyl-CoA carboxylase α).

[0042] Figure 7 The effects of Tgal on the fecal efflux of cholesterol and the transcriptional levels of efflux-related genes in hyperlipidemic mice were shown: (A) Comparison of cholesterol levels in serum and feces, (B) LXR-α (liver X receptor α), (C) ABCG5 (ATP binding box subfamily G member 5), (D) ABCG8 (ATP binding box subfamily G member 8).

[0043] Figure 8 The effect of Tgal on the transcriptional levels of inflammation-related genes in hyperlipidemic mice was shown: inflammatory factors (A) TNF-α (tumor necrosis factor α), (B) IL-1β (interleukin-1β), (C) IL-6 (interleukin-6), and anti-inflammatory factor (D) IL-10 (interleukin-10).

[0044] Figure 9 The effect of Tgal on oxidative stress levels in hyperlipidemic mice: serum (A) MPO activity, (B) SOD activity.

[0045] Figure 10 The effect of Tgal on liver tissue lesions in hyperlipidemic mice: (A) H&E staining images of representative sections from each group, with a scale bar of 200 μm; (B) Statistical analysis of liver vacuolar area in each group.

[0046] Figure 11 The effect of Tgal on lipid deposition in the liver of hyperlipidemic mice is shown. (A) Oil Red O staining images of representative sections from each group, with a scale bar of 200 μm. (B) Statistical analysis of the lipid droplet area shown by Oil Red staining.

[0047] Figure 12 Showing HFD feeding builds ApoE - / - A schematic diagram of a mouse model of atherosclerosis and grouping of mice for drug administration.

[0048] Figure 13 This study demonstrates the effect of Tgal on lipid plaques (Oil Red staining) on ​​the overall aortic wall of atherosclerotic mice.

[0049] Figure 14 The effects of Tgal on blood lipids in atherosclerotic mice are shown: (A) serum TC content, (B) low-density lipoprotein LDL-C, and (C) high-density lipoprotein HDL-C.

[0050] Figure 15 The effects of Tgal on aortic tissue-related inflammatory factors in atherosclerotic mice are shown: (A) inflammatory factor IL-1β, (B) inflammatory factor IL-6, (C) anti-inflammatory factor IL-4, (D) anti-inflammatory factor IL-13.

[0051] Figure 16 The effect of Tgal on the aortic root cross-sectional plaque in atherosclerotic mice is shown. (A) Oil Red O staining, H&E staining and Masson staining images of aortic root sections from each group of mice, with a scale bar of 500 μm; (B) ImageJ quantitative statistics of Oil Red O staining of aortic root sections; (C) Statistical analysis of Masson staining of aortic root sections.

[0052] Figure 17 The effects of Tgal on major markers of aortic root atherosclerosis (AS) in mice are shown: (A) Immunofluorescence staining images of major markers VCAM-1, CD68, and αSMA (red fluorescence) in the aortic root of mice in each group, with a scale bar of 500 μm; (B) ImageJ statistics of VCAM-1 immunofluorescence staining; (C) Statistical analysis of CD68 immunofluorescence staining; (D) Statistical analysis of αSMA immunofluorescence staining.

[0053] Figure 18 Physiological hemostasis experiments are shown: (A) Schematic diagram of wound healing experiment and grouping scheme, (B) Schematic diagram of tail bleeding experiment and grouping scheme.

[0054] Figure 19 Showing (A) photos of mouse back wounds at different time points, (B) a schematic diagram of wound area at different time points, and (C) the wound healing rate on day 9.

[0055] Figure 20 The display shows (A) the time to stop bleeding in the tail hemorrhage experiment, and (B) the amount of bleeding (hemoglobin concentration). Figure 21 The effects of Tgal on four coagulation functions in rats were shown: (A) prothrombin time (PT), (B) activated partial thromboplastin time (APTT), (C) thrombin time (TT), and (D) fibrinogen (FIB).

[0056] Figure 22 This diagram shows the Tgal acute toxicity test and grouping scheme.

[0057] Figure 23 The effects of high-dose Tgal on mouse body weight and food intake are shown: (A) change in average body weight of mice over 14 days, (B) average feed intake, and (C) average water intake.

[0058] Figure 24 The effects of high-dose Tgal on blood lipids and liver and kidney function in mice were shown: (A) total triglycerides (TG) in serum, (B) total cholesterol (TC), (C) low-density lipoprotein cholesterol (LDL-C), and (D) high-density lipoprotein cholesterol (HDL-C).

[0059] Figure 25 The effects of high-dose Tgal on liver and kidney function in mice were shown: (A) uric acid (UA), (B) creatinine (CRE), (C) aspartate aminotransferase (AST), and (D) alanine aminotransferase (ALT).

[0060] Figure 26 The effects of high-dose Tgal on the organ coefficients of the heart, liver, spleen, lungs, and kidneys in mice are shown: (A) Heart coefficient, (B) Liver coefficient, (C) Spleen coefficient, (D) Lung coefficient, and (E) Kidney coefficient.

[0061] Figure 27 The effects of high-dose Tgal on tissue sections (H&E staining) of the heart, liver, spleen, lung, and kidney are shown: the scale bar for the heart, liver, spleen, and kidney sections is 200 μm, and the scale bar for the lung section is 600 μm; (A) ♂ Photographs of heart, liver, spleen, lungs, and kidney sections from the male control group (B). ♂ Photographs of heart, liver, spleen, lungs, and kidney sections from male Tgal group; (C) Photographs of heart, liver, spleen, lungs, and kidney sections from female control group; (D) Photographs of heart, liver, spleen, lungs, and kidney sections from female Tgal group. Detailed Implementation

[0062] The present invention will be described in detail below with reference to the embodiments, but the implementation of the present invention is not limited thereto. The embodiments described below are only some embodiments of the present invention. For those skilled in the art, the technology implemented based on the above content of the present invention is within the protection scope of the present invention.

[0063] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All references to this specification are incorporated herein by reference to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail. To more clearly illustrate the advantages, objectives, and technical solutions of this invention, this section provides a clear and complete description of the methods, techniques, and materials used in the exemplary embodiments of this invention. Where specific conditions are not specified in the exemplary embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. All reagents and instruments without a specified manufacturer can be purchased commercially available conventional products with equivalent functions.

[0064] I. Experimental Materials and Equipment (1) Experimental reagents Tyrosol-β-D-galactoside (Tgal) was prepared in our laboratory (purity > 99%); control feed and high-fat, high-cholesterol feed for hyperlipidemia were purchased from Jiangsu Xietong Biotechnology Co., Ltd.; rosuvastatin calcium (purity > 99%) and lovastatin (purity > 99%) were purchased from MedChemExpress; 4% paraformaldehyde solution was purchased from Nanjing Senbeijia Biotechnology Co., Ltd.; physiological and biochemical detection kits for TG, TC, HDL-C, LDL-C, AST, ALT, MPO, SOD, and TBA were purchased from Nanjing Jiancheng Bioengineering Institute; total RNA rapid extraction reagent, one-step cDNA first-strand rapid synthesis kit (genomic de-generated), and 2×SYBR Green Fast qPCR Master Mix-HR were purchased from Nanjing Yifeixue Biotechnology Co., Ltd.

[0065] (2) Experimental equipment The main instruments used in this study include: electronic balance (Sartorius Shanghai Trading Co., Ltd.); TGrinder third-generation high-speed tissue grinder kit (Tiangen Biotech (Beijing) Co., Ltd.); mini centrifuge (Qun'an Scientific Instrument (Zhejiang) Co., Ltd.); gene amplifier (Hangzhou Bioer Technology Co., Ltd.); ABI STEPONE PLUS real-time fluorescent quantitative PCR instrument (Thermo Fisher Scientific Inc.); Tanon-4600 fully automatic chemiluminescence image analysis system (Shanghai Tanon Technology Co., Ltd.); -80°C ultra-low temperature refrigerator (Panasonic Corporation, Japan).

[0066] (3) Experimental animals and feeds SPF grade adult male and female C57BL / 6J mice were used for hyperlipidemia and acute toxicity experiments, which were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. (Animal Certificate No.: SCXK (Zhejiang) 2024-0001). For AS experiments, 36 SPF grade 8-week-old male ApoE - / - C57BL / 6J (purchased from Cyagen Biosciences Inc.) and 9 8-week-old male C57BL / 6J mice were used as the normal control group (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). Feeds related to the hyperlipidemia model: purified control diet and purified high-cholesterol high-fat diet (1.25% cholesterol, 20% fat, 0.5% cholic acid) were provided by Jiangsu Xietong Biological Technology Co., Ltd. Feeds related to the atherosclerosis model: purified control diet and purified high-fat diet (0.15% cholesterol, 21% fat) were provided by Jiangsu Xietong Biological Technology Co., Ltd. Referring to the laboratory animal feeding standards, the mice were reared in an environment with a room temperature of about 25°C, a relative humidity of 40%-60%, and a 12 h light-dark alternating cycle, and the mice had free access to food and water.

[0067] Experimental methods, analysis and statistics The Tgal used in the following exemplary embodiments is all the compound shown in molecular structural formula I, with a purity of >99%, which was independently synthesized and purified by this laboratory (Example 1). The present invention is carried out around pathological models of hyperlipidemia and atherosclerosis, and also includes safety tests, ①. Hyperlipidemia model induced by high-fat and high-cholesterol diet (1.25% cholesterol, 20% fat, 0.5% cholic acid) (Example 2). ②. Apolipoprotein E gene knockout (ApoE - / -) Mouse model of atherosclerosis (Example 3) [Ilyas I et al., Mouse models of atherosclerosis in translational research [J]. Trends in Pharmacological Sciences, 2022, 43(11):920–39. ③ Physiological hemostasis test (wound healing test + tail bleeding test) (Example 4). ④ Acute toxicity test (Example 5).

[0068] (1) Mouse weight records (Example 2, Example 5) During the experimental period, the weight and food intake of the mice were recorded at regular intervals, and the average weight change curve and the food intake change curve of each group were plotted using GraphPad Prism 8.

[0069] (2) Detection of physiological and biochemical indicators (Example 2, Example 3, Example 5) Blood was collected from the eyes of mice in each group, with a volume of 0.8-1.0 mL. The blood was allowed to coagulate naturally at room temperature for 3 hours, then centrifuged at 3000 r / min for 10 min. The serum was separated and stored at -80℃ for later use. A suitable amount of serum was used to detect physiological and biochemical indicators. Serum lipid levels such as TC, TG, LDL-C, and HDL-C, as well as transaminase levels and oxidative stress levels, were measured using a kit. Example 3 only measured the levels of TC, LDL-C, and HDL-C in the serum.

[0070] (3) Liver tissue and liver coefficient analysis, bile sampling (Example 2) After the last administration, feces were collected from each mouse and placed in a nuclease-free tube, stored at -80°C. Mice were fasted and allowed free access to water for 6 hours, then weighed, anesthetized, and blood was collected from the eye. The mice were then dissected, and the liver and bile were removed. The liver was washed with physiological saline, dried, and photographed; the liver coefficient (%) was calculated as (liver weight / body weight after fasting) × 100. Bile was stored in nuclease-free tubes at -80°C.

[0071] (4) Determination of cholesterol content in feces (Example 2) At the end of the experiment, after fasting for 6 hours, feces were collected from each mouse and weighed using an analytical balance. Based on the fecal weight of each mouse, anhydrous ethanol was added at a ratio of 1 mg feces / 0.01 mL anhydrous ethanol to prepare a fecal suspension. The suspension was centrifuged at 2500 rpm for 2 min, and the supernatant was collected. The TC content in the supernatant was determined using a total cholesterol (TC) assay kit.

[0072] (5) Transcriptional analysis of key enzymes, regulators, inflammatory factors and vascular inflammatory factors in hepatic lipid synthesis (Examples 2 and 3) During dissection, 0.1 g samples were taken from the same location in the liver of each mouse. Total RNA was extracted from the liver according to the kit instructions, and the RNA was reverse transcribed to synthesize cDNA for subsequent qPCR experiments. The qPCR experiments included the transcriptional levels of genes involved in the synthesis of inflammatory factors TNF-α, IL-1β, IL-6, and IL-10, as well as key enzymes and factors in the synthesis of cholesterol and fatty acids in vivo: HMGCR, Acaca, and SREBF.

[0073] Total RNA extraction and inflammatory factor analysis of blood vessels: After dissecting the aorta during dissection, the aortic arch junction was cut off, minced, and 300 μL of total RNA rapid extraction reagent (containing RNApure) was added. Total RNA was extracted according to the kit instructions, reverse transcribed into cDNA, and then RT-qPCR was performed to detect the expression levels of aortic vascular-related inflammatory factors TNF-α and IL-1β.

[0074] (6) Tissue section preparation and staining analysis (Examples 2, 3, and 5) Organ sectioning and staining (liver as an example): During dissection, samples were taken from the same location on the left lobe of the liver of each mouse. After washing with physiological saline, the liver tissue was fixed in 10 times its volume of 4% paraformaldehyde solution. The fixed liver tissue was dehydrated and embedded in paraffin. The paraffin-embedded liver tissue was sectioned into sections approximately 5 μm thick using a microtome and stained with hematoxylin and eosin (H&E) staining. The sections were observed and photographed under an optical microscope to assess the pathological changes in the mouse liver. Separately, samples were taken from the same location on the left lobe of the liver of each mouse, washed with physiological saline, and immediately stored at -80°C for subsequent frozen section preparation. Frozen sectioning was used to prepare liver tissue sections approximately 10 μm thick, and stained according to the standard Oil Red O staining procedure. After staining, the sections were mounted and observed and photographed under an optical microscope to assess lipid deposition in the mouse liver.

[0075] Vascular sectioning and staining: The cut sections of the heart and aortic arch were fixed by immersing in 4% paraformaldehyde at room temperature. The tissue was then embedded in a special box, submerged in OTC embedding medium, and flash-frozen in liquid nitrogen. The tissue was then cut into 5 μm thick sections using a cryostat. Oil Red O staining, H&E staining, and Masson staining were then performed. After photographing, the sections were mounted using preheated glycerol-gelatin mounting medium.

[0076] (7) Aortic dissection and Oil Red staining analysis of aortic intima plaques in ApoE- / - mice (Example 3) Mice in each group were anesthetized by intraperitoneal injection of 0.5% sodium pentobarbital after the experiment. After blood collection and euthanasia, the thoracic and abdominal cavities were exposed. Cardiac perfusion was performed, and excess organs and viscera were removed. The entire aorta was carefully dissected, and the outer layer of fat and connective tissue was thoroughly removed under a stereomicroscope. The junction of the heart and aortic arch was cut off and fixed in 4% paraformaldehyde at room temperature for at least 48 hours. The aorta was then rinsed three times with PBS. The entire aorta was longitudinally cut with dissecting scissors and thoroughly stained with Oil Red dye at 37°C for 1 hour. After staining, the aorta was differentiated with 60% isopropanol until only the adipose tissue remained red. After washing, the aorta was photographed under a stereomicroscope.

[0077] (8) Immunofluorescence staining analysis of AS-related markers in the aortic intima of ApoE- / - mice (Example 3) After being rapidly frozen in liquid nitrogen as described in 2.4, the tissue was cut into 5 μm thick sections using a cryostat. Primary antibodies against AS-related markers (including CD68, VCAM-1, and α-SMA) were added to each section, and the sections were incubated at 37°C for 2 hours. The sections were then washed three times with 1×PBS for 5 minutes each time. The fluid around the tissue was aspirated, and secondary fluorescent antibodies were added. The sections were incubated at room temperature in the dark for 1 hour, and then washed three times with 1×PBS for 5 minutes each time. Hoechst fluorescent dye was added to label the cell nuclei, and the sections were incubated at room temperature for 15-20 minutes. The sections were then washed twice with 1×PBS for 5 minutes each time. Finally, a small amount of fluorescent quenching mounting medium was added to the tissue. After mounting, the sections were observed and photographed under a fluorescence microscope. In the photographs, positive CD68, VCAM-1, and α-SMA showed red fluorescence.

[0078] (9) Complete blood count (Example 5) This drug is used to assess the effects of Tgal on the blood system of animals and verify its safety. Specific indicators include white blood cell, red blood cell, and platelet counts; and multiple blood routine analyses in rats.

[0079] (10) Wound healing detection (Example 4) To evaluate the effect of Tgal on wound healing in animals, mice were observed for 14 consecutive days after modeling, and the degree and speed of wound healing were recorded.

[0080] (11) Tail bleeding experiment (Example 4) The effects of Tgal on physiological hemostasis in animals were evaluated, with specific indicators being tail vein bleeding time and bleeding volume in mice.

[0081] Quantifying mouse bleeding volume by hemoglobin content [Liu Y et al., Standardizing a simpler, more sensitive and accurate tail bleeding assay in mice [J]. World journal of experimental medicine, 2012, 2(2): 30–6]: Blood-containing saline was collected and centrifuged at 4000r for 15 min to obtain red blood cells. The red blood cells were resuspended in 5 ml of saline, lysed with 15 ml of lysis buffer, centrifuged again, and the supernatant was collected. The absorbance value at 600 nm was measured. The hemoglobin content of each mouse was calculated according to the hemoglobin standard curve, thereby quantifying the bleeding volume of the mouse.

[0082] (12) Statistical processing All results from Examples 2 to 5 are expressed as Mean ± SD (n=6). #p < 0.05 (significant statistical difference), ##p < 0.01 (very significant statistical difference), ###p < 0.001 (extremely significant statistical difference), ####p < 0.0001 (highly extremely significant statistical difference) compared to the normal group; *p < 0.05 (significant statistical difference), **p < 0.01 (very significant statistical difference), ***p < 0.001 (extremely significant statistical difference), ****p < 0.0001 (highly extremely significant statistical difference) compared to the model group.

[0083] Example 1. Preparation of tyrosol-β-D-galactoside Tgal (purity > 99%) The preparation of tyrosol-β-D-galactoside Tgal uses tyrosol and lactose as raw materials and is synthesized by enzymatic catalysis. The reaction steps are simple, highly selective, and the purification process is convenient. The cost of raw materials and reagents used in the enzymatic reaction and purification process is about 5 yuan / gram (99% Tgal).

[0084] 1.1. Enzymatic catalytic synthesis of Tgal: Enzymatic synthesis of tyrosol-β-D-galactosidase (Tgal): The galactosidase is described in patent CN108148824A. A fermented galactosidase is used as the catalyst. The reaction system contains tyrosol at a concentration of 0.29 M (40 g / L) and lactose at a concentration of 0.87 M. After thorough mixing, β-galactosidase (3-10 U / mL) is added. The reaction is carried out at 40°C for approximately 24 hours, then rapidly heated to 90-100°C to terminate the reaction. Denatured proteins are removed by centrifugation, and the supernatant is the reaction solution containing Tgal. A schematic diagram of the enzymatic catalytic synthesis of Tgal is shown below. Figure 1The molar yield of Tgal to the substrate tyrosol was approximately 40-50%. Tgal accounted for 90% of the tyrosol glycosylation product, while tyrosol digalactoside accounted for 10% (the molecular weight was estimated to be Tgal+162 by LC-MS). The supernatant after the reaction was used for further purification.

[0085] 1.2 Purification and preparation of Tgal: After the enzymatic catalytic reaction was completed, ethyl acetate was added at a volume ratio of 1:3, which was 3 times the total volume of the reaction solution. After thorough mixing and centrifugation, most of the tyrosol entered the ethyl acetate layer, while the aqueous layer contained most Tgal and lactose. The ethyl acetate extraction was repeated once. Subsequently, 4 times the volume of n-butanol was added to the aqueous phase for extraction. Tgal was enriched in the n-butanol phase. The n-butanol extraction was repeated once, and the n-butanol phases were combined. The n-butanol phase containing Tgal was removed by rotary evaporation. After adding a small amount of pure water, the n-butanol was removed by rotary evaporation, leaving about 1 / 10 of the original volume of concentrate. 100 ml of the distillation concentrate containing Tgal (containing about 10 g of Tgal) was added to a D101 macroporous resin column (50*1000 mm) for column chromatography purification. The elution conditions were: pure water was used to wash away monosaccharides and unreacted disaccharides, and elution was continued until the reducing sugar test in the eluent was negative. The target product was eluted with 20% ethanol. Finally, the 20% ethanol eluent was freeze-dried, and the purity of Tgal was evaluated by HPLC using a C18 column. Figure 2 The results showed that the main component, Tgal, had a content of approximately 99.29%, with only trace impurities (approximately 0.71%), indicating that the chromatographic purity of Tgal in the sample was >99%. This Tgal was then used in subsequent pharmacological and efficacy experiments.

[0086] The tyrosol-β-D-galactoside Tgal prepared in this embodiment has the structure shown in formula (I): Formula (I).

[0087] Example 2. Application of Tgal in the prevention and treatment of hyperlipidemia This is the first time a compound has been discovered that has a dual regulatory effect of inhibiting cholesterol synthesis in the body and promoting cholesterol excretion from the intestines.

[0088] In this embodiment, a hyperlipidemia model was established in mice by feeding them a high-cholesterol, high-fat diet (HCHFD). After 6 weeks of feeding with the HCHFD diet, the mice were fed for another 8 weeks. After 6 weeks, the mice were administered drugs for 8 consecutive weeks. Rosuvastatin RSV and lovastatin LOV, which are commonly used lipid-lowering drugs in clinical practice, were used as positive control drugs. The dosage was based on the references [FENG D et al., Isoflavones enhance pharmacokinetic exposure of active lovastatin acid via the upregulation of carboxylesterase in high-fat diet mice after oral administration of Xuezhikang capsules [J]. Acta Pharmacologica Sinica, 2018, 39(11): 1804–15; and WEI X et al., Bavachin protects against diet-induced hepatic steatosis and obesity in mice [J]. Acta Pharmacologica Sinica, 2023, 44(7): 1416–28]. The results showed that Tgal had a significantly better lipid-lowering effect than RSV and LOV, and for the first time, a compound with dual cholesterol regulation, tyrosol-β-D-galactoside Tgal, was reported. Tgal effectively inhibits the in vivo synthesis of lipids such as cholesterol and promotes the excretion of cholesterol through the intestine, thereby achieving effective regulation of blood lipids such as TC and LDL-C, while inhibiting TG, which can broaden the application of mixed hyperlipidemia.

[0089] Preliminary experiments showed that Tgal had good bioavailability when administered orally or intraperitoneally. However, considering patient compliance, all experiments in this disclosure used oral administration.

[0090] 2.1. Establishment of an animal model of hyperlipidemia and instructions for grouping and administration of drugs Fifty-six 8-week-old, 25±2 g SPF-grade adult male C57BL / 6J mice were acclimatized for one week and then randomly divided into the following groups: a control group (Control), Model-1 (rosuvastatin solvent), a rosuvastatin positive control group (RSV-10, 10 mg / kg), Model-2 (lovastatin solvent), a lovastatin positive control group (LOV-10, 10 mg / kg), and Tgal-50 (50 mg / kg) and Tgal-100 (100 mg / kg) treatment groups. The mean weight difference between each group was kept to be <0.5 g, with 8 mice in each group. A schematic diagram of hyperlipidemia modeling and grouping / administration is shown below. Figure 3 The blank control group was fed a normal diet, while the other groups were fed a high-cholesterol, high-fat diet (HCHFD, 1.25% cholesterol, 20% fat, 0.5% bile acids). After 6 weeks, the diet was continued for another 8 weeks, for a total of 14 weeks, to establish a mouse model of hyperlipidemia. Simultaneously, starting from week 6, each group was administered positive control drugs via gavage: rosuvastatin (RSV) and lovastatin (LOV), along with the test drug Tgal. These were administered once daily via gavage at the dosages specified in the above groupings for 8 consecutive weeks. The blank control group received saline, model group 1 received saline via gavage, and model group 2 received LOV in a 10% DMSO + 90% (20% SBE-β-CD in Saline) solution. All other feeding conditions remained the same. After the experiment, the mice were sacrificed, and blood and liver samples were collected for further analysis.

[0091] 2.2 Tgal significantly improved liver coefficient and liver transaminase activity in hyperlipidemic mice. Figure 4 Figures A and B show the liver condition and average weight changes of mice in each group, respectively. The livers of mice in the normal control group are bright red and shiny, while the livers of mice in the hyperlipidemia model group are lighter in color and enlarged, indicating severe fat deposition in the liver. The livers of the two positive drug groups are reddish-brown. The livers of mice in the test drug Tgal-50 and Tgal-100 groups are bright red, and their appearance is similar to that of the normal control group. Figure 4 C represents the liver coefficient of each group of mice. Compared with the normal control group, the liver coefficients of Model-1 (p<0.01) and Model-2 (p<0.05) fed with high-cholesterol, high-fat diet HCHFD were significantly increased. There were no significant differences between the two positive drug groups, rosuvastatin and lovastatin, and their corresponding model groups. However, the liver coefficients of the Tgal-50 and Tgal-100 groups were significantly improved (p<0.01). Figure 4D and E represent liver function indicators: Compared with the blank control group, the activities of transaminases AST and ALT in the model group mice were significantly increased (Model-1, p < 0.001; Model-2, p < 0.05). The two positive control drugs RSV-10 and LOV-10 showed no statistically significant difference in liver transaminases AST and ALT compared with the corresponding model groups. Treatment with Tgal showed significant improvement in a dose-dependent manner, and Tgal-100 significantly reduced or reversed AST (p < 0.01) and ALT (p < 0.001) activities.

[0092] 2.3 Tgal significantly reduced blood lipid levels in hyperlipidemic mice. Figure 5 As shown in the AC results: Compared with the blank control group, the concentrations of TC, TG, and LDL-C in the blood lipids of mice in the two hyperlipidemia model groups (Model-1, Model-2) were significantly increased (p < 0.0001); Compared with the corresponding model groups, the two positive control drugs RSV-10 and LOV-10 only showed significant relief for TC, and the two positive control drugs did not show significant differences in LDL-C and TG; Compared with the model group, the concentrations of TC, TG, and LDL-C of Tgal-50 and Tgal-100 in the treatment group showed significant improvement in a dose-dependent manner; LDL-C is a key indicator for the development of hyperlipidemia into AS and other cardiovascular diseases. In addition, the effective regulation of TG by Tgal shows that the drug also has significant advantages in mixed hyperlipidemia. Figure 5 The HDL-C concentrations shown in Figure D are as follows: Compared with the blank control group, both Model-1 (p < 0.01) and Model-2 (p < 0.001) mice in the model group showed a significant compensatory increase, indicating that the increase in HDL-C is beneficial to promoting cholesterol metabolism. Compared with the model group, the two positive drugs RSV-10 and LOV-10 did not show significant changes, while the test drugs Tgal-50 and Tgal-100 showed extremely significant increases (p < 0.0001), indicating that Tgal can significantly promote cholesterol from the periphery to the liver for metabolism (or efflux), thereby reducing the impact on other tissues.

[0093] 2.4 Tgal significantly inhibited the in vivo synthesis of cholesterol and fatty acids in hyperlipidemic mice. 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGCR) is a key rate-limiting enzyme in cholesterol biosynthesis; SREBF is a sterol regulatory element (SRE); and acetyl-CoA carboxylase (ACC) is a key enzyme in the synthesis of fatty acids and triglycerides. Figure 6As shown in the AC, compared with the blank group, the expression levels of HMGCR (p<0.0001), SREBF (p<0.0001), and Acaca (p<0.001) in the liver of the model group were significantly increased, indicating that the HCHFD diet significantly promoted the synthesis of cholesterol and fatty acids in mice; statins are typical HMGCR inhibitors, compared with the corresponding model group. RSV-10 (p < 0.0001) and LOV-10 (p < 0.01) showed significant inhibition of HMGCR enzyme, validating their ability to regulate multiple lipid pathways. The two positive control drugs had no significant regulatory effect on SREBF and Acaca, indicating that statins have little impact on fatty acid synthesis, resulting in insignificant efficacy in treating mixed hyperlipidemia. In contrast, the Tgal treatment group showed highly effective inhibition of cholesterol and fatty acid synthesis in hyperlipidemic mice in a dose-dependent manner. Tgal-100 significantly downregulated the expression levels of HMGCR (p < 0.0001), SREBF (p < 0.0001), and Acaca (p < 0.001), comprehensively inhibiting lipid synthesis in hyperlipidemic mice. The downregulation of the key cholesterol synthesis enzyme HMGCR (p < 0.0001) by Tgal-100 was even greater than that of the two positive control drugs.

[0094] 2.5 Tgal significantly promoted the fecal excretion of cholesterol and the expression of cholesterol efflux factors in hyperlipidemic mice. Figure 7 Figure A shows the comparison of cholesterol content in feces (red) and serum cholesterol content (blue) in each group of mice: the cholesterol content in the feces of the blank control mice was extremely low; the two model groups showed a compensatory increase in fecal cholesterol content under high serum TC conditions; compared with the corresponding model groups, the amount of cholesterol excreted in feces by the two positive drug RSV and LOV groups decreased or showed no significant difference; the two Tgal treatment groups, even when serum cholesterol was significantly controlled or even lower than the blank control group level, still significantly promoted the excretion of cholesterol in feces of HCHFD-fed mice in a dose-dependent manner; compared with the model group, Tgal-100 (p<0.01) significantly increased the amount of cholesterol excreted in feces of mice.

[0095] LXR-α is an important nuclear receptor in the liver. It acts like an intracellular "cholesterol sensor" to promote the transport of cholesterol from peripheral tissues back to the liver for processing or excretion, thereby alleviating hyperlipidemia and cardiovascular diseases such as AS. ABCG5 and ABCG8 are heterodimers. The ABCG5 / ABCG8 complex mediates the transport of cholesterol from the liver to bile, which is then released into the intestinal lumen and finally excreted through feces. Figure 7As shown in the BD diagram, compared with the control group, the expression levels of LXR-α, ABCG5, and ABCG8 were reduced in both model groups (Model-1, Model-2), indicating that cholesterol was not actively excreted in either model group. Compared with the corresponding model groups, there was no significant difference in the expression levels of LXR-α, ABCG5, and ABCG8 in the two statin-positive drug groups, except for a significant upregulation of ABCG8 in the RSV-10 group (p < 0.01). After treatment with Tgal-50 and Tgal-100, the expression of cholesterol efflux-related genes LXR-α, ABCG5, and ABCG8 was significantly increased in a dose-dependent manner. The Tgal-100 group significantly increased the expression levels of LXR-α (p < 0.001), ABCG5 (p < 0.0001), and ABCG8 (p < 0.0001), indicating that Tgal treatment can significantly upregulate the fecal efflux of cholesterol in HCHFD-fed mice by regulating efflux-related regulatory factors.

[0096] 2.6 Tgal significantly inhibited the expression of inflammatory factors in hyperlipidemic mice. like Figure 8 As shown in the AC results, compared with the blank control group, the pro-inflammatory factors TNF-α, IL-1β, and IL-6 in the livers of Model-1 and Model-2 mice in the model group were significantly increased, indicating that the livers of hyperlipidemic mice exhibited severe inflammation. Compared with the model group, the positive control drugs RSV-10 and LOV-10 only showed extremely significant inhibition of TNF-α, while having no significant effect on the inflammatory factors IL-1β and IL-6. Treatment with Tgal-50 and Tgal-100 comprehensively and significantly inhibited the mRNA levels of pro-inflammatory factors TNF-α, IL-1β, and IL-6 in a dose-dependent manner. Tgal-100 significantly inhibited the expression of TNF-α (p < 0.0001), IL-1β (p < 0.01), and IL-6 (p < 0.01), effectively improving the inflammation of the livers of hyperlipidemic mice. Figure 8 The D values ​​show the anti-inflammatory factor IL-10 in the livers of mice in each group. Compared with the blank control group, there were no significant changes in IL-10 levels in the two model groups (Model-1 and Model-2). Compared with the corresponding model groups, there were also no significant changes in IL-10 levels in the positive control groups RSV-10 and LOV-10. Treatment with Tgal significantly increased the level of the anti-inflammatory factor IL-10 in a dose-dependent manner, especially Tgal-100 (p < 0.0001), which significantly increased the IL-10 level, indicating that the test drug Tgal has excellent anti-inflammatory effects.

[0097] 2.7 Tgal significantly increased the antioxidant level in hyperlipidemic mice. Myeloperoxidase (MPO) can fight the invasion of foreign pathogens under physiological conditions, but it can cause tissue damage under pathological conditions such as inflammation. Superoxide dismutase (SOD) is an important antioxidant enzyme in the body that catalyzes the dismutation reaction of superoxide anions, reducing oxidative stress damage. Figure 9 As shown in Figures A and B, compared with the control group, the serum MPO level of the hyperlipidemic model Model-1 mice was significantly increased (p < 0.01), while the serum SOD level was significantly decreased (p < 0.0001); Model-2 mice showed a significant decrease in SOD (p < 0.01), indicating a significantly low antioxidant capacity; compared with the corresponding model groups, the two statin positive control drugs did not show a significant effect on MPO and SOD; after treatment with Tgal, the levels of MPO and SOD in hyperlipidemic mice were significantly improved in a dose-dependent manner, and Tgal-100 significantly inhibited the MPO level (p < 0.0001) and significantly increased the SOD level (p < 0.0001), significantly enhancing the antioxidant capacity of the mice.

[0098] 2.8 Tgal significantly reduced liver tissue damage and vacuolar area in hyperlipidemic mice. Figure 10 Figure A shows the results of hematoxylin and eosin (H&E) staining of liver tissue sections from each group of mice. Figure 10 B represents the statistical analysis of vacuolar area in liver tissue sections: Compared with the blank control group, the number of vacuolars Model-1 (p < 0.01) and Model-2 (p < 0.001) in hepatocytes of the model group was significantly increased, indicating severe liver damage and vacuolar accumulation in both model groups. Compared with the corresponding model groups, there was no statistically significant difference in vacuolar area in liver tissue of the RSV-10 group, while LOV-10 (p < 0.05) reduced vacuolar area in liver tissue to some extent. After treatment with Tgal, liver tissue damage and vacuolar area in hyperlipidemic mice were significantly reduced in a dose-dependent manner. Compared with the model group, the vacuolar area in liver tissue of the Tgal-50 and Tgal-100 groups was significantly reduced (p < 0.01) and similar to that of the blank control group, significantly alleviating liver tissue damage in hyperlipidemic mice.

[0099] 2.9 Tgal significantly reduced or reversed lipid deposition in the liver tissue of hyperlipidemic mice. Oil Red O staining results of liver tissue sections from each group of mice Figure 11 Figure A shows that the liver tissue of mice in the blank control group was light blue overall, with less fat accumulation in hepatocytes; compared with the blank control group, large areas of red lipid droplets were visible in the liver sections of mice in the model groups (Model-1 and Model-2) after staining. Figure 11B showed that the proportion of oil droplet area in liver tissue of both model groups (Model-1, p < 0.0001 and Model-2, p < 0.001) was significantly increased, indicating severe lipid deposition and lesions. Compared with the corresponding model groups, the red lipid droplet area in liver slices of mice treated with the two positive drugs RSV-10 and LOV-10 was significantly reduced (RSV-10, p < 0.01 and LOV-10, p < 0.01). Tgal showed excellent therapeutic effects. Compared with the model groups, the red lipid droplets in liver slices of mice in the Tgal-50 and Tgal-100 groups tended to disappear in a dose-dependent manner. The proportion of oil droplet area in the Tgal-50 (p < 0.001) and Tgal-100 (p < 0.0001) groups showed a significant decrease, and the degree of lipid deposition relief was significantly better than that of the two positive drugs, indicating that the lipid deposition in the liver tissue of hyperlipidemic mice treated with Tgal-50 and Tgal-100 was similar to that in the control group.

[0100] Example 3: Application of Tgal in the prevention and treatment of atherosclerotic diseases Apolipoprotein E (ApoE) is the most important lipid transport protein, involved in the transport of cholesterol and triglycerides. When the ApoE gene is deficient in mice, ApoE protein synthesis is inhibited, leading to elevated levels of cholesterol and triglycerides in the blood, especially LDL-C. Mice can spontaneously develop atherosclerosis. Combined with high-fat diet (HFD), this can accelerate the pathogenesis and progression of atherosclerosis. Feeding mice with apolipoprotein E gene knockout (ApoE) can further enhance the therapeutic effect. - / - The mouse model has been proven to be the most commonly used and reliable model of atherosclerosis.

[0101] This embodiment uses ApoE. - / - A mouse model of atherosclerosis was established by feeding mice a high-fat diet (HFD). Rosuvastatin was used as a positive control to investigate the preventive and therapeutic effects of Tgal on atherosclerosis. Results showed that in the model group, the average area ratio of fatty plaques in the longitudinal section of the aorta reached 50.1%, and the aortic wall fibrosis was severe. In the positive control group (RSV-10), the average area ratio of fatty plaques decreased to 24.5%. The average area ratios of fatty plaques in the test drugs Tgal-50 and Tgal-100 decreased to 19.3% and 11.7%, respectively. Furthermore, Tgal significantly improved the degree of fibrosis surrounding aortic fatty plaques compared to the positive control group. Therefore, Tgal has significant potential for development into a drug for the treatment and prevention of atherosclerosis.

[0102] 3.1 Establishment of an animal model of atherosclerosis and instructions on grouping and administration. 36 8-week-old male ApoE - / -C57BL / 6J mice and nine 8-week-old male C57BL / 6J mice (Control) were randomly assigned to groups after one week of acclimatization feeding. Specific protocols and grouping details are as follows: Figure 12 As shown, ApoE - / - Mice were randomly divided into a model group, a rosuvastatin group (Rsv-10, 10 mg / kg), a tyrosol-β-D-galactosidase (Tgal) treatment group (Tgal-50, 50 mg / kg), and a Tgal-100 group (Tgal-100, 100 mg / kg); control C57BL / 6J mice were given a purified control diet throughout the treatment; ApoE - / - After a week of acclimatization, C57BL / 6J mice were switched to a purified high-fat diet (HFD, 0.15% cholesterol, 21% fat) starting from the second week until the feeding endpoint. Starting from the second week, the mice were administered the test drug via gavage once daily, following the dosages used in the positive control group and the model group. The model group received an equal volume of physiological saline. The administration lasted for 12 weeks, and the mice were sacrificed and samples were collected at week 14. After the experiment, the mice were sacrificed, and blood, liver, and aortic samples were collected for further analysis.

[0103] 3.2 Tgal significantly inhibited the formation of total intimal lipid plaques in the aorta of atherosclerotic mice. Figure 13 Image A shows longitudinal cross-sections (Oil Red staining) of the entire aorta of mice after dissection in each group. The fatty plaques on the inner wall of the aorta appear bright red. Figure 13 B shows the statistical distribution of fatty plaque area on the aortic endothelial wall: Compared with the model group, Tgal-50 (P < 0.0001), Tgal-100 (P < 0.0001), and the positive control drug RSV-10 (P < 0.001) all significantly inhibited the formation of fatty plaque on the aortic endothelial wall, and Tgal showed a dose-dependent effect; Figure 13 In the blank control group (C57BL / 6J mice) shown in Figures A and B, no fatty plaques formed on the inner wall of the aorta after being fed with normal feed. In the model group, the average area ratio of fatty plaques in the longitudinal section of the aorta reached 50.1%. In the positive drug RSV-10 group, the average area ratio of fatty plaques in the aorta decreased to 24.5%. In the test drug Tgal-50 and Tgal-100 groups, the average area ratio of fatty plaques in the aorta decreased to 19.3% and 11.7%, respectively. Tgal significantly improved the aortic fatty plaques better than the positive drug RSV.

[0104] 3.3 Tgal significantly reduced blood lipid levels in atherosclerotic mice. Figure 14 AC values ​​showed the blood lipid levels of TC, LDL-C, and HDL-C in each group of mice. Compared with the blank control group, mice fed with HFD and ApoE... - / -In the mouse model of atherosclerosis, the levels of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) were significantly increased (p < 0.0001), while the level of high-density lipoprotein cholesterol (HDL-C) remained unchanged. Compared with the model group, the test drugs Tgal-50, Tgal-100, and the positive control drug RSV-10 all significantly inhibited the concentration of total cholesterol (TC) (p < 0.0001). However, the inhibitory effect of the test drug Tgal-100 was significantly better than that of the positive control drug RSV. Figure 14 B shows the concentration of LDL-C, a key indicator of atherosclerosis. Compared with the model group, Tgal-50 and Tgal-100 showed extremely significant downregulation of LDL-C concentration (p < 0.0001), while RSV-10 only showed significant downregulation (p < 0.01). Tgal was significantly better than the positive control drug RSV in relieving LDL-C. Figure 14 The results showed that the HDL-C levels in both the control group and the model group were low. Compared with the model group, the levels of the test drugs Tgal-50, Tgal-100 and the positive control drug RSV-10 were all significantly upregulated (P < 0.0001).

[0105] 3.4 Tgal significantly improved the levels of vascular inflammatory factors in atherosclerotic mice. Depend on Figure 15 As shown in A and B, compared with the blank control group, ApoE after HFD feeding was significantly higher. - / - The expression levels of pro-inflammatory factors IL-1β and IL-6 in the aorta of mice (model group) were significantly upregulated (P < 0.0001). Figure 15 As shown in C and D, the expression levels of anti-inflammatory factors IL-4 (P < 0.01) and IL-13 (P < 0.0001) in the aorta of the model group were significantly downregulated, indicating that HFD-fed ApoE - / - Mice showed severe inflammatory cell infiltration of the aortic intima. Compared with the model group, the test drugs Tgal-50, Tgal-100, and the positive control drug RSV-10 all significantly inhibited the expression levels of IL-1β and IL-6 (p < 0.0001). In addition, the test drugs Tgal-50, Tgal-100, and the positive control drug RSV-10 all significantly increased the expression level of the anti-inflammatory factor IL-4 (P < 0.0001). Tgal-100 also significantly increased the expression level of the anti-inflammatory factor IL-13 (P < 0.0001). The ameliorative effects of Tgal-100 on the above-mentioned pro-inflammatory and anti-inflammatory factors in atherosclerotic mice were significantly better than those of RSV-10.

[0106] 3.5 Tgal significantly improved lipid plaque and fibrosis formation in the aortic root intima of atherosclerotic mice. Figure 16 Figure A shows the results of Oil Red O staining, H&E staining, and Masson staining of aortic root sections from each group of mice. Figure 16 B shows the statistical analysis of lipid deposition in the aortic root section after Oil Red O staining. Figure 16 C-staining showed the degree of fibrosis in the aortic root after Masson staining: H&E staining showed that the aortic intima of mice in the blank control group was thin and tight, while that of mice fed with HFD and ApoE was thin and tight. - / - Mice (model) exhibited a highly convex and undulating aortic intima, forming typical AS symptoms. After treatment with RSV-10, Tgal-50, and Tgal-100, the convex intima became smoother. Oil Red O staining showed no lipid deposition in the aortic intima of the blank control group mice. Compared with the control group, the model group had a large amount of lipid deposition between the aortic intima and smooth muscle cells. Lipid deposition in the model group was significantly increased (P < 0.0001). After treatment with RSV-10 (P < 0.001), lipid deposition significantly decreased. The test drugs Tgal-50 (P < 0.0001) and Tgal-100 (P < 0.0001) showed superior therapeutic effects, with both drugs significantly reducing lipid deposition in a dose-dependent manner. Masson staining showed the degree of fibrosis in the aortic root tissue; collagen fibers appeared blue, and smooth muscle cells appeared red. Figure 16 A showed that only the blank control group had a thin intima with blue circles, while the model group showed severe fibrosis at the lipid deposition sites between the aortic intima and smooth muscle cells. Figure 16 The C-line showed a significant increase in fibrosis at the aortic root in the model group (P < 0.0001). Although both RSV and Tgal significantly inhibited fibrosis after treatment (P < 0.0001), Tgal was significantly superior to the positive control drug RSV in inhibiting lipid deposition and fibrosis in the vascular wall, a result consistent with the degree of arterial plaque formation.

[0107] 3.6 Tgal significantly improved the accumulation of AS-related markers in the aortic root of atherosclerotic mice. VCAM-1 is a vascular smooth muscle cell adhesion molecule. In the progression of ankylosing spondylitis (AS), VCAM-1 is one of the key factors promoting the recruitment of monocytes and inflammatory factors. CD68 is a lysosome-associated membrane protein, mainly found in macrophages and monocytes. Macrophage infiltration and phagocytosis of lipids to form foam cells is a core component of AS. α-SMA is a vascular smooth muscle protein that migrates from the tunica media to the tunica intima and proliferates during AS development.

[0108] Figure 17Figure A shows the double-antibody specific staining (positive staining with red fluorescence) of atherosclerosis (AS)-related markers VCAM-1, CD68, and α-SMA in aortic root sections from mice in each group. No obvious red fluorescence was observed in the aortic root sections of mice in the blank control group; Figure 17 As shown in Figure BD, the fluorescence area statistics of VCAM-1, CD68, and α-SMA in each group are displayed. Compared with the control group, the aortic intima VCAM-1 (P < 0.0001), CD68 (P < 0.0001), and α-SMA (P < 0.0001) in the model group were all significantly increased. After treatment with RSV-10, Tgal-50, and Tgal-100, the fluorescence intensity of VCAM-1 was significantly decreased (P < 0.0001). After treatment with RSV-10 (P < 0.001) and Tgal-50 (P < 0.001), CD68 was significantly improved, and after treatment with Tgal-100 (P < 0.0001), CD68 was significantly decreased. The RSV-10 treatment group did not show a significant effect on α-SMA, while Tgal-50 (P < 0.05) and Tgal-100 (P < 0.0001) significantly downregulated α-SMA. Tgal-50 and Tgal-100 significantly inhibited the accumulation of AS-related markers in the aortic root and were significantly superior to the positive control drug RSV.

[0109] Example 4. Effects of Tgal on physiological hemostasis—Wound healing experiment + tail hemostasis experiment This embodiment assesses bleeding risk by constructing mouse wound models and mouse tail hemorrhage models. Both wound healing experiments and mouse tail hemostasis experiments are important validations for analyzing the risk of drug-induced internal bleeding. The results show that Tgal has no significant effect on wound healing rate, while the anticoagulant aspirin significantly reduces the wound healing rate; Tgal has no significant effect on mouse tail hemostasis time and bleeding volume, while aspirin significantly prolongs mouse tail hemostasis time and increases bleeding volume; indicating that Tgal does not affect physiological hemostasis and poses no bleeding risk.

[0110] 4.1 Establishment of a mouse model for physiological hemostasis (wound healing experiment + tail bleeding experiment) and instructions for grouping and administration. ① Wound healing experiment: Eight-week-old male C57 mice were used, such as... Figure 18 As shown in Figure A, a 1 cm long wound model was created on the backs of all mice, and they were divided into the following groups: control group, positive control group (Aspirin Asp-10, 10 mg / kg), Tgal-25 (25 mg / kg), Tgal-50 (50 mg / kg), and Tgal-100 (100 mg / kg), with 6 mice in each group. The drugs were administered by gavage once a day for 13 consecutive days according to the dosage of each group. The control group was given an equal volume of physiological saline. The wound healing status of each group was recorded and analyzed.

[0111] ② Mouse tail hemorrhage experiment: Balb / c male mice aged 7-8 weeks were used, such as... Figure 18 Group B was designated as the control group and the positive control group, consisting of aspirin Asp-10 (10 mg / kg), Tgal-50 (50 mg / kg), Tgal-100 (100 mg / kg), and Tgal-400 (400 mg / kg). The mice were administered the drugs by gavage at the recommended doses. After 30 minutes, the tail vein of the mice was incised and immediately placed in physiological saline at 37°C. The hemostasis time and amount of bleeding in the mice were then analyzed.

[0112] 4.2 Effects of Tgal on wound healing on the back of mice Figure 19 Figure A shows photographs of mouse back wounds at different time points. Figure 19 B shows the corresponding wound area diagram: Compared with the control group, the wound healing in the positive drug aspirin group was slower. On day 13, only the aspirin group still had a clearly visible wound. The Tgal dosage groups (25, 50, 100 mg / kg) all showed similar healing effects to the control group. Figure 19 The C-value showed the healing rate on day 9. Compared with the control group, there was no significant difference in wound healing rate among the Tgal administration groups on day 9, while the wound healing rate in the aspirin group was significantly lower (p < 0.01).

[0113] 4.3 Effects of Tgal on tail hemorrhage in mice Figure 20 A shows the time to hemostasis after tail bleeding in mice. Figure 20 B shows the amount of bleeding (expressed as hemoglobin concentration): Compared with the control group, there were no significant changes in hemostasis time and bleeding volume in the Tgal dosage groups (50, 100, 400 mg / kg) (bleeding volume in mice was quantified by hemoglobin concentration), while the typical anticoagulant aspirin group significantly prolonged bleeding time (p < 0.01) and increased bleeding volume (p < 0.01); indicating that Tgal administration in the range of 50-400 mg / kg does not affect physiological hemostasis and has no risk of internal bleeding.

[0114] 4.4 Effects of Tgal on four coagulation function parameters in rats Figure 21In a rat deep vein thrombosis model, Tgal was used to measure four coagulation parameters, including prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), and fibrinogen (FIB), to evaluate its effect on physiological coagulation. Compared with the sham-operated group, the positive control group (Hep-200 U / kg) significantly prolonged APTT and TT (P < 0.001), while the Tgal administration groups (40, 80, and 120 mg / kg) had no significant effect on the four coagulation parameters (PT, APTT, TT, and FIB) in rats, indicating that Tgal has no significant effect on physiological hemostasis.

[0115] Example 5. Acute toxicity test of high doses of Tgal. To verify the safety of Tgal as a pharmaceutical or functional composition (such as a dietary supplement or food additive), this embodiment conducted an acute toxicity assessment of a high dose of Tgal 4500 mg / kg (45 times the effective therapeutic dose disclosed herein).

[0116] High-dose Tgal administration did not affect the mental state, blood lipids, or liver and kidney function in mice. Blood routine tests remained within normal ranges. It did not affect the organ coefficients of the heart, liver, spleen, lungs, and kidneys, nor did it cause any pathological changes. This demonstrates that high-dose Tgal has no acute toxicity. The acute toxicity test was conducted according to the "Technical Guidelines for Acute Toxicity Testing of Chemical Drugs" formulated by the National Medical Products Administration, referring to the NMPA maximum dose method.

[0117] 5.1 Acute toxicity test, grouping, and administration instructions Twenty adult male and female C57BL / 6J mice, aged 8 weeks and weighing 22±2g, were selected and fed according to experimental animal feeding standards. After one week of acclimatization, the mice were divided into two groups according to sex: a male control group (Control) and a male Tgal group (4.5g / kg); and a female control group (Control) and a female Tgal group (4.5g / kg), with 5 mice in each group. The mice were administered the above dosage by gavage for one day, divided into three doses (1.5g / kg each time, every 4 hours). The control group was given an equal volume of physiological saline in the same manner. Mouse behavior was observed. After fasting for 12 hours on the evening of day 13, blood was collected from the heart under anesthesia on day 14 for analysis of complete blood count and other physiological and biochemical indicators. The heart, liver, spleen, lungs, and kidneys of the mice were dissected to assess the acute toxicity of Tgal. A diagram illustrating the grouping and administration of Tgal in the acute toxicity test is shown below. Figure 22 .

[0118] 5.2 Effects of high-dose Tgal on mental indicators in mice After administration of high-dose Tgal (4.5 g / kg) by gavage, mice were observed for 14 consecutive days. The mental indicators of each group of mice during this period are shown in Table 1. Neither male nor female mice in the high-dose Tgal (4.5 g / kg) administration group showed any abnormal behavior compared with the pure water control group. The number of mice with corresponding behavioral abnormalities in the control group and the high-dose Tgal (4.5 g / kg) administration group was zero after 14 days.

[0119] 5.3 Effects of high-dose Tgal on body weight and food intake in mice Figure 23 The results show that there were no significant changes in mean body weight (A), mean feed intake (B), and mean water consumption (C) between the male control group and the male Tgal group, and between the female control group and the female Tgal group, during the 14-day observation period. This indicates that high-dose Tgal had no effect on body weight, feed intake, or water consumption in either male or female mice.

[0120] 5.4 Effects of high-dose Tgal on routine blood parameters in mice Table 2 shows the complete blood count (CBC) results of mice on day 14 after administration of high-dose Tgal (4.5 g / kg). The results indicate that the CBC results of the male control group and the male Tgal administration group, as well as the female control group and the female Tgal administration group, were all within the normal range (only the red blood cell distribution width was slightly higher overall, possibly due to sample transportation). Compared with the control group, there were no significant differences in the number of white blood cells, lymphocytes, neutrophils, red blood cells, hemoglobin, and the number and ratio of eosinophils in the Tgal administration groups (including both male and female groups). Compared with the control group, the percentage of monocytes and the number of platelets in the Tgal administration groups (including both male and female groups) were significantly decreased, but were still basically within the middle range of the normal range.

[0121] 5.5 Effects of high-dose Tgal on blood lipids and liver and kidney function in mice Figure 24 The results showed that, compared with the male and female control groups, the total triglycerides (TG) (A), total cholesterol (TC) (B), low-density lipoprotein cholesterol (LDL-C) (C), and high-density lipoprotein cholesterol (HDL-C) (D) in the Tgal group mice did not change significantly, indicating that high-dose Tgal had no effect on blood lipids in normal mice.

[0122] Figure 25The results showed that, compared with the male and female control groups, there were no significant changes in uric acid (A) and creatinine (B) in both the male and female Tgal-treated groups, indicating that high-dose Tgal had no effect on renal function indicators in normal mice. There were also no significant changes in the activities of aspartate aminotransferase (AST) (C) and alanine aminotransferase (ALT) (D) in mice, indicating that high-dose Tgal had no effect on liver function indicators in normal mice.

[0123] 5.6 Effects of high-dose Tgal on organ coefficients in mice (heart, liver, spleen, lungs, kidneys, etc.) like Figure 26 As shown: Compared with the male control group and the female control group, respectively, after high-dose Tgal administration, there were no significant differences in the heart coefficient (A), liver coefficient (B), spleen coefficient (C), lung coefficient (D), and kidney coefficient (E) between the male Tgal group and the female Tgal group.

[0124] 5.7 Effects of high-dose Tgal on tissue sections of mouse heart, liver, spleen, lung, and kidney Figure 27 The images show sections of the heart, liver, spleen, lungs, and kidneys of mice in each group (H&E staining): Compared with the male control group (A) and the female control group (C), the sections of the heart, liver, spleen, lungs, and kidneys in the high-dose male Tgal group (B) and the female Tgal group (D) showed no significant changes, indicating that high-dose Tgal did not produce any pathological changes in the heart, liver, spleen, lungs, and kidneys of normal mice. This verifies that a dose of 4500 mg / kg Tgal has no significant toxicity to the heart, liver, spleen, lungs, and kidneys of normal mice.

[0125] Table 1. Mental health scores of mice 14 days after administration of Tgal (4.5 g / kg). .

[0126] Table 2 Effects of Tgal (4.5 g / kg) on ​​routine blood parameters in female / male C57 mice .

Claims

1. Use of tyrosol-β-D-galactoside or its solvate or hydrate in the preparation of medicaments for the treatment and / or prevention of hyperlipidemia or atherosclerosis, wherein said tyrosol-β-D-galactoside has the structure shown in Formula I: Formula (I).

2. The use according to claim 1, wherein the hyperlipidemia is selected from hypercholesterolemia, hypertriglyceridemia, mixed hyperlipidemia, and low high-density lipoprotein cholesterolemia.

3. The use according to claim 2, wherein the hyperlipidemia is characterized by an abnormally high level of low-density lipoprotein cholesterol.

4. The use according to any one of claims 1-3, wherein the tyrosol-β-D-galactoside reduces serum total cholesterol, reduces total triglycerides, reduces low-density lipoprotein cholesterol, and increases high-density lipoprotein cholesterol by inhibiting cholesterol and fatty acid synthesis and promoting cholesterol excretion in feces.

5. The use according to any one of claims 1-3, wherein the tyrosol-β-D-galactoside can improve inflammatory markers and enhance the body's antioxidant capacity.

6. The use according to any one of claims 1-3, wherein the tyrosol-β-D-galactoside can inhibit the formation of lipid plaques and fibrosis in the aortic intima and improve vascular inflammatory markers of atherosclerosis.

7. Use of tyrosol-β-D-galactoside or a solvate or a hydrate thereof in the manufacture of a pharmaceutical composition for lowering blood lipids and / or improving atherosclerosis, wherein, The pharmaceutical composition comprises tyrosol-β-D-galactoside or its solvate or hydrate and one or more pharmaceutically acceptable carriers, excipients or excipients.

8. The use according to claim 7, wherein, Tyrosol-β-D-galactoside or its solvate or hydrate is the main active ingredient, with a content of 1 to 100%.

9. The use according to claim 7, wherein, The dosage form of the pharmaceutical composition is tablets, pills, powders, capsules, injections, ointments, sprays, or pills.

10. Use of tyrosol-β-D-galactoside or its solvate or hydrate in the preparation of food compositions for assisting in maintaining the body's antioxidant levels, assisting in maintaining healthy blood lipids, and / or assisting in improving hyperlipidemia and / or atherosclerosis, wherein said tyrosol-β-D-galactoside has the structure shown in Formula I: Formula (I) The food composition is not intended for therapeutic use.

11. The use according to claim 10, wherein the food composition is a health food, functional food, special dietary food, solid beverage, compressed candy, capsule, granule, oral liquid, powder or powder.

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