Application of hesperidin in preparation of product for relieving atherosclerosis

Abstract

The invention discloses application of hesperidin in preparation of a product for relieving atherosclerosis, and belongs to the technical field of food and medicine. The invention proves that the hesperidin can play a role in resisting atherosclerosis through an axial mechanism of regulating intestinal flora, promoting BCAA degradation and reducing the BCAA level of a system. After the hesperidin enters the intestinal tract of the host, the microflora structure of the intestinal tract can be remodeled, the abundance of effective flora is improved, and the microbiological degradation metabolism of branched chain amino acid is promoted, so that the level of metabolite causing atherosclerosis in the host is reduced. Meanwhile, hesperidin has multiple effects of resisting inflammation, resisting oxidation, regulating blood fat and blood sugar, relieving liver injury, reducing atherosclerotic atherosclerotic plaque area and the like. The hesperidin breaks through the traditional cognition that the existing hesperidin only focuses on inflammation resistance and oxidation resistance, and provides a new lead compound and an action target for targeted intestinal microecology prevention and treatment of cardiovascular diseases.

Description

Technical Field

[0001] This invention relates to the field of food and pharmaceutical technology, and in particular to the application of hesperidin in the preparation of products that alleviate atherosclerosis. Background Technology

[0002] Atherosclerosis (AS) is one of the major threats to cardiovascular health and is closely related to poor dietary habits. Currently, common dietary interventions mainly include controlling fat intake and increasing dietary fiber. However, the following limitations exist in the dietary approach to maintain cardiovascular health: (1) Most functional foods and dietary supplements focus only on single efficacy pathways such as lowering blood lipids and antioxidation, resulting in a narrow target and limited overall improvement effect on multifactorial diseases such as atherosclerosis; (2) Most neglect the systematic regulation of the "gut microbiota-host metabolism" axis and lack effective food means to reduce key risk metabolites by precisely regulating specific gut microbiota; (3) Functional raw materials such as hesperidin are used in a crude manner, mostly as ordinary antioxidants or flavoring ingredients, lacking dosage design, formulation research and mechanism-oriented application schemes aimed at cardiovascular health; (4) The efficacy of a large number of related food ingredients lacks full-chain empirical data support such as animal models, and there is a lack of systematic verification of physiological indicators, pathological plaques, gut microbiota and metabolites, resulting in vague efficacy claims and low credibility.

[0003] Therefore, providing a product that can regulate the balance of gut microbiota and reduce the level of potential risk metabolites in the body through dietary supplementation, thereby alleviating the occurrence and development of atherosclerosis, is an urgent problem to be solved in this field. Summary of the Invention

[0004] To address the aforementioned problems in existing technologies, this invention provides the application of hesperidin in the preparation of products that alleviate atherosclerosis. Hesperidin can regulate the "gut flora-branched chain amino acid (BCAA)-host" axis, reducing the levels of various atherosclerotic metabolites in the host, ultimately achieving multi-target relief of atherosclerosis.

[0005] The technical solution of the present invention is as follows:

[0006] The first aspect of this invention protects the use of hesperidin in the preparation of products that alleviate atherosclerosis.

[0007] Preferably, the atherosclerosis includes atherosclerosis induced by a high-fat diet, a high-cholesterol diet, or metabolic disorders.

[0008] Preferably, the method for alleviating atherosclerosis includes: reshaping the gut microbiota structure, increasing the relative abundance of beneficial bacteria, and promoting the microbial degradation and metabolism of branched-chain amino acids in the gut; The beneficial bacteria include Verrucous Microbes and Bacteroidetes; The branched-chain amino acids include valine, leucine, and isoleucine.

[0009] Preferably, the relief of atherosclerosis includes: reducing the area of ​​atherosclerotic plaques in the aorta.

[0010] Preferably, the method for alleviating atherosclerosis includes at least one of the following: (1) Lowering fasting blood glucose; (2) It lowers triglyceride, total cholesterol, and low-density lipoprotein cholesterol levels, and upregulates high-density lipoprotein cholesterol levels; (3) Reduces serum AST and ALT activity; (4) Reduces superoxide dismutase in the liver; (5) Reduce total bile acids in the liver; (6) Reduce the levels of inflammatory markers of atherosclerosis.

[0011] Preferably, the inflammatory markers of atherosclerosis include oxidized low-density lipoprotein, TNF-α, and IL-6.

[0012] Preferably, the product includes pharmaceuticals, health foods, and dietary supplements.

[0013] A second aspect of this invention protects a drug whose active ingredient includes hesperidin.

[0014] Preferably, the drug comprises hesperidin, a pharmaceutically acceptable carrier, and / or excipients; The dosage forms of the drug include oral liquid, injection, tablet, pill, dispersant, capsule, drop pill, granule, suspension, and emulsion.

[0015] The third aspect of this invention protects the use of the drug described in the second aspect in the preparation of products for relieving atherosclerosis.

[0016] The beneficial technical effects of this invention are as follows: This invention discloses and confirms that hesperidin exerts its anti-atherosclerotic effect through a coherent axial mechanism of "regulating gut microbiota - promoting BCAA degradation - reducing systemic BCAA levels." After entering the host gut, hesperidin can reshape the gut microbiota structure, significantly increasing the abundance of beneficial bacteria such as Verrucous and Bacteroidetes, promoting the microbial degradation and metabolism of branched-chain amino acids such as valine, leucine, and isoleucine, thereby reducing the levels of atherosclerotic metabolites in the host body. Simultaneously, hesperidin possesses multiple effects including anti-inflammatory, antioxidant, lipid and glucose regulation, liver damage reduction, and reduction of aortic atherosclerotic plaque area, as well as ApoE activity. - / -The intervention showed a definite and significant effect in model mice, breaking through the traditional understanding of hesperidin's effects as limited to anti-inflammatory and antioxidant properties. It provides a new lead compound and target for targeting the gut microbiota to prevent, alleviate and treat cardiovascular diseases.

[0017] In addition, hesperidin is derived from citrus plants such as tangerine peel. It is a natural flavonoid compound with the advantages of wide availability, high safety and no obvious toxic side effects from long-term consumption. It can be formulated into various dosage forms with pharmaceutically or food-acceptable excipients and can be widely developed into pharmaceuticals, health foods or dietary supplements, with good prospects for clinical translation and market application. Attached Figure Description

[0018] Figure 1 The change in body weight of the mice in Example 1.

[0019] Figure 2 The fasting blood glucose level of the mice in Example 1 is shown.

[0020] Figure 3 (Top left) shows the level of total cholesterol (TC) in the serum of mice in Example 1; Figure 3 (Top right) shows the level of triglycerides (TG) in the serum of mice in Example 1; Figure 3 (Bottom left) shows the level of high-density lipoprotein cholesterol (HDL-C) in the serum of mice in Example 1; Figure 3 (Bottom right) shows the level of low-density lipoprotein cholesterol (LDL-C) in the serum of mice in Example 1.

[0021] Figure 4 Anatomical observation of the heart and aorta of the mice in Example 1; in, Figure 4 Image A is an Oil Red O staining image of a cross-section of the mouse aorta. Figure 4 B is an H&E staining image of the aortic root of a mouse.

[0022] Figure 5 (Top left) shows the level of aspartate aminotransferase (AST) in mouse serum in Example 1; Figure 5 (Top right) shows the level of alanine aminotransferase (ALT) in mouse serum in Example 1; Figure 5 (Bottom left) shows the level of superoxide dismutase (SOD) in mouse liver samples in Example 1.

[0023] Figure 6 (Top left) shows the total bile acid (TBA) level in the liver of mice in Example 1; Figure 6 (Top right) shows the level of oxidized low-density lipoprotein (oX-LDL) in mouse serum in Example 1; Figure 6 (Bottom left) shows the TNF-α level in mouse serum in Example 1; Figure 6 (Bottom right) shows the IL-6 level in mouse serum in Example 1.

[0024] Figure 7 The Venn diagrams for the CD group, HFD group, and HFD+Hes group in Example 1 are shown.

[0025] Figure 8 This is an α-diversity assessment diagram for the CD group, HFD group, HFD+Hes group, and HFD+AT group in Example 1.

[0026] Figure 9 This is a principal coordinate analysis diagram of the CD group, HFD group, HFD+Hes group, and HFD+AT group in Example 1.

[0027] Figure 10 The Jaccard Anosim analysis graphs are for the CD group, HFD group, HFD+Hes group, and HFD+AT group in Example 1.

[0028] Figure 11 This is a diagram showing the phylum-level composition of the mouse gut microbiota in Example 1.

