New application of donkey-hide gelatin

Abstract

The invention provides new application of donkey-hide gelatin, relates to application of donkey-hide gelatin in functional foods, medicines and foods, and is used for preparing pharmaceutical preparations and / or health care products and / or foods for improving atherosclerosis complicated with hyperlipemia. According to the new application of the donkey-hide gelatin disclosed by the invention, the levels of serum total cholesterol (TC), triglyceride (TG) and low-density lipoprotein (LDL) can be reduced; the level of high density lipoprotein (HDL) is increased; eT secretion, Ang II generation and iNOS enzyme synthesis are effectively inhibited, so that NO synthesis and release are promoted; the activity of antioxidant enzymes such as superoxide dismutase and catalase can be improved, and the content of malondialdehyde (MDA) is reduced; secretion of inflammatory factors such as IL-6, CRP, TNF-alpha and IFN-gamma can be remarkably reduced, and the inflammatory state is improved; the aortic plaque area is significantly reduced, and lipid deposition and plaque formation are improved.

Description

Technical Field

[0001] The present invention relates to the use of Ejiao in the fields of functional foods, medicine, and food, and particularly to the application of Ejiao in the preparation of drugs, health foods, and foods for improving atherosclerosis complicated with hyperlipidemia. Background Art

[0002] Atherosclerosis and hyperlipidemia are common cardiovascular diseases, and the two often occur together, seriously threatening human health. The occurrence and development of atherosclerosis start with various harmful stimuli damaging the endothelial cells (EC) of the arterial wall. Platelet adhesion, aggregation, release of bioactive substances, and smooth muscle cell proliferation are the main links, and lipid infiltration, inflammatory reaction, oxidative stress, and destruction of elastic fibers in the arterial wall lead to the pathological outcome of arterial lumen stenosis.

[0003] Currently, the main strategy for preventing and treating atherosclerosis is lipid-lowering. Clinically, statins, fibrates and other drugs are mainly used for treatment, but these drugs have certain side effects, such as liver function damage, muscle pain, etc. At present, for the patient group of patients with atherosclerosis complicated with hyperlipidemia, there is a lack of effective traditional Chinese medicine in clinical practice. Therefore, it is of great significance to develop a natural drug that is safe, effective and has few side effects and can effectively prevent and treat atherosclerosis complicated with hyperlipidemia.

[0004] Since ancient times, Ejiao has been a relatively precious Chinese herbal medicine and also a famous traditional Chinese medicine in China, with a medicinal history of more than 2,500 years. It is a glue block made from the skin of Equus asinus after removing the hair and is mostly composed of collagen and its partial hydrolysis products; nature and flavor: sweet, flat; entering the lung, liver, and kidney meridians. As a traditional Chinese medicine, Ejiao has the effects of enriching blood, nourishing yin, and moistening dryness, etc., but its application in improving atherosclerosis complicated with hyperlipidemia has not been fully studied. Summary of the Invention

[0005] The purpose of the present invention is to provide a new use of Ejiao in the preparation of pharmaceutical preparations and / or health products and / or foods for improving atherosclerosis complicated with hyperlipidemia.

[0006] The purpose of the present invention is achieved by the following technical solutions: A new use of Ejiao for the preparation of pharmaceutical preparations and / or health products and / or foods for improving atherosclerosis complicated with hyperlipidemia.

[0007] Preferably, the weight-average molecular weight (Mw) of the Ejiao is 16,001 - 16,654 Da, where the proportion of molecules > 50 kDa is 8.34 - 8.38%, the proportion of molecules 10 - 50 kDa is 47.74 - 49.68%, the proportion of molecules 5 - 10 kDa is 13.21 - 13.75%, and the proportion of molecules < 5 kDa is 28.71 - 29.89%.

[0008] Preferably, the pharmaceutical preparation includes: tablets, granules, pills, emulsions, granules, soft capsules, hard capsules or microcapsules.

[0009] Preferably, the health product includes: tablets, granules, pills, emulsions, granules, soft capsules, hard capsules or microcapsules.

[0010] Preferably, the food includes one or more of: beverages, paste foods, candied fruits and pastry foods.

[0011] Preferably, the pharmaceutical preparation includes Ejiao and a pharmaceutically acceptable excipient or carrier.

[0012] Preferably, the health product includes Ejiao and an excipient or carrier acceptable in the field of health products.

[0013] Preferably, the food includes Ejiao and an excipient or food additive acceptable in the field of food.

[0014] The present invention also provides a preparation method of the above-mentioned Ejiao, which includes the following steps: a. Raw material treatment: Select high-quality donkey hides, after rinsing to remove impurities, depilating and defatting, cut them into 5×5 cm squares, soak for 12 - 24 h, and change water 2 - 3 times during soaking to remove blood and odor; b. Decoction: Add water in a ratio of 1:5, decoct the donkey hides at a low temperature (40 - 60°C) and under vacuum (-0.08 ~ -0.10 Mpa) for 12 - 24 h to avoid damage to active ingredients by high temperature; c. Filtration and concentration: After the decoction is completed, filter with gauze or a filter screen to remove residues; use an ultrafiltration membrane or nanofiltration membrane to concentrate the filtrate, remove small molecule impurities, and retain large molecule active ingredients; d. Freeze-drying: Freeze the concentrated solution and perform sublimation drying under vacuum conditions to obtain freeze-dried Ejiao powder with high active ingredients.

[0015] Compared with the prior art, the present invention has the following effects: The present invention first reveals that Ejiao improves atherosclerosis complicated with hyperlipidemia through the following mechanism: (1)Lipid metabolism regulation: reducing the levels of serum total cholesterol (TC), triglyceride (TG) and low-density lipoprotein (LDL); increasing the level of high-density lipoprotein (HDL); (2)Protecting vascular endothelium: effectively inhibiting the secretion of ET, the generation of Ang II and the synthesis of iNOS enzyme, thereby promoting the synthesis and release of NO; (3)Oxidative stress: increasing the activities of antioxidant enzymes such as superoxide dismutase and catalase, and reducing the level of malondialdehyde (MDA); (4)Anti-inflammatory mechanism: reducing the secretion of inflammatory factors such as IL-6, CRP, TNF-α, IFN-γ, and improving the inflammatory state; (5)Plaque area: significantly reducing the aortic plaque area, and improving lipid deposition and plaque formation. Description of the Drawings

[0016] In order to more clearly illustrate the content of the present invention, the following further describes the present invention in detail according to specific embodiments in conjunction with the drawings, where: Figure 1 It is a diagram showing the influence results of Ejiao on the blood lipid levels of rats with atherosclerosis combined with hyperlipidemia model, where A is total cholesterol, B is triglyceride, C is low-density lipoprotein, and D is high-density lipoprotein; Figure 2 It is a diagram showing the influence results of Ejiao on vasoactive substances in rats with atherosclerosis combined with hyperlipidemia model, where A is endothelin, B is nitric oxide, C is angiotensin II, and D is inducible nitric oxide synthase; Figure 3 It is a diagram showing the influence results of Ejiao on serum enzyme activities of rats with atherosclerosis combined with hyperlipidemia model, where A is superoxide dismutase, B is catalase, and C is malondialdehyde; Figure 4 It is a diagram showing the influence results of Ejiao on inflammatory factors in rats with atherosclerosis combined with hyperlipidemia model, where A is interleukin 6, B is serum C-reactive protein, C is tumor necrosis factor α, and D is interferon γ; Figure 5 It is a diagram showing the influence results of Ejiao on plaque deposition in the aortic tissue of rats with atherosclerosis combined with hyperlipidemia model, where a is the oil red O staining diagram, and b is the calculation result diagram of the positive rate of aortic lipid deposition; Figure 6 It is a HE staining diagram showing the influence results of Ejiao on the aortic blood vessels of rats with atherosclerosis combined with hyperlipidemia model. Detailed Description of the Invention

[0017] Example 1 Preparation of Ejiao a. Raw material treatment: Select high-quality donkey skins. After rinsing to remove impurities, depilating and defatting, cut them into 5×5 cm squares and soak for 12 h, changing water twice during this period to remove blood and odor. b. Decoction: Add water in a ratio of 1:5 and decoct the donkey skins at 40°C under a vacuum of -0.08 Mpa for 12 h to avoid damage to active ingredients caused by high temperature. c. Filtration and concentration: After the decoction is completed, filter with gauze or a filter screen to remove residues; use an ultrafiltration membrane or nanofiltration membrane to concentrate the filtrate, remove small molecule impurities, and retain large molecule active ingredients. d. Freeze-drying: Freeze the concentrated solution and perform sublimation drying under vacuum conditions to obtain freeze-dried Ejiao powder with high active ingredients. Determined by high performance liquid chromatography, the weight average molecular weight of the freeze-dried Ejiao is 16328 Da, among which the proportion of molecules >50 kDa is 8.51%, the proportion of molecules 10 - 50 kDa is 48.71%, the proportion of molecules 5 - 10 kDa is 13.48%, and the proportion of molecules <5 kDa is 29.30%.

[0018] Example 2 Animal experiment verification Experimental materials 1.1 Experimental animals: SPF-grade female SD rats, animal production license number: SCXK(Shandong) 2022 0006, animal quality certificate number: No.370726240101851852.

[0019] 1.2 Test drugs: Ejiao obtained in Example 1; Vitamin D3.

[0020] 1.3 Reagents and consumables: Detection kits for blood lipid indicators, vasoactive substances, and serum antioxidant enzyme activities, etc., detection kits for serum inflammatory factors, etc., Oil Red O staining solution (B0023), HE staining solution (BH0001), and other reagents are of analytical purity.

[0021] Experimental methods 2.1 Animal grouping Purchase SPF-grade SD rats. After three days of adaptive feeding, they were randomly divided into a negative control group, a model group, a low-dose donkey-hide gelatin group, and a high-dose donkey-hide gelatin group, with 10 rats in each group. Rats in the negative control group were fed with basal diet throughout the experiment, while rats in the other three groups (i.e., the model groups) were fed with high-fat diet throughout the experiment. The formula of the high-fat diet was: 3% cholesterol, 0.5% sodium cholate, 0.2% propylthiouracil, 5% white sugar, 10% lard, and 81.3% basal diet. Rats in the model groups were intraperitoneally injected with vitamin D3 at a dose of 500,000 IU / kg within the first week, and 200,000 IU / kg at the 3rd, 5th, and 7th weeks respectively to establish an atherosclerotic combined hyperlipidemia model. After the model was established, except for the negative control group fed with normal diet, the model group and the donkey-hide gelatin dose groups continued to be fed with high-fat diet. The difference was that the negative control group and the model group were intragastrically administered with equal volumes of double-distilled water every day, while the donkey-hide gelatin dose groups were intragastrically administered with donkey-hide gelatin solutions at different concentrations (0.75 g / kg / day and 1.5 g / kg / day respectively) every day, and the intragastric administration continued for 30 days until the experiment ended.

[0022] 2.2 Detection indicators 2.2.1 Blood lipid detection After the experiment ended, blood was collected from the rats to measure the contents of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) in the serum, and the differences among groups were compared.

[0023] 2.2.2 Detection of vasoactive substances After the experiment ended, blood was collected from the rats. Using an ELISA kit, the contents of endothelin (ET), angiotensin II (AII), NO content, thromboxane stable metabolite (TXB2), humoral factors CD31, CD34, CD105, etc. in the serum were measured according to the instructions of the kit. The OD value at a wavelength of 450 nm was measured using a Thermo Fisher Multiskan SkyHigh full-wavelength microplate reader and calculated.

[0024] 2.2.3 Determination of serum enzyme activity After the experiment ended, blood was collected from the rats. Using an ELISA kit, the activities of inducible nitric oxide synthase (iNOS), superoxide dismutase (SOD), malondialdehyde (MDA) content, catalase content, etc. in the serum were measured according to the instructions of the kit. The OD value at a wavelength of 450 nm was measured using a Thermo Fisher Multiskan SkyHigh full-wavelength microplate reader and calculated.

[0025] 2.2.4 Determination of serum inflammatory factors After the experiment, blood was collected from the rats. Using an ELISA kit, the contents of inflammation-related factors such as C-reactive protein (CRP), interleukin IL-6, interleukin IL-1β, IFN-γ, and tumor necrosis factor-α (TNF-α) in the serum were measured according to the instructions of the kit. The OD value at a wavelength of 450 nm was measured using a Thermo Fisher Multiskan SkyHigh full-wavelength microplate reader and calculations were performed.

[0026] 2.2.5 Histopathological observation Oil Red O staining: After the experiment, the rats were sacrificed, and the proximal aorta was taken and fixed in 10% formalin for Oil Red O staining. Images were collected, and the red-stained lipid area / total lumen area of the aortic sinus was analyzed using Image-Pro Plus 6.0 software.

[0027] HE staining: After the experiment, the rats were sacrificed, and the proximal aorta was taken and fixed in 10% formalin for HE staining. The tissue sections were observed under a light microscope to compare the histopathological morphology of rats in each group.

[0028] 2.2.6 Statistical analysis SPSS 19.0 software was used for statistical analysis. The data were expressed as mean ± standard deviation; the t-test was used for comparison between two groups; one-way ANOVA was used for comparison among three groups or more groups; P < 0.05 was considered statistically significant.

[0029] 2.2 Experimental results 2.2.1 Blood lipid detection results Blood lipids are the general term for neutral fats (cholesterol and triglycerides), phospholipids, etc. in plasma. Among them, the levels of cholesterol and triglycerides are closely related to clinical diseases. Serum total cholesterol (TC) refers to the sum of cholesterol contained in all lipoproteins in the blood, and its concentration can be used as a marker of lipid metabolism. Its increase may directly lead to atherosclerosis. There are mainly two sources of triglycerides (TG) in plasma: ① Exogenous: Fats ingested from food in the intestine are absorbed by the intestinal mucosa under the action of bile acids and lipases and are synthesized into triglycerides in intestinal mucosal epithelial cells. ② Endogenous: Triglycerides synthesized in the body are mainly in the liver, followed by adipose tissue. An increase in triglycerides is a risk factor for cardiovascular diseases.

[0030] Lipoproteins in plasma are mainly divided into chylomicrons (CM), intermediate density lipoproteins (IDL), low density lipoproteins (LDL), and high density lipoproteins (HDL). Among them, when low density lipoprotein, especially oxidized modified low density lipoprotein (OX-LDL), is in excess, the cholesterol it carries accumulates on the arterial wall and is likely to cause arteriosclerosis over a long time. Therefore, reducing LDL is the basis for preventing cardiovascular diseases. High density lipoprotein (HDL) is mainly synthesized in the liver and is an anti-atherosclerotic lipoprotein that can transport cholesterol from extrahepatic tissues to the liver for metabolism and excrete it out of the body through bile. The level of its plasma content is negatively correlated with the risk of cardiovascular disease. Therefore, high density lipoprotein is an anti-atherosclerotic plasma lipoprotein, undertaking the work of cholesterol "scavenger", being negatively correlated with the occurrence of cardiovascular diseases, being "good cholesterol", and being a protective factor for coronary heart disease.

[0031] The ELISA kit was used to detect the blood lipid levels in the plasma of rats. Figure 1 The results showed that: compared with the negative control group, the contents of total cholesterol (TC), triglyceride (TG), and low density lipoprotein (LDL) in the model group were significantly increased (P < 0.05 or P < 0.01), while the content of high density lipoprotein (HDL) was extremely significantly decreased (P < 0.01). Compared with the model group, the low-dose donkey-hide gelatin group could significantly reduce the contents of triglyceride and low density lipoprotein (P < 0.05), but there was no significant difference in reducing total cholesterol and increasing the content of high density lipoprotein (P > 0.05); the high-dose donkey-hide gelatin group could extremely significantly reduce the contents of total cholesterol, triglyceride, and low density lipoprotein (P < 0.01), and at the same time could significantly increase the content of high density lipoprotein (P < 0.01). It can be seen from this that donkey-hide gelatin has a positive effect on improving the blood lipids of rats with atherosclerosis complicated with hyperlipidemia.

[0032] 2.2.2 Detection results of vasoactive substances The occurrence and development of atherosclerosis (AS) start with the injury of arterial wall endothelial cells (EC), with platelet adhesion and aggregation, release of bioactive substances, and proliferation of smooth muscle cells (SMC) as the main links, lipid infiltration, and destruction of elastic fibers in the arterial wall, leading to the pathological outcome of arterial lumen stenosis.

[0033] Vascular endothelium is not only a mechanical barrier but also has multiple functions such as substance transport, autocrine, and paracrine, playing an important role in the physiological and pathological processes of cardiovascular and cerebrovascular diseases such as wound repair, angiogenesis, and thrombosis. However, vascular endothelium is also a vulnerable functional interface and can undergo morphological and biochemical changes in response to various different pathophysiological stimuli.

[0034] Endothelin (ET) is the most potent vasoconstrictor known so far, with a long-lasting effect. It is not antagonized by α-receptor, H1-receptor, and 5-HT receptor blockers, but can be inhibited by hormones such as isoproterenol, atrial natriuretic peptide, and calcitonin gene-related peptide. Endothelin not only exists in vascular endothelium but also widely exists in various tissues and cells. It is an important factor regulating cardiovascular function and plays an important role in maintaining basal vascular tone and cardiovascular system homeostasis. Nitric oxide (NO) has the functions of dilating blood vessels, reducing blood pressure, inhibiting platelet adhesion and aggregation, and is important for maintaining normal cardiac output, controlling the opening of collateral circulation, adjusting local tissue blood perfusion, and maintaining myocardial blood perfusion. It can antagonize the vasoconstrictive effect of ET and is an endogenous myocardial protective substance. The different biological effects of the two are involved in the regulation of cardiovascular function and the pathogenesis of coronary heart disease. Therefore, protecting vascular endothelial damage, effectively inhibiting ET secretion, promoting NO release, and regulating the plasma concentration balance of the two to improve myocardial blood supply and oxygen supply are important ways to treat coronary heart disease.

[0035] Angiotensin II can activate angiotensin receptors to cause vasoconstriction, thereby increasing blood pressure. Angiotensin II in local tissues and blood circulation can directly act on cardiomyocytes and non-cardiomyocytes, and can enhance myocardial contractility and increase heart rate by promoting the release of norepinephrine from cardiac sympathetic nerve endings.

[0036] Nitric oxide synthase (NOS) is an isoenzyme that exists in endothelial cells, macrophages, neurophagocytes, and nerve cells respectively. There are three subtypes of nitric oxide synthase isoenzymes, namely neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) expressed under normal conditions, and inducible nitric oxide synthase (iNOS) induced to express after injury. Nitric oxide synthase derived from inducible nitric oxide synthase and neuronal nitric oxide synthase has neurotoxic effects, while nitric oxide derived from endothelial nitric oxide synthase has neuroprotective effects.

[0037] Use a detection kit to measure the content of vasoactive substances in rats. Figure 2The results showed that compared with the negative control group, the model group showed significantly increased endothelin (ET), angiotensin II (Ang II), and inducible nitric oxide synthase (iNOS) levels (P < 0.01), while nitric oxide (NO) levels were significantly decreased (P < 0.01). After oral administration of donkey-hide gelatin, the low-dose donkey-hide gelatin group significantly improved ET, NO, and iNOS levels (P < 0.05 or P < 0.01), while the high-dose donkey-hide gelatin group significantly improved ET, Ang II, and iNOS levels (P < 0.05 or P < 0.01). This suggests that donkey-hide gelatin can effectively inhibit ET secretion, Ang II production, and iNOS synthesis, thereby promoting NO synthesis and release, and playing a protective and repairing role against vascular endothelial damage.

[0038] 2.2.3 Serum enzyme activity test results Enzymes are biological catalysts produced by cells in organisms. Antioxidant enzymes can slow down the rate of oxidation. Antioxidant enzymes can effectively inhibit the body's oxidative stress and prevent the harm caused by peroxidation.

[0039] Superoxide dismutase (SOD) is an antioxidant metalloenzyme present in organisms. It can catalyze the dismutation of superoxide anion free radicals to produce oxygen and hydrogen peroxide. It plays a vital role in the body's oxidation and antioxidant balance. It is closely related to the occurrence and development of many diseases and has significant therapeutic effects in the treatment of inflammation, autoimmunity, cardiovascular and cerebrovascular diseases caused by free radicals.

[0040] Catalase (CAT) is a ubiquitous antioxidant enzyme found in nearly all organisms, primarily found in the chloroplasts, mitochondria, and endoplasmic reticulum of plants, and in the liver and erythrocytes of animals. A peroxisome marker enzyme, CAT catalyzes the decomposition of hydrogen peroxide into water and oxygen, clearing it from the body and protecting cells from H2O2 toxicity. It is a key enzyme in the biological defense system, providing the body with an antioxidant defense mechanism.

[0041] In vivo, free radicals act on lipids to produce peroxidation reactions. The end product is malondialdehyde (MDA), which can cause cross-linking and polymerization of macromolecules such as proteins and nucleic acids, and is cytotoxic. MDA content is an important parameter reflecting the body's antioxidant potential. It can reflect the rate and intensity of lipid peroxidation and indirectly indicate the degree of tissue peroxidation damage.

[0042] After the rats were killed, the serum was collected by centrifugation, and the antioxidant enzyme system and MDA content in the serum of different groups were detected according to the kit instructions. Figure 3The results showed that compared with the negative control group, the activities of superoxide dismutase (SOD) and catalase (CAT) in the model group were extremely significantly decreased (P < 0.05), and the content of malondialdehyde (MDA) was extremely significantly increased (P < 0.01). After intragastric administration of donkey-hide gelatin, compared with the model group, the low-dose donkey-hide gelatin group could significantly increase the activity of CAT and decrease the content of MDA (P < 0.05); while the high-dose donkey-hide gelatin group could significantly increase the activities of SOD and CAT (P < 0.05 or P < 0.01), and could extremely significantly decrease the content of MDA (P < 0.01). It can be seen from this that donkey-hide gelatin can significantly improve the activities of serum antioxidant enzymes in rats with atherosclerosis combined with hyperlipidemia and has certain antioxidant properties.

[0043] 2.2.4 Detection results of serum inflammatory factors Inflammation exists throughout the process of atherosclerosis (AS) combined with hyperlipidemia. Lipid accumulation in macrophages induces inflammation, which promotes and enhances the development of AS, and a positive feedback loop is formed between inflammation and the development of AS lesions. Therefore, anti-inflammatory treatment is one of the effective ways to delay the development of AS plaques and stabilize advanced lesions.

[0044] Interleukin 6 (IL-6) is the most typical cytokine related to inflammation. It plays an important role in host defense by regulating immune and inflammatory responses. IL-6 can be produced by lymphoid and certain non-lymphoid cells, such as lymphoid cells like T lymphocytes and B lymphocytes, and non-lymphoid cells like macrophages, monocytes, dendritic cells, and mast cells.

[0045] Serum C-reactive protein (CRP) is a relatively sensitive indicator reflecting various infections and non-infectious inflammations in the body and is a non-specific marker of systemic inflammation. C-reactive protein (CRP) is a protein synthesized by the liver and is an acute-phase reaction protein that increases significantly during infection. It directly participates in inflammations and cardiovascular diseases such as atherosclerosis, and is the most powerful predictor and risk factor for cardiovascular diseases.

[0046] Inflammatory cytokines can mediate all stages of atherosclerosis combined with hyperlipidemia. Among them, tumor necrosis factor α (TNF-α) can effectively up-regulate the trans-endothelial cell transport of low-density lipoprotein (LDL) and promote the retention of LDL in the vascular wall, thereby accelerating the occurrence and development of atherosclerosis, and this process is the result of the interaction between nuclear factor κB (NF-κB) and peroxisome proliferator-activated receptor γ (PPAR-γ).

[0047] Interferon gamma (IFN-γ), also known as immune interferon, has relatively low antiviral activity but strong immunomodulatory and anti-cell proliferation effects. It is a potent activator of macrophages, NK cells, and vascular endothelial cells. IFN-γ enhances antigen presentation, activates T lymphocytes, interacts with various pro-inflammatory factors, promotes the inflammatory response at atherosclerotic lesions, and exacerbates lesion progression. As a regulator of lipid metabolism-related enzymes, IFN-γ induces the formation of foam cells and activates endothelial cells to promote atherosclerosis.

[0048] From Figure 4 The experimental data showed that compared with the negative control group, the levels of cytokines such as interleukin-6 (IL-6), C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) in the serum of the model group were significantly increased (P < 0.05 or P < 0.01). Compared with the model group, both the low-dose and high-dose donkey-hide gelatin groups could significantly reduce the levels of cytokines such as IL-6, CRP, TNF-α, and IFN-γ (P < 0.05 or P < 0.01). It can be seen from this that donkey-hide gelatin can effectively reduce the levels of inflammatory factors such as IL-6, CRP, TNF-α, and IFN-γ in the serum of rats with atherosclerosis combined with hyperlipidemia, and improve the inflammatory state of atherosclerosis combined with hyperlipidemia.

[0049] 2.2.5 Histopathological observation Oil Red O staining results: Fast the animals for 12 h with water available before sampling, draw blood from the abdominal aorta, separate the serum, and dissect the aorta. After removing the surrounding adipose tissue, fix it in 4% paraformaldehyde for more than 24 h, immerse it in Oil Red staining solution for staining, and observe the plaque area of the aortic tissue in each group. From Figure 5 The staining results showed that: The aortic wall of the negative control group mice was translucent and smooth, without obvious red lipid plaque deposition; compared with the negative control group, a large number of obvious red lipid depositions could be seen on the aortic vascular wall of the model group mice, with a clear boundary with the vascular wall, mainly distributed in the abdominal aorta; compared with the model group, the aortic plaque deposition area in the low-dose and high-dose donkey-hide gelatin groups was significantly reduced, especially in the high-dose donkey-hide gelatin group, with a more obvious improvement.

[0050] Figure 6HE staining results: In the negative control group, the structure and layers of the aortic intima of rats were clear, the intima was smooth, the vascular endothelium was intact and continuous, and no obvious lipid deposition or plaque formation was observed; spindle-shaped and neatly arranged vascular smooