Composition for reducing blood fat and preventing atherosclerosis, combined product and application thereof

Abstract

The invention relates to the field of foods and medicines, in particular to a citrus and dietary fiber composition and a combined product for reducing blood fat and preventing atherosclerosis, application of the composition in preparation of medicines, common foods, functional foods or health-care foods, and application of the combined product in preparation of medicines.

Description

Technical Field

[0001] The invention relates to the field of food and medicine, and in particular to a citrus and dietary fiber composition and application thereof in reducing blood lipids and preventing atherosclerosis. Background Art

[0002] At present, cardiovascular disease has become the main risk of death worldwide, and the incidence rate is increasing year by year, bringing huge health and economic burden to society. Dyslipidemia is one of the main risk factors for cardiovascular disease. Although modern medicine has a variety of lipid-lowering drugs, most of them have side effects and unstable efficacy.

[0003] Atherosclerosis (As) is an inflammatory disease that affects the arterial wall and is characterized by the accumulation of lipids and inflammatory cells in the intima of large arteries. Atherosclerosis can lead to atherosclerotic cardiovascular disease (ASCVD). Clinically diagnosed ASCVD diseases mainly include: acute coronary syndrome, stable or unstable angina, atherosclerotic stroke or transient ischemic attack, etc. ASCVD has become the leading cause of death worldwide and seriously endangers health. In my country, the incidence of atherosclerosis has also shown a clear upward trend and is more common in middle-aged and elderly people. Clinical drugs for the treatment of atherosclerosis mainly include: (1) lipid-lowering drugs: statins, ezetimibe, PCSK9 inhibitors, etc.; (2) antiplatelet drugs: aspirin, clopidogrel, etc. There are also some Chinese patent medicines for clinical anti-atherosclerosis, such as Compound Danshen Dropping Pills, Qishen Yiqi Dropping Pills, Naoxintong Capsules, etc. Atherosclerosis requires a treatment plan under the guidance of a doctor and long-term medication. Different drugs can cause different degrees of side effects on the human body, such as liver and muscle damage, gastrointestinal reactions, etc., so a more natural and safe way of intervention is needed.

[0004] Citrus (Citrus reticulata Blanco) is a small tree in the genus Citrus of the family Rutaceae. Its fruit is usually oblate to nearly spherical, light yellow, scarlet or dark red, and the flesh is sour, sweet, bitter, or has a special smell. Citrus is divided into citrus and orange, and its origin is widely distributed all over the world with many varieties, mainly including tomato, Fumin trifoliate orange, kumquat, arrow leaf orange, tangerine, Mangshan wild orange, four-season orange, sour orange, sweet orange, citron, etc.

[0005] Dietary fiber usually refers to carbohydrates that are not easily digested and absorbed by the human body and are found in plant foods. The World Health Organization points out that dietary fiber is a polysaccharide containing 10 or more monomer units, which is not hydrolyzed by endogenous hormones in the small intestine and plays an important role in human health. According to solubility, dietary fiber can be divided into soluble dietary fiber and insoluble dietary fiber. The main sources of soluble dietary fiber are fruits and vegetables, which are mainly composed of a variety of non-cellulose polysaccharides and oligosaccharides; the main sources of insoluble dietary fiber are grains and whole grain products, which are mainly composed of cellulose, lignin, and hemicellulose.

[0006] Citrus fruits and dietary fiber each have high edible and medicinal value, but there is still limited research on their combined use to lower blood lipids and prevent atherosclerosis. SUMMARY OF THE INVENTION

[0008] The inventors of the present application have found through extensive experimental studies that the combination of citrus (or citrus extract) and dietary fiber has excellent effects in lowering blood lipids and preventing atherosclerosis.

[0009] To this end, in a first aspect of the present invention, the present invention provides a composition comprising:

[0010] A first component, the first component being citrus or a citrus extract; and

[0011] The second component is dietary fiber.

[0012] In a second aspect of the invention, the invention provides a combination product comprising:

[0013] A first product, the first product being the aforementioned composition;

[0014] The second product is a component for preventing blood lipid diseases or atherosclerotic diseases.

[0015] In the third aspect of the present invention, the present invention provides the use of the aforementioned composition in the preparation of medicines (such as Chinese medicines, Chinese patent medicines, natural medicines), ordinary foods, functional foods or health foods, and the medicines (such as Chinese medicines, Chinese patent medicines, natural medicines), ordinary foods, functional foods or health foods are used to prevent blood lipid diseases or atherosclerotic diseases.

[0016] In the fourth aspect of the present invention, the present invention provides the use of the aforementioned combination product in the preparation of a drug (such as a traditional Chinese medicine, a Chinese patent medicine, a natural medicine), wherein the drug (such as a traditional Chinese medicine, a Chinese patent medicine, a natural medicine) is used to prevent blood lipid diseases or atherosclerotic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0018] Figure 1 The experimental animal groups are shown;

[0019] Figure 2 The weight and food intake of mice are shown, and the values ​​are mean ± SD, n = 10 or 12; a: compared with group 1, P < 0.05; b: compared with group 2, P < 0.05; c: compared with group 3, P < 0.05;

[0020] Figure 3 Mouse organ masses are shown;

[0021] Figure 4 The functional status of mouse pancreatic islets is shown;

[0022] Figure 5 The serum and liver inflammation status of mice are shown;

[0023] Figure 6 The results of HE staining of liver tissues of mice in each group after the intervention are shown;

[0024] Figure 7 Liver damage and hepatic lipid metabolism are shown;

[0025] Figure 8 The results of Oil Red O staining of the whole aorta of mice in each group are shown;

[0026] Fig. 9 Aortic histopathological staining results are shown;

[0027] Fig.10 The results of aortic CD68 immunohistochemical staining and image propuls analysis are shown;

[0028] Fig.11 The results of F4 / 80 immunohistochemical staining and image propuls analysis of aorta are shown;

[0029] Fig.12 The results of liver F4 / 80 immunohistochemical staining and image propuls analysis are shown;

[0030] Fig.13 The results of aortic ICAM-1 immunohistochemical staining and image propuls analysis are shown;

[0031] Fig.14 The results of aortic VCAM-1 immunohistochemical staining and image propuls analysis are shown;

[0032] Fig.15 The expression of inflammation-related genes and proteins and mitochondrial-related genes and proteins in liver tissue is shown;

[0033] Fig.16 The expression of proteins and genes related to liver lipogenesis is shown;

[0034] Fig.17 The expression of proteins and genes related to hepatic lipid metabolism is shown. DETAILED DESCRIPTION

[0035] Composition

[0036] The present invention has found that feeding dyslipidemia model mice with a combination of citrus (or citrus extract) and dietary fiber can effectively improve their dyslipidemia.