[0029] Figure 12 This is a diagram showing the genus-level composition of the mouse gut microbiota in Example 1.

[0030] Figure 13 This is a diagram showing the species differences between the model group and the normal control group in Example 1.

[0031] Figure 14 This is a diagram showing the species differences between the model group and the hesperidin group in Example 1.

[0032] Figure 15 The value represents the BCAA content in mouse feces in Example 1. Detailed Implementation

[0033] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0034] This invention discloses the application of hesperidin in the preparation of products that alleviate atherosclerosis.

[0035] Preferably, the product includes pharmaceuticals, health foods, or dietary supplements.

[0036] The health food products include hesperidin and pharmaceutically or food-acceptable excipients.

[0037] The dietary supplements include hesperidin and pharmaceutically or food-acceptable excipients.

[0038] The hesperidin mentioned is hesperidin that meets the standards for medicinal or edible use.

[0039] Preferably, the excipients include fillers, disintegrants, lubricants, and flavoring agents.

[0040] Preferably, the filler includes at least one of starch and microcrystalline cellulose.

[0041] Preferably, the flavoring agent includes at least one of citrus flavoring and sweetener.

[0042] Health foods and dietary supplements can be prepared into dosage forms such as tablets, capsules, granules, powders, or oral liquids using conventional pharmaceutical processes.

[0043] The effective dose range for oral administration of hesperidin according to the present invention is 75~225 mg / kg / d (based on mice), and the corresponding human equivalent dose (HED) is approximately 6.1~18.3 mg / kg / d.

[0044] Preferably, the effective dose of hesperidin for oral administration is 150 mg / kg / day (based on mice), corresponding to an equivalent human dose of approximately 12.2 mg / kg / day. At this dose, hesperidin simultaneously achieves optimal metabolic improvement, plaque inhibition, and gut microbiota regulation, with good safety profile.

[0045] Example 1: Hesperidin's effect on ApoE - / - Intervention effect on atherosclerosis in mice The experimental materials and methods are as follows: Experimental animals and grouping: 30 male ApoE 6-8 weeks old - / - Mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were randomly divided into 3 groups (n=10): (1) Model control group (HFD): fed with high-fat, high-cholesterol diet; (2) Hesperidin group (HFD+Hes): fed with high-fat, high-cholesterol diet and administered 150 mg / kg / d of hesperidin by gavage; (3) Positive drug control group (HFD+AT): fed with high-fat, high-cholesterol diet and administered 10 mg / kg / d atorvastatin calcium by gavage.

[0046] All high-fat, high-cholesterol feed was purchased from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd.

[0047] Ten age-matched male C57BL / 6J mice were set up as a normal control group (CD) and fed with ordinary feed.

[0048] Ten age-matched male C57BL / 6J mice were set up as a hesperidin supplementation control group (CD+Hes), fed with normal feed and administered 150 mg / kg / d of hesperidin by gavage.

[0049] Test substance: hesperidin (purity >95%, batch number: HSD20231001, purchased from Shanghai Yuanye Biotechnology Co., Ltd.), which was dissolved in 0.5% sodium carboxymethyl cellulose (CMC-Na) solution before use.

[0050] Experimental period and sampling: The intervention lasted for 12 weeks, with weekly weighing. Fresh feces were collected in week 11 for storage at -80°C. After the last administration, mice were fasted for 12 hours, anesthetized, and blood was collected by enucleation. Serum was separated. The heart and aortic arch were completely dissected, with some parts fixed in 4% paraformaldehyde and others flash-frozen in liquid nitrogen and stored at -80°C. Liver tissue was collected.

[0051] The detection indicators and methods are as follows: Physiological and metabolic indicators: Serum triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels were measured using a fully automated biochemical analyzer (model: AU480, Beckman Coulter Trading (China) Co., Ltd.) and corresponding reagent kits (Ningbo Zhenhai Baichuan Biotechnology Co., Ltd.). Fasting blood glucose (FBG) was measured using a blood glucose meter (Roche Diagnostics GmbH, Germany).

[0052] Liver function and oxidative stress indicators: Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were measured using a kit (as above). Superoxide dismutase (SOD) activity, total bile acids (TBA), and oxidized low-density lipoprotein (oX-LDL) levels in liver homogenates were measured using an enzyme-linked immunosorbent assay (ELISA) kit (Shanghai Enzyme-Link Biotechnology Co., Ltd., operated according to the product instructions).

[0053] Systemic inflammatory markers: The concentrations of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in serum were measured using the ELISA method (kit source as above).

[0054] Pathological analysis: The fixed aortic arch was stained with Oil Red O, and the heart root was paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H&E). Observation was performed using an optical microscope (Nikon Eclipse Ci), and the area of ​​atherosclerotic plaques at the aortic sinus was quantitatively analyzed using Image J 1.53k software.

[0055] Gut microbiota analysis: 16S rRNA gene sequencing was commissioned to Hangzhou Leading Gene Technology Co., Ltd. Total DNA from fecal microorganisms was extracted using a kit, and the V3-V4 regions of the 16S rRNA gene were amplified and sequenced on the Illumina NovaSeq 6000 platform. Bioinformatics analysis, including ASV clustering, species annotation, and diversity calculation, was performed using the QIIME2 platform.

[0056] Branched-chain amino acid (BCAA) content determination: The contents of valine (Val), leucine (Leu), and isoleucine (Ile) in feces were determined by Shanghai MetaVision Biomedical Technology Co., Ltd. using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The instrument was a SCIEX QTRAP 6500+ system, and the chromatographic column was an AdvanceBio MS Spent Media column. Data acquisition and processing were performed using AB Sciex OS software.

[0057] The experimental results are as follows: Figure 1 This represents the change in body weight in mice. According to... Figure 1 It can be seen that the body weight gain trend of the model control group (HFD) mice was relatively rapid, with a rate of change of approximately 12.96%. While there was a certain difference compared to the rate of change in the normal control group (CD) (12.48%), it was not statistically significant. However, the body weight changes were significantly different from those in the hesperidin group (HFD+Hes) and the positive drug control group (HFD+AT). Specifically, the rate of body weight change in the HFD+Hes and HFD+AT mice decreased to 11.08% and 7.12%, respectively.

[0058] Before euthanizing the mice, they were deprived of water for 12 hours to detect and record fasting blood glucose levels in each group. The results are as follows: Figure 2 As shown. According to Figure 2 It can be seen that the fasting blood glucose level of the model control group (HFD) mice (11.97±1.2 mmol / L) was significantly higher than that of the normal control group (CD) mice (9.6±0.8 mmol / L). The fasting blood glucose levels of the HFD+Hes group mice (8.16±0.7 mmol / L) and the HFD+AT group mice (7.29±0.6 mmol / L) were both statistically significantly improved compared to the HFD group.

[0059] Figure 3 The levels of total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in mouse serum were respectively measured. Figure 3It can be seen that the CD group had a TC content of approximately 2.85, a TG content of approximately 0.69, an LDL-C content of approximately 1.57, and an HDL-C content of approximately 5.45. In contrast, the HFD group showed significantly elevated serum TC, TG, and LDL-C levels, approximately 30.64, 1.9, and 14.72, respectively, but a significantly decreased serum HDL-C level, down to 2.02. Furthermore, the experimental data from the HFD+Hes group indicate that, compared to the HFD group, hesperidin intervention significantly reduced serum TC, TG, and LDL-C levels and upregulated HDL-C.

[0060] Figure 4 This was for anatomical observation of the heart and aorta in mice. Based on... Figure 4 It can be seen that the arterial walls of mice in the HFD group were significantly thickened, the intimal space was widened, and cholesterol and lipid deposition and aortic calcification were observed, with a plaque area reaching 0.212 μm. 2 In the HFD+Hes and HFD+AT groups, arterial wall thickness was improved, cholesterol and lipid deposition was reduced, and plaque area decreased to 0.087 μm. 2 and 0.063μm 2 .

[0061] Figure 5 (Top left) shows the level of aspartate aminotransferase (AST) in mouse serum. Figure 5 (Top right) shows the level of alanine aminotransferase (ALT) in mouse serum; Figure 5 (Bottom left) shows the level of superoxide dismutase (SOD) in mouse liver samples. Figure 5 It can be seen that the AST and ALT levels in the HFD group mice were significantly higher than those in the CD group. However, the AST and ALT levels in the HFD+Hes group were improved compared to the HFD group, decreasing to approximately 88±1.171 U / L and 7.06±0.96 U / L, respectively. p<0.01); the HFD+AT groups were 35.64±1.51 U / L and 6.45±1.40 U / L, respectively. p<0.01).

[0062] Superoxide dismutase (SOD) is a key antioxidant enzyme in the liver, protecting hepatocytes by scavenging superoxide free radicals. It works synergistically with other antioxidant systems to maintain liver homeostasis; its expression is significantly upregulated under oxidative stress to alleviate related damage. The restoration of SOD levels to the normal range signifies the re-establishment of redox balance. SOD level detection in liver samples showed that the SOD activity in the HFD group was significantly increased compared to the CD group. However, the SOD level in the HFD+Hes group was significantly lower than that in the HFD group. The increased SOD activity in the HFD group is a compensatory response to severe oxidative stress, but this response is insufficient. In contrast, the decreased SOD activity in the HFD+Hes group does not reflect impaired antioxidant capacity, but rather indicates that hesperidin treatment effectively alleviated oxidative stress, thereby reducing the need for SOD upregulation. This further demonstrates that hesperidin has a reversible effect on atherosclerosis-induced liver damage.

[0063] Figure 6 (Top left) shows the total bile acid (TBA) level in mouse liver. Figure 6 (Top right) shows the level of oxidized low-density lipoprotein (oX-LDL) in mouse serum. Figure 6 (Bottom left) shows the TNF-α level in mouse serum. Figure 6 (Bottom right) shows the level of IL-6 in mouse serum.

[0064] The conversion of cholesterol to total bile acids plays a crucial role in maintaining cholesterol homeostasis and preventing the accumulation of cholesterol, triglycerides, and toxic metabolites. Furthermore, TBA levels are associated with cardiovascular disease risk in both animals and humans. According to... Figure 6 It can be seen that the TBA level in the CD group mice was 9.29±1.56 μmol / g, while the TBA level in the HFD group mice was significantly increased to 25.79±4.35 μmol / g, showing a statistically significant difference compared to the CD group (## p<0.01). In contrast, the TBA level in the HFD+Hes group mice was significantly lower than that in the HFD group, decreasing to 16.14±3.243 μmol / g. Furthermore, the TBA level in the HFD+AT group mice was approximately 16.78±2.61 μmol / g, also significantly lower than that in the model group. This indicates that hesperidin has an ameliorative effect on atherosclerosis in mice.

[0065] Oxidized low-density lipoprotein (oX-LDL) is closely related to the severity of atherosclerosis, is a key factor in foam cell formation, and participates in the formation of atherosclerotic plaques. According to... Figure 6It can be seen that the oX-LDL level in the HFD group mice was significantly higher than that in the CD group, while after hesperidin intervention, the oX-LDL level in the HFD+Hes group mice (157.4±8.64 μg / mL) was significantly lower than that in the HFD group. Furthermore, the detection results of inflammatory factors in mouse blood showed that the elevation of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in atherosclerotic mice was inhibited after hesperidin gavage intervention. The elevation of inflammatory factor levels is also an important indicator for detecting atherosclerosis. This further confirms the antioxidant and anti-inflammatory properties of hesperidin, indicating its clear therapeutic effect in alleviating high-fat / high-cholesterol diet-induced atherosclerosis in mice.

[0066] The gut microbiome is closely related to the progression of atherosclerosis. This invention analyzed the fecal microbiota of ApoE- / - mice supplemented with or without hesperidin using 16S rRNA gene sequencing (Tables 1 and 2).

[0067] Table 1 16S rRNA data

[0068] Table 2: α-diversity index for each sample

[0069] Figure 7 The Venn diagrams for the CD group, HFD group, and HFD+Hes group in Example 1 are shown. Figure 8 This is an α-diversity assessment diagram for the CD group, HFD group, HFD+Hes group, and HFD+AT group in Example 1. Figure 9 This is a principal coordinate analysis diagram of the CD group, HFD group, HFD+Hes group, and HFD+AT group in Example 1. Figure 10 The Jaccard Anosim analysis graphs are for the CD group, HFD group, HFD+Hes group, and HFD+AT group in Example 1.

[0070] Venn plot analysis revealed unique amplicon sequence variants for each group: the CD group, HFD group, and HFD+Hes group contained 458, 407, and 283 unique ASVs, respectively. Figure 7 The dilution curve tends to flatten, indicating sufficient sequencing depth and stable α-diversity estimates. Figure 8 Although not statistically significant (p>0.05), Jaccard Anosim analysis ( Figure 10The results showed certain differences in gut microbiota composition among the CD, HFD, HFD+Hes, and HFD+AT groups. Principal coordinate analysis further supported this result: the CD group was clearly separated from the HFD group, while the HFD+Hes and HFD+AT groups showed partially independent and distinguishable clustering trends relative to the HFD group. Figure 9 This indicates that changes in gut microbiota composition are related to hesperidin supplementation.

[0071] Figure 11 This is a diagram showing the phylum-level composition of the mouse gut microbiota in Example 1. Figure 12 This is a diagram showing the genus-level composition of the mouse gut microbiota in Example 1. Figure 13 This is a diagram showing the species differences between the model group and the normal control group in Example 1. Figure 14 This is a diagram showing the species differences between the model group and the hesperidin group in Example 1.

[0072] At the phylum level, 16S rRNA sequencing results showed that the gut microbiota of all experimental mice mainly consisted of Verrucous Microbes, Firmicutes, Bacteroidetes, Actinobacteria, Dethiobacteria, and Proteobacteria. Figure 11 Compared to the CD group, the HFD group showed a significantly increased relative abundance of Firmicutes, while the relative abundance of Verrucomicrobia and Bacteroidetes was significantly decreased. The HFD + Hes and HFD + AT groups mitigated these diet-induced changes to some extent, resulting in decreased Firmicutes abundance and increased Verrucomicrobia and Bacteroidetes abundance compared to the HFD group; further analysis at the genus level ( Figure 12 The results showed that, compared with the CD group, the relative abundance of Akkermansia and Lactobacillus decreased in the HFD group, while the abundance of Trichophyton increased.

[0073] LEfSe analysis showed statistically significant differences in gut microbiota composition among the experimental groups. Figure 13 , 14 As shown, specific bacterial taxa exhibited distinct enrichment patterns: in group CD, p_Bacteroidota, c_Bacteroidia, o_Bacteroidales, f_Muribaculaceae, and s_Paramuribaculum_intestinale had relatively high abundance. In contrast, p_Firmicutes, c_Bacilli, and p_Actinobacteriota were significantly enriched in group HFD.

[0074] Furthermore, certain differences existed between the HFD group and the HFD+Hes group mice. In the HFD group mice, c_Bacili, o_Laciobacillales, and f_Lactobacillaceae were the most abundant, playing important roles; in the HFD+Hes mice, the key bacteria were f_Atopobiaceae, g_Coriobacteriaceae_UCG-002, and sCoriobacteriaceae UCG-002_unclassified. These data suggest that hesperidin can alter the gut microbiota composition in atherosclerotic mice, potentially influencing disease progression by regulating the abundance of specific bacterial groups. This change may be related to the anti-inflammatory, antioxidant, and cardiovascular protective effects of hesperidin, indirectly affecting host health by improving gut microbiota balance.

[0075] Valine, leucine, and isoleucine are essential amino acids, collectively known as branched-chain amino acids (BCAAs). They can serve as direct or indirect nutritional signals. Multiple studies have shown that BCAA levels in the body play a crucial role in the dietary composition and metabolism of patients with cardiovascular disease, and elevated levels indicate an increased risk of cardiovascular disease. Analysis of BCAA content in mouse feces revealed that the levels of valine, leucine, and isoleucine in the feces of mice in the HFD group were 395.57, 275.4, and 190.8 μmol / g, respectively, all significantly higher than the corresponding levels in the CD group. After treatment with hesperidin, the levels of these three amino acids in the feces of mice in the HFD+Hes group decreased to 287.09, 137.55, and 113.03 μmol / g, respectively. Figure 15 Compared with HFD mice, HFD+Hes mice showed a 27.4%, 50.1%, and 40.8% reduction in fecal valine, leucine, and isoleucine levels, respectively (p<0.05).

[0076] Example 2: Effects of different doses of hesperidin on ApoE - / - Intervention effect on atherosclerosis in mice The experimental materials and methods are as follows: Experimental animals and grouping: 50 male ApoE 6-8 weeks old - / - Mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were randomly divided into 5 groups (n=10): (1) Model control group (HFD): fed with high-fat, high-cholesterol diet; (2) Low-dose hesperidin group (HFD+Hes-L): fed with high-fat, high-cholesterol feed and administered 75 mg / kg / d of hesperidin by gavage; (3) Standard dose group of hesperidin (HFD+Hes-M): fed with high-fat and high-cholesterol feed and administered 150 mg / kg / d of hesperidin by gavage; (4) High-dose hesperidin group (HFD+Hes-H): fed with high-fat, high-cholesterol feed and administered 225 mg / kg / d of hesperidin by gavage; (5) Positive drug control group (HFD+AT): fed with high-fat, high-cholesterol diet and administered 10 mg / kg / d atorvastatin calcium by gavage.

[0077] All high-fat, high-cholesterol feed was purchased from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd.

[0078] Ten age-matched male C57BL / 6J mice were set up as a normal control group (CD) and fed with ordinary feed.

[0079] Test substance: hesperidin (purity >95%, batch number: HSD20231001, purchased from Shanghai Yuanye Biotechnology Co., Ltd.), which was dissolved in 0.5% sodium carboxymethyl cellulose (CMC-Na) solution before use.

[0080] The experimental period and sampling method were the same as in Example 1.

[0081] The detection indicators and methods are the same as in Example 1.

[0082] The experimental results are as follows: Mice in the HFD+Hes-L group showed a moderate improvement trend. Specifically, compared with the HFD group, serum TC and LDL-C in the HFD+Hes-L group decreased by 22.5% and 30.8%, respectively (p<0.05), but the effect was weaker than that in the HFD+Hes-M group. The plaque area decreased to (0.152 ± 0.020) mm² (a reduction of approximately 28.3%, p<0.05). The increase in beneficial bacteria in the gut microbiota and the decrease in fecal BCAAs were both smaller than those in the standard dose group, but still statistically significant (e.g., a 22.3% reduction in Leu, p<0.05). The results indicate that the 75 mg / kg / d dose has some activity, but 150 mg / kg / d is a better choice.

[0083] In terms of improving blood lipids, blood glucose, inflammation, and reducing plaque area, the HFD+Hes-H group showed comparable effects to the HFD+Hes-M group, with no significant dose-escalation effect. The plaque area was (0.082 ± 0.014) mm. 2However, in terms of gut microbiota regulation and BCAA reduction, its effects were comparable to the standard dose group. Mice in all dose groups exhibited normal behavior and appetite throughout the experiment, and serum AST and ALT levels were significantly improved compared to the model group. No drug-related liver or kidney dysfunction was observed, indicating that hesperidin has good safety within this dose range.

[0084] The above description is merely a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and variations that are directly derived or conceived by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included within the protection scope of the present invention.

Claims

1. Application of hesperidin in the preparation of products that alleviate atherosclerosis.

2. The application according to claim 1, characterized in that, The atherosclerosis mentioned includes atherosclerosis induced by high-fat diets, high-cholesterol diets, and metabolic disorders.

3. The application according to claim 1, characterized in that, The methods for alleviating atherosclerosis include: reshaping the gut microbiota structure, increasing the relative abundance of beneficial bacteria, and promoting the microbial degradation and metabolism of branched-chain amino acids in the gut. The beneficial bacteria include Verrucous Microbes and Bacteroidetes; The branched-chain amino acids include valine, leucine, and isoleucine.

4. The application according to claim 1, characterized in that, The relief of atherosclerosis includes: reducing the area of ​​atherosclerotic plaques in the aorta.

5. The application according to claim 1, characterized in that, The relief of atherosclerosis includes at least one of the following: (1) Lowering fasting blood glucose; (2) It lowers triglyceride, total cholesterol, and low-density lipoprotein cholesterol levels, and upregulates high-density lipoprotein cholesterol levels; (3) Reduces serum AST and ALT activity; (4) Reduces superoxide dismutase in the liver; (5) Reduce total bile acids in the liver; (6) Reduce the levels of inflammatory markers of atherosclerosis.

6. The application according to claim 5, characterized in that, The inflammatory markers of atherosclerosis include oxidized low-density lipoprotein, TNF-α, and IL-6.

7. The application according to claim 1, characterized in that, The products include pharmaceuticals, health foods, and dietary supplements.

8. A drug, characterized in that, The active ingredient in the drug includes hesperidin.

9. The medicament according to claim 8, characterized in that, The drug includes hesperidin, a pharmaceutically acceptable carrier, and / or excipients; The dosage forms of the drug include oral liquid, injection, tablet, pill, dispersant, capsule, drop pill, granule, suspension, and emulsion.

10. The use of the medicament according to claim 8 or 9 in the preparation of a product for relieving atherosclerosis.

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