[0037] In one aspect, the present invention provides a composition comprising:

[0038] A first component, the first component being citrus or a citrus extract; and

[0039] The second component is dietary fiber.

[0040] In some embodiments, the weight ratio of the first component to the second component is 1:20-20:1, for example, 1:20, 1:15, 1:10, 1:5, 1:2, 1:1, 2:1, 5:1, 10:1, 15:1 or 20:1.

[0041] In some embodiments, the citrus extract is obtained by one of the following extraction methods: heating extraction, immersion extraction, enzymatic ultrasonic extraction, ultra-high pressure extraction, and subcritical extraction.

[0042] In some embodiments, the dietary fiber is obtained by one of the following extraction methods: acid-base extraction, water extraction, enzymatic extraction, microwave extraction, and membrane separation extraction.

[0043] In some embodiments, the composition is in the form of fresh fruit, juice, jam, fruit powder (such as freeze-dried powder), candy (such as compressed candy), oral liquid, capsule, tablet, dried fruit, preserved fruit, fruit wine or cake.

[0044] In a preferred embodiment, the composition is in the form of fruit powder, which comprises citrus fruit powder and dietary fiber powder.

[0045] In some embodiments, the composition is in the form of medicine (such as traditional Chinese medicine, Chinese patent medicine), ordinary food or health food.

[0046] In some embodiments, the drug further contains a pharmaceutically acceptable excipient.

[0047] In the present invention, "pharmaceutically acceptable excipients" refer to substances contained in pharmaceutical preparations in addition to active ingredients when producing drugs. Examples of pharmaceutically acceptable excipients include, but are not limited to, fillers, colorants, flavoring agents, plasticizers, binders, disintegrants, etc.

[0048] In some embodiments, the citrus is selected from the group consisting of tomato, citrus aurantium, kumquat, orange, tangelo, mandarin orange, orange, sour orange, citron, and the like.

[0049] In some embodiments, the dietary fiber includes cellulose, hemicellulose, pectin, lignin, oligosaccharides, and resistant starch. Preferably, the dietary fiber is selected from the group consisting of resistant dextrin, polydextrose, isomaltooligosaccharide, inulin, fructooligosaccharide, xylo-oligosaccharide, and galacto-oligosaccharide.

[0050] Combination Products

[0051] In yet another aspect, the present invention provides a combination product comprising:

[0052] A first product, the first product being the aforementioned composition;

[0053] The second product is a component for preventing blood lipid diseases or atherosclerotic diseases.

[0054] In some embodiments, the lipid disorder is selected from hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, combined hyperlipidemia, low-high-density lipoproteinemia, and the like.

[0055] In some embodiments, the component for preventing blood lipid disorders is selected from atorvastatin, simvastatin, rosuvastatin, niacin, fenofibrate, linoleic acid, Omega-3 fatty acids, ezetimibe, and the like.

[0056] In some embodiments, the atherosclerotic disease is selected from atherosclerosis of the aorta and its main branches, coronary artery atherosclerosis, cerebral artery atherosclerosis, renal artery atherosclerosis, mesenteric artery atherosclerosis, limb artery atherosclerosis, etc.

[0057] In some embodiments, the components for preventing atherosclerotic diseases are selected from statins (such as rosuvastatin, atorvastatin, simvastatin), ezetimibe, PCSK9 inhibitors, aspirin, clopidogrel, colchicine, canakinumab, probucol, idebenone, etc., and Chinese patent medicine preparations.

[0058] In some embodiments, the weight ratio of the first product to the second product is 1000:1-1:1000, for example, 1000:1, 800:1, 600:1, 500:1, 400:1, 200:1, 100:1, 80:1, 60:1, 50:1, 40:1, 20:1, 10:1, 5:1, 2:1, 1:1, 1:2, 1:5, 1:10, 1:20, 1:40, 1:50, 1:60, 1:80, 1:100, 1:200, 1:400, 1:500, 1:600, 1:800, 1:1000.

[0059] In another aspect, the present invention provides the use of the aforementioned composition in the preparation of medicines (such as Chinese medicines, Chinese patent medicines, natural medicines), ordinary foods, functional foods or health foods, and the medicines (such as Chinese medicines, Chinese patent medicines, natural medicines), ordinary foods, functional foods or health foods are used to prevent lipid disorders or atherosclerotic diseases.

[0060] In another aspect, the present invention provides the use of the aforementioned combination product in the preparation of a drug (such as a traditional Chinese medicine, a Chinese patent medicine, a natural medicine) for preventing blood lipid disorders or atherosclerotic diseases.

[0061] In the present invention, "ordinary food" refers to finished products and raw materials for human consumption or drinking, as well as items that are traditionally both food and medicine, but does not include items for therapeutic purposes.

[0062] In the present invention, "health food" refers to food that claims to have specific health functions or is intended to supplement vitamins and minerals, that is, food that is suitable for consumption by specific groups of people, has the function of regulating body functions, is not intended to treat diseases, and does not cause any acute, subacute or chronic harm to the human body. Functional food is mainly a term used in Europe, America and Japan to refer to food that can improve physical health or reduce illness, which is similar to the definition of health food in China.

[0063] In the present invention, "Chinese medicine" refers to medicines that are collected, processed, and prepared according to the theory of traditional Chinese medicine, explain the mechanism of action, and guide clinical application. Chinese medicines are mainly derived from natural medicines and their processed products, including herbal medicines, animal medicines, mineral medicines, and some chemical and biological medicines, with herbal medicines being the majority.

[0064] In the present invention, "Chinese patent medicine" refers to a Chinese medicine product of a certain dosage form that is made of Chinese medicinal materials as raw materials and processed according to prescribed prescriptions and preparation processes under the guidance of traditional Chinese medicine theory for the purpose of preventing and treating diseases. It is a type of commercialized Chinese medicine preparation approved by the national drug supervision and administration department.

[0065] In the present invention, "natural medicine" refers to animal medicine, plant medicine and mineral medicine that have been proven to have certain pharmacological activity by the modern medical system. Natural medicine is not equivalent to traditional Chinese medicine or Chinese herbal medicine. Natural medicine refers to natural medicinal substances and their preparations used under the guidance of modern medical theory. Its sources include plants, animals and minerals, and generally do not include substances derived from genetically modified animals and plants, substances fermented by microorganisms or modified by chemicals, etc.

[0066] Example

[0067] The present invention is further described below by specific examples and comparative examples. However, it should be understood that these examples and comparative examples are only used for more detailed and specific description and should not be construed as limiting the present invention in any form.

[0068] In the present invention, ApoE- / - mice were fed with a high-fat and high-cholesterol diet to establish an atherosclerosis (AS) mouse model, and olive fruit powder, citrus powder, dietary fiber combination, and a combination of the two were used to observe their lipid-lowering and cardiovascular disease prevention effects, and to explore their mechanism of action.

[0069] 1. Test samples

[0070] The test substance citrus powder was provided by Shanghai Youwen Chemical Industry Co., Ltd.; olive fruit powder was provided by Shanghai Tongyuan Food Technology Co., Ltd.; dietary fiber (Fibersol-2) was provided by ADM Company of the United States.

[0071] 2. Experimental animals and groups

[0072] The experimental animals were 108 male ApoE- / - mice and 10 C57bl / 6J mice.

[0073] 108 male ApoE- / - mice and 10 C57bl / 6J mice were housed in an SPF barrier animal room at Suzhou Medical College, Soochow University. The animal room was kept quiet, clean, ventilated and properly illuminated, with a temperature of (22±2)℃, a humidity of 40% to 70%, and a light-dark cycle of 12h. The animals were free to drink water and eat. After one week of adaptive feeding, they were fed a high-fat, high-cholesterol diet for 12 weeks.

[0074] Ten C57bl / 6J mice were used as the normal mouse control group, and 108 ApoE- / - mice were randomly divided into 9 groups according to body weight, with 12 mice in each group. The experiment set up 1 blank control group, 1 model control group and 7 experimental groups. The detailed grouping is shown in Table 1 and Figure 1 .

[0075] Table 1 Experimental animal grouping design

[0076]

[0077] Among them, Fibersol-2: total dietary fiber >90% [calculated as 100%]; citrus powder: [calculated as 100%]; olive fruit powder: hydroxytyrosol and tyrosol 10-12% [calculated as 12%].

[0078] The standard feed in groups 1 and 2 was purchased from Dyets, catalog number: AIN93G.

[0079] The high-fat, high-cholesterol diet (45% fat + 1% cholesterol) in groups 3-10 was purchased from Dyets, item number: HF45.

[0080] "Fibersol-2 (3.9 g / kg)" in Group 4 means that the content of "Fibersol-2" in the "high-fat and high-cholesterol feed" is 3.9 g / kg.

[0081] Similarly, “citrus powder (0.39 g / kg)” in Group 5 indicates that the content of “citrus powder” in “high-fat and high-cholesterol feed” is 0.39 g / kg; “olive fruit powder (0.65 g / kg)” in Group 10 indicates that the content of “olive fruit powder” in “high-fat and high-cholesterol feed” is 0.65 g / kg; other similar expressions have similar meanings.

[0082] Sample collection and test indicators: At the end of the experiment, blood, aorta, liver, kidney, spleen, pancreas, subcutaneous fat, epididymal fat, perirenal fat, and feces were dissected and tested for the following indicators.

[0083] Blood: fasting blood glucose, OGTT, insulin, four blood lipid items (TG, TC, LDL, HDL), free fatty acids (NEFA), oxidized LDL and AST, ALT, tumor necrosis factor-α (TNFα), interleukin-1β (IL1β), and C-reactive protein (CRP).

[0084] Liver: TC, TG, TNFα, IL1β, IL-6, SOD, Catalase, liver pathological sections (HE, Oil red staining and inflammation index F4 / 80), protein and mRNA level detection: NFκB, GSTP, NLRP3; lipid synthesis indicators: FAS, SREBP1c, LXRα, SCD-1, CYP7A1, HMG-CoA reductase.

[0085] Heart: Aortic root pathological sections (oil red staining), aortic root pathological immunohistochemistry adhesion factor indicators ICAM-1, VCAM-1 and inflammatory indicators F4 / 80, CD68.

[0086] Experimental Results

[0087] 1. Effects of food intake and body weight

[0088] Before the experiment, the weights of mice in the C57 group, Apoe group, Model group and experimental groups were (24.12±1.20) g, (23.08±1.51) g, (23.15±1.01) g, (22.54±1.32) g, (22.27±1.53) g, (22.53±1.14) g, (22.42±1.21) g, (22.73±1.52) g, (22.75±1.20) g, (22.73±1.42) g, respectively. There were no significant differences among the groups (P>0.05). As the intervention progressed, the weight of mice in each group increased. Figure 2 As shown in the figure, compared with the Model group, the body weight of mice in the F group, L-orange+F group, and H-orange+F group was lower than that in the Model group from the 9th week, and the difference was statistically significant (P < 0.05). During the intervention period, there was no significant statistical difference in the average food intake of mice in each group. Since the 10 groups of mice had the same food intake, the energy intake was consistent with the food intake. It can be seen that long-term consumption of dietary fiber or a combination of dietary fiber and citrus can have the effect of weight loss.

[0089] 2. Organ quality

[0090] The weights of liver, kidney, spleen, and white fat (subcutaneous fat, perirenal fat, and epididymal fat) were measured in each group of mice and are shown in Table 1. Figure 3 There was no significant statistical difference in the results (P>0.05). Compared with the Model group, the white fat weight of the H-orange+F group, H-orange group, and H-olive group showed a decreasing trend.

[0091] 3. Blood lipid levels

[0092] In order to study the effects of citrus powder, olive fruit powder and dietary fiber on lipid metabolism, the levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL-c), oxidized low-density lipoprotein (ox-LDL), high-density lipoprotein (HDL-c) and free fatty acid (FFA) in the serum of each group of mice were measured. As shown in Table 2, compared with the Model group, the serum lipid profile of the H-orange+F group, H-orange group and H-olive group showed a significant trend of improvement, especially in the H-orange+F group, whose serum TC, LDL-c and ox-LDL were significantly lower than those of the Model group, and HDL-c was significantly higher than that of the Model group (P<0.05), and TG also showed a downward trend. The above results show that dietary supplementation with high-dose citrus powder and dietary fiber combination can effectively improve the abnormal serum lipid profile of mice with atherosclerosis.

[0093] Table 2 Blood lipid status of mice in each group after intervention (mmol / L)

[0094]

[0095] 4. Pancreatic islet function

[0096] Figure 4 The data related to the pancreatic islet function of mice are shown. Compared with the C57 group, the fasting insulin level of the Model group increased, the HOMA-β index reflecting the function of pancreatic β cells decreased, and the insulin resistance (IR) index increased, indicating impaired glucose metabolism. Compared with the Mdoel group, after intervention, the fasting insulin levels of the F group, L-orange+F group, H-orange+F group, L-olive+F group, H-olive+F group, and H-olive group decreased, and the difference was statistically significant. The HOMA-β index of the H-orange group increased, and the difference was statistically significant. The HOMA-IR index of the H-olive group increased, and the difference was statistically significant (p < 0.05).

[0097] 5. Serum and liver inflammatory status

[0098] Atherosclerosis is usually accompanied by chronic inflammation. IL-6, IL-1β, TNF-α and CRP are all pro-inflammatory cytokines. Figure 5 The results show the inflammatory status of mouse serum and liver. The results show that the content of IL-6 (p<0.05) in the serum of the Model group mice was significantly higher than that of the C57 group. The serum IL-6 in the H-olive+F group decreased significantly, and the difference was statistically significant compared with the model group. The serum IL-1β in the H-orange+F group decreased significantly, and the difference was statistically significant compared with the model group. The serum CRP of the Model group mice increased significantly; after intervention, H-orange+F, H-olive+F and H-olive decreased significantly. The content of IL-1β and TNF-α (p<0.05) in the liver of the Model group mice was significantly higher than that of the Apoe group, and the liver TNF-α in the H-orange+F group decreased significantly, and the difference was statistically significant compared with the model group (p<0.05). The liver IL-1β in the H-olive+F group, H-orange group and H-olive group decreased significantly, and the difference was statistically significant compared with the model group (p<0.05). The above results show that the combination of high doses of citrus powder and dietary fiber can reduce inflammation, indicating that the trend of atherosclerosis is slowed down.

[0099] 6. Liver Histopathology

[0100] Figure 6The results of HE staining of liver tissues of mice in each group after the intervention were shown. A large number of round vacuoles of different sizes appeared in the liver tissues of mice in the Model group, and the liver sinusoids became narrower and balloon-like, showing obvious fatty degeneration of the liver. The fatty degeneration of the livers of mice in the H-orange+F group, H-olive+F group, and H-orange group improved to varying degrees, and the fat vacuoles were significantly smaller and less, and the NAS scores (NAFLD activity scores, non-alcoholic fatty liver disease activity scores) were statistically significant compared with the Model group (p<0.05). The above results show that the combination of high doses of citrus powder and dietary fiber can improve the degree of fatty degeneration of the liver.

[0101] 7. Liver damage and hepatic lipid accumulation

[0102] Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are two main indicators of liver function. The levels of AST and ALT in serum were further analyzed to understand the liver damage. The results are shown in Figure 7 In the model group, the serum ALT level of mice showed a significant upward trend, indicating liver damage. Compared with the model group, the serum ALT levels of the H-orange+F group, the H-orange group, and the H-olive group decreased significantly, and the difference between the H-orange+F group and the H-orange group was statistically significant (p<0.05). The levels of oxidized low-density lipoprotein (ox-LDL), TC, and TG in the liver were also detected. The levels of ox-LDL, TC, and TG in the model group showed a significant upward trend, indicating that lipid accumulation also occurred in the liver. After the intervention, compared with the model group, the liver lipid metabolism of the F group, the L-orange+F group, and the H-orange+F group was significantly improved, and the difference was statistically significant (p<0.05). The above results show that the combination of high-dose citrus powder and dietary fiber effectively reduces liver damage, and the combination of dietary fiber and citrus powder improves liver lipid metabolism.

[0103] 8. Aortic atherosclerosis

[0104] The effects of whole aorta oil red O staining and aortic sinus oil red O staining on atherosclerosis were studied. The results of whole aorta oil red O staining are as follows: Figure 8 As shown, compared with the model group, the plaque area of ​​the aorta of mice in the L-orange+F group and the H-orange+F group was reduced. This result indicates that the combination of dietary fiber and citrus powder can effectively improve the condition of atherosclerosis and inhibit its development.

[0105] 9. Aortic Histopathology

[0106] Fig. 9The histopathological photos of the mouse aorta are shown. Under low magnification, compared with the mice in the C57 group, the aortic vascular wall of the mice in the Model group was significantly thickened, resulting in stenosis of the arterial lumen. Under high magnification, the structures of the various layers of the aortic wall of the mice in the C57 group were normal, the endothelium was intact and smooth, and the smooth muscle cells in the middle layer were neatly arranged. The intima of the aortic arch of the mice in the Model group was incomplete, protruding from the lumen to form a fibrous cap, the smooth muscle cells degenerated, necrotic and focally aggregated, the boundaries between the intima and the middle membrane were unclear and obviously irregularly thickened, and the cells had lipid precipitation accompanied by the generation of a large number of foam cells. These conditions of the mice in the L-orange+F group, the H-orange+F group, and the L-olive+F group were improved to a certain extent, indicating that the combination of dietary fiber and citrus powder can improve the aortic lesions of mice to varying degrees.

[0107] 10. Macrophage infiltration in the aorta and liver

[0108] The number of macrophages is closely related to the development of inflammation. A large number of literature reports that the severity of AS is positively correlated with the number of macrophages. In the early stages of atherosclerosis, the activation of endothelial cells can induce the increased expression of a large number of adhesion molecules, which together with the destruction of endothelial barrier function promote the aggregation of leukocytes and monocytes in the vascular wall. After migrating to the subendothelium of the blood vessels, monocytes rapidly differentiate into macrophages and phagocytize oxidized low-density lipoproteins to eventually form foam cells. This process promotes the development of the lipid core of atherosclerotic plaques. In order to study the infiltration of macrophages in the aorta and liver of mice, CD68 and F4 / 80 are highly expressed by blood monocytes and tissue macrophages, which are surface markers of macrophages. Therefore, CD68 and F4 / 80 immunohistochemical staining was performed on the paraffin sections of mouse liver tissue and aorta tissue. Figure 10-12 Shown are aorta, liver staining and analysis results.

[0109] Image pro puls software statistical analysis found that the proportion of macrophages in the aorta and liver of mice in the Model group was significantly higher than that in the C57 group, and the difference was statistically significant (p<0.05). After intervention, compared with the Model group, the number of macrophages in the F group, L-orange+F group, and H-orange+F group decreased, and the difference in the H-orange+F group was statistically significant (p<0.05). It can be seen that the combination of dietary fiber and high-dose citrus powder can significantly reduce the number of macrophages, indicating that the degree of atherosclerosis is reduced.

[0110] 11. Expression of VCAM-1 and ICAM-1 in aorta

[0111] Vascular cell adhesion molecule (VCAM-1) and intercellular adhesion molecule (ICAM-1) are two members of the immunoglobulin superfamily, which are mainly secreted by vascular endothelial cells and smooth muscle cells, and play an important role in the adhesion of blood cells to the vascular endothelium, respectively. In the early stages of AS, VCAM-1 and ICAM-1 mainly mediate the adhesion of monocytes to endothelial cells. As AS progresses, the expression levels of VCAM-1 and ICAM-1 gradually decrease, and promote the transformation of monocytes into macrophages. Macrophages phagocytize lipids to form foam cells, which accelerates the formation of AS plaques. At the same time, VCAM-1 and ICAM-1 can also promote the migration of vascular smooth muscle to the intima, further promoting the progression of AS. In addition, soluble cell adhesion molecules are considered to be predictors of cardiovascular events in healthy people and patients with various ischemic heart diseases. Therefore, VCAM-1 and ICAM-1 are important molecules in the early stages of AS. Immunohistochemical staining of ICAM-1 and VCAM-1 was performed on mouse aortic tissue, and the results are shown in Fig.13 , 14 .

[0112] Image pro puls software statistical analysis found that the expression of VCAM-1 and ICAM-1 in the Model group mice increased. After intervention, compared with the Model group, the expression of VCAM-1 and ICAM-1 in the F group and H-orange+F group mice was significantly reduced, and the difference in ICAM-1 between the F group and H-orange+F group mice was statistically significant (p<0.05), that is, the combination of dietary fiber and high-dose citrus powder inhibited the expression of VCAM-1 and ICAM-1 and slowed down the development of AS.

[0113] 12. Expression of liver inflammation-related and mitochondrial-related genes and proteins

[0114] Fig.15The expression of inflammation-related genes, proteins and mitochondrial-related genes and proteins in liver tissue is shown. The results in the figure show that after intervention, the Model group showed increased expression of inflammation-related mRNA and proteins such as NFκB and NLRP3 inflammasome. Compared with the Model group, the mRNA expression of NF-κB in the F group, L-orange+F group, H-orange+F group, L-olive+F group, H-olive+F group, H-orange group, and H-olive group was downregulated, and the difference was statistically significant (p < 0.05). Compared with the Model group, the mRNA expression of NLRP3 in the H-orange+F group and H-olive+F group was downregulated, and the difference was statistically significant (p < 0.05). Compared with the Model group, the protein expression of NF-κB in the L-orange+F group, L-olive+F group, and H-olive+F group was downregulated, and the difference was statistically significant (p < 0.05). Compared with the Model group, the protein expression of NLRP3 in the H-olive+F group was upregulated (p < 0.05). Compared with the Model group, the expression of glutathione S-transferase P1 (GSTP1) protein in the H-olive+F group and the H-orange group was significantly downregulated (p < 0.05). Therefore, the combination of citrus powder and dietary fiber can improve the expression of liver inflammation.

[0115] 13. Expression of proteins and genes related to liver lipogenesis

[0116] Fatty acid synthase (FAS) is one of the key enzymes in fatty acid synthesis, and stearoyl-CoA1 (SCD-1) is an important metabolic enzyme in the formation of monounsaturated fatty acids during fatty acid synthesis. Fig.16The expression of proteins and genes related to liver fat synthesis was shown. The study found that the expression of liver fat synthesis in the Model group mice increased at the protein and gene levels. Compared with the Model group, the expression of Fas protein in the L-orange+F group, H-orange+F group, L-olive+F group, H-orange group, and H-olive group was statistically significant (p < 0.05). The expression of SCD-1 protein in the L-orange+F group, H-orange+F group, L-olive+F group, H-olive+F group, H-orange group, and H-olive group was statistically significant (p < 0.05). The expression of SCD-1 gene in the H-orange+F group was statistically significant. The expression of sterol regulatory element binding protein 1C (SREBP-1C) protein in the L-orange+F group, H-orange+F group, L-olive+F group, H-olive+F group, H-orange group, and H-olive group was statistically significant (p < 0.05). There was a statistically significant difference in SREBP-1C gene expression between the H-orange+F group and the L-olive+F group (p<0.05). The above results indicate that the combination of citrus powder and dietary fiber is beneficial to inhibit liver fat synthesis.

[0117] 14. Expression of proteins and genes related to liver lipid metabolism

[0118] CYP7A1 is the rate-limiting enzyme that catalyzes the conversion of animal liver cholesterol into bile acid. Fig.17The results show the expression of proteins and genes related to liver lipid metabolism, from which it can be seen that the expression of CYP7A1 in mice in the Model group increased. After intervention, compared with the Model group, the expression of CYP7A1 protein in the F group, H-orange+F group, and H-olive group was upregulated, and the difference was statistically significant (p < 0.05). LXR-α plays an important role in regulating cholesterol homeostasis. The study found that the expression of LXR-α in mice in the Model group increased. Compared with the Model group, the expression of LXR-α protein in the L-orange+F group, L-olive+F group, and H-olive+F group was downregulated, and the difference was statistically significant (p < 0.05). As a transmembrane glycoprotein, HMGCR protein is involved in the control of cholesterol biosynthesis. The expression of HMGCR in mice in the Model group increased. Compared with the Model group, the expression of HMGCR protein in the L-orange+F group, H-orange+F group, L-olive+F group, H-olive+F group, and H-orange group was downregulated, and the difference was statistically significant (p < 0.05). Similar results were found for the mRNA expression of CYP7A1, LXR-α, and HMGCR, but there was no significant difference in CYP7A1 compared with the Mdoel group (p>0.05). It can be seen that the combination of dietary fiber and high-dose citrus powder can promote cholesterol conversion, the combination of dietary fiber and low-dose citrus powder can help regulate cholesterol homeostasis, and the combination of high-dose citrus powder and citrus powder and dietary fiber can inhibit cholesterol synthesis and help lower cholesterol.

[0119] Analysis and discussion

[0120] The pathological mechanism of atherosclerosis (AS) is complex, and multiple risk factors accelerate this process, such as abnormal lipid metabolism, hypertension, diabetes, smoking, unhealthy lifestyle and aging. AS is a chronic inflammatory pathological process, and impaired endothelial function is the initial step of atherosclerosis. In the early stage of atherosclerotic plaques, damaged endothelial cells can release adhesion molecules, cytokines, etc. to recruit monocytes and T lymphocytes in the blood circulation. After migrating to the intima, monocytes transform into macrophages. Lipids accumulated under the intima are phagocytosed by macrophages to form foam cells, which together with the infiltration of inflammatory cells aggravate the occurrence of AS plaques. In this study, ApoE- / - mice were fed with a high-fat and high-cholesterol diet to establish an AS mouse model. Olive fruit powder, citrus powder, dietary fiber combination, and the combination of the two were used to observe their lipid-lowering and cardiovascular disease prevention effects, and to explore their mechanism of action.

[0121] ApoE- / - mice are an ideal model for AS modeling. ApoE- / - mice can spontaneously develop hypercholesterolemia and then evolve into AS, which well simulates the occurrence and development process of human AS. After ApoE- / - mice were fed a high-fat and high-cholesterol diet for 12 weeks, large areas of red-stained AS lesions appeared on the inner wall of the aorta, indicating that the AS modeling was successful. After intervention, although the plaque area of ​​each experimental group decreased, there was no statistically significant difference compared with the Model group. Among them, the F group, H-orange+F group, H-orange group, and H-olive group had better improvement effects. The aortic HE pathological results showed that under low magnification, compared with the mice in the C57 group, the aortic vascular wall of the mice in the Model group was significantly thickened, causing stenosis of the arterial lumen. Under high magnification, the structures of the aortic walls of the mice in the C57 group were normal, the endothelium was intact and smooth, and the smooth muscle cells in the middle layer were neatly arranged. The intima of the aortic arch of mice in the Model group was incomplete, with a fibrous cap formed in the lumen, degeneration, necrosis and focal aggregation of smooth muscle cells, unclear boundaries between the intima and the media, and obvious irregular thickening, lipid precipitation in the cells and the generation of a large number of foam cells. The conditions of mice in the L-orange+F, H-orange+F and L-olive+F groups were improved to a certain extent.

[0122] This study found that ApoE- / - mice fed a high-fat, high-cholesterol diet developed lipid metabolism disorders. The serum TG, TC, and LDL-c levels of mice in the Model group were significantly increased, but the serum TG, TC, and LDL-c levels of mice in the H-orange+F, H-orange, and H-olive groups showed a significant improvement trend, among which the H-orange+F group had the best improvement effect.

[0123] The fasting insulin of ApoE- / - mice fed with high-fat and high-cholesterol diet was detected, and it was found that the insulin level of the Model group mice was increased and the pancreatic islet function was impaired. After intervention, the fasting insulin levels of the F, L-orange+F, H-orange+F, L-olive+F, H-olive+F and H-olive groups decreased significantly. The HOMA-β index of the H-orange group increased significantly, and the HOMA-IR index of the H-olive group decreased significantly.

[0124] After mice were fed a high-fat and high-cholesterol diet, a large number of round vacuoles of varying sizes appeared in the livers of the mice in the Model group, the hepatic sinusoids became narrower, and balloon-like changes occurred, showing obvious fatty degeneration of the liver. After intervention, the fatty degeneration of the livers of the mice in the H-orange+F, H-olive+F and H-orange groups improved to varying degrees, the fat vacuoles were significantly smaller and fewer, and the NAS scores were significantly lower than those in the Model group. Serum AST and ALT levels reflect liver damage. The serum ALT levels of the mice in the Model group showed a significant upward trend. The serum ALT levels of the H-orange+F, H-orange and H-olive groups decreased significantly, among which the H-orange+F and H-orange groups had the best effects. In addition, the TC and TG levels of the livers of the mice in the Model group also showed a significant upward trend, indicating that lipid accumulation occurred in the liver. After intervention, the lipid metabolism of the livers of the F, L-orange+F and H-orange+F groups was improved, among which the H-orange+F group had the best improvement effect. This study found that the body weight of mice in group F, group L-orange+F, and group H-orange+F decreased after intervention, indicating that it also has a certain effect on weight loss.

[0125] Most of the fatty acids required for animal body fat deposition come from de novo fatty acid synthesis, that is, the synthesis of fatty acids from acetyl-CoA and malonyl-CoA catalyzed by fatty acid synthase (Fas). The expression of Fas mRNA and protein in ApoE mice and ApoE mice fed with a high-fat and high-cholesterol diet (Model group) were higher than those in normal mice. After intervention, the expression of Fas protein in the H-orange+F, L-olive+F, H-orange and H-olive groups was downregulated, and the downregulation in the H-orange group was the most obvious. Stearoyl-coA desaturase-1 (SCD-1) is a class of endoplasmic reticulum transmembrane proteins that catalyze the formation of monounsaturated fatty acids from saturated fatty acids. It is widely expressed in various tissues in the body, with the liver and adipose tissue expressing more, and is a gene downstream of SREBP-1c. SREBP-1c and SCD-1 regulate the synthesis and absorption of lipids such as triglycerides and cholesterol through a series of pathways, and play a key role in the development of diabetic liver lesions. Increased expression of SREBP-1c and SCD-1 will cause lipid metabolism disorders, leading to fatty degeneration of non-fat tissues such as the liver, and clinical manifestations such as hepatic steatosis. After feeding mice with a high-fat and high-cholesterol diet, the mRNA and protein expressions of SREBP-1c and SCD-1 in the liver of the Model group mice were significantly upregulated. Compared with the Model group, the mRNA expression of SCD-1 in the H-orange+F group mice was significantly downregulated; the protein expression of SCD-1 in the L-orange+F, H-orange+F, L-olive+F, H-olive+F, H-orange and H-olive groups was significantly downregulated. Compared with the Model group, the mRNA expression of SREBP-1C in the H-orange+F and L-olive+F groups was significantly downregulated; the protein expression of SREBP-1C in the L-orange+F, H-orange+F, L-olive+F, H-olive+F, H-orange and H-olive groups was significantly decreased. In summary, the H-orange+F and L-olive+F groups had the best improvement effect.

[0126] Macrophages promote the release of proinflammatory factors and growth factors, and the release of these inflammatory factors in turn promotes the local proliferation of macrophages in the inflammatory response. This cycle will further aggravate the process of inflammatory response. The aggravation of the inflammatory response will chemotaxis and recruit more immune cells to the subintimal membrane, and promote the expression of collagen fibers and the production of plaque necrosis core, further promoting the occurrence of atherosclerosis. Therefore, macrophages enhance the inflammatory response by promoting the release of inflammatory factors, and ultimately induce the formation of AS plaques. The results of CD68 and F4 / 80 immunohistochemistry showed that feeding mice with a high-fat and high-cholesterol diet led to increased macrophage infiltration in the aortic root plaques of mice, indicating that high fat and high cholesterol can induce the aggregation of macrophages during the occurrence of AS. After intervention, the number of macrophages in mice in the F, L-orange+F and H-orange+F groups decreased, among which the H-orange+F group had the best improvement effect. AS is a chronic inflammatory pathological process. After mice were fed a high-fat, high-cholesterol diet, the inflammatory factors IL-6, TNF-α, IL-1β and CRP in the serum and liver tissues of the Model group mice increased. After intervention, the serum IL-6 in the H-olive+F group decreased significantly; the serum IL-1β in the H-orange+F group decreased significantly; the CRP in the H-orange+F, H-olive+F and H-olive groups decreased significantly. The IL-1β and TNF-α levels in the liver of the Model group mice were significantly higher than those in the Apoe group, and the liver IL-1β in the H-orange+F group decreased significantly; the liver IL-1β in the H-olive+F, H-orange and H-olive groups decreased significantly. In summary, the H-orange+F and H-olive+F groups had the best effects.

[0127] ox-LDL is an important inflammatory stimulus in the development of atherosclerosis, leading to chronic inflammatory response in blood vessels. After mice were fed a high-fat and high-cholesterol diet, the ox-LDL in the liver of the Model group mice increased significantly, and the ox-LDL levels in the L-orange+F and H-orange+F groups decreased significantly after intervention. With the progression of AS, VCAM-1 and ICAM-1 promote the transformation of monocytes into macrophages. Macrophages phagocytize lipids to form foam cells, which accelerates the formation of AS plaques. In addition, soluble cell adhesion molecules are predictors of cardiovascular events in healthy people and patients with various ischemic heart diseases. Therefore, VCAM-1 and ICAM-1 are important molecules in the early stages of AS. Feeding mice with a high-fat and high-cholesterol diet increased the expression of VCAM-1 and ICAM-1 in plaques. After intervention, the expression of ICAM-1 in mice in the F and H-orange+F groups was significantly reduced, and the expression of VCAM-1 was reduced. In the process of AS, ox-LDL can stimulate monocytes / macrophages to release inflammatory factors. Once released, inflammatory factors can increase cholesterol uptake by activating NF-κB and promoting the expression of acyl-CoA cholesterol acyltransferase l, thereby promoting the formation of foam cells. According to the results of WB and RT-PCR, it can be found that after mice were fed a high-fat and high-cholesterol diet, the mRNA and protein expressions of NFκB and NLRP3 in the Model group were upregulated. After intervention, the mRNA expression of NF-κB in F, L-orange+F, H-orange+F, L-olive+F, H-olive+F, H-orange and H-olive groups was significantly downregulated; the protein expression of NF-κB in L-orange+F, L-olive+F and H-olive+F groups was significantly downregulated. After intervention, the mRNA expression of NLRP3 in H-orange+F and H-olive+F groups was significantly downregulated; the protein expression of GSTP1 in H-olive+F and H-orange groups was significantly downregulated. That is, the L-orange+F group, L-olive+F group and H-olive+F group had the best effect.

[0128] in conclusion

[0129] The above experimental results are summarized in Table 3 below. It can be seen that high-dose citrus powder + dietary fiber has an improvement effect on various indicators of atherosclerosis mice, with the best effect, followed by high-dose citrus powder and high-dose olive fruit powder.

[0130] Table 3 Effects of olive fruit powder, citrus powder, dietary fiber combination, and their combination on AS mice

[0131]

[0132] Note: √ indicates that the relevant experimental results are relatively good

[0133] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details based on all the teachings disclosed, and these changes are within the scope of protection of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. A composition comprising: A first component, the first component being citrus or a citrus extract; and The second component is dietary fiber.

2. The composition of claim 1, wherein The weight ratio of the first component to the second component is 1:20-20:1, preferably the weight ratio of the first component to the second component is 1:20, 1:15, 1:10, 1:5, 1:2, 1:1, 2:1, 5:1, 10:1, 15:1 or 20:

1.

3. The composition of claim 1, wherein The citrus extract is obtained by one of the following extraction methods: heating extraction, leaching extraction, enzymatic hydrolysis and ultrasonic extraction, ultra-high pressure extraction, and subcritical extraction; Alternatively, the dietary fiber is obtained by one of the following extraction methods: acid-base extraction, water extraction, enzymatic extraction, microwave extraction, and membrane separation extraction.

4. The composition of claim 1, wherein The composition is in the form of fresh fruit, juice, jam, fruit powder (such as freeze-dried powder), candy (such as compressed candy), oral liquid, capsule, tablet, dried fruit, preserved fruit, fruit wine or cake; Alternatively, the composition is in the form of a medicine (such as traditional Chinese medicine, Chinese patent medicine), a common food or a health food. Preferably, the medicine further contains a pharmaceutically acceptable excipient.

5. The composition of claim 1, wherein The citrus is a fruit of a plant of the genus Citrus in the Rutaceae family. Preferably, the citrus is selected from the group consisting of: tomato, Fumin trifoliate orange, kumquat, arrow leaf orange, tangelo, Mangshan wild orange, four-season orange, sour orange, fragrant orange, and citron; Alternatively, the dietary fiber includes cellulose, hemicellulose, pectin, lignin, oligosaccharides, and resistant starch. Preferably, the dietary fiber is selected from the group consisting of resistant dextrin, polydextrose, isomaltooligosaccharide, inulin, fructooligosaccharide, xylo-oligosaccharide, and galacto-oligosaccharide.

6. A combination product comprising: A first product, wherein the first product is the composition according to any one of claims 1 to 5; The second product is a component for preventing blood lipid diseases or atherosclerotic diseases.

7. The combination product of claim 6, wherein The blood lipid disease is selected from hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, combined hyperlipidemia, and low-high-density lipoproteinemia; Alternatively, the component for preventing blood lipid disorders is selected from atorvastatin, simvastatin, rosuvastatin, niacin, fenofibrate, linoleic acid, omega-3 fatty acids, and ezetimibe; Wherein, the atherosclerotic disease is selected from atherosclerosis of the aorta and its main branches, coronary artery atherosclerosis, cerebral artery atherosclerosis, renal artery atherosclerosis, mesenteric artery atherosclerosis, and limb artery atherosclerosis; Alternatively, the component for preventing atherosclerotic diseases is selected from statins (such as rosuvastatin, atorvastatin, simvastatin), ezetimibe, PCSK9 inhibitors, aspirin, clopidogrel, colchicine, canakinumab, probucol, idebenone, and Chinese patent medicine preparations.

8. The combination product of claim 6, wherein The weight ratio of the first product to the second product is 1000:1-1:1000, and preferably the weight ratio of the first product to the second product is 1000:1, 800:1, 600:1, 500:1, 400:1, 200:1, 100:1, 80:1, 60:1, 50:1, 40:1, 20:1, 10:1, 5:1, 2:1, 1:1, 1:2, 1:5, 1:10, 1:20, 1:40, 1:50, 1:60, 1:80, 1:100, 1:200, 1:400, 1:500, 1:600, 1:800, 1:1000.

9. Use of the composition according to any one of claims 1 to 5 in the preparation of medicines (such as Chinese medicines, Chinese patent medicines, natural medicines), ordinary foods, functional foods or health foods, wherein the medicines (such as Chinese medicines, Chinese patent medicines, natural medicines), ordinary foods, functional foods or health foods are used to prevent blood lipid diseases or atherosclerotic diseases.

10. Use of the combination product according to any one of claims 6 to 8 in the preparation of a drug (such as a Chinese medicine, a Chinese patent medicine, or a natural medicine), wherein the drug (such as a Chinese medicine, a Chinese patent medicine, or a natural medicine) is used to prevent blood lipid disorders or atherosclerotic diseases.

11. The use according to claim 9 or 10, wherein The blood lipid disease is selected from hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, combined hyperlipidemia, and low-high-density lipoproteinemia; The atherosclerotic disease is selected from atherosclerosis of the aorta and its main branches, coronary artery atherosclerosis, cerebral artery atherosclerosis, renal artery atherosclerosis, mesenteric artery atherosclerosis, and limb artery atherosclerosis.

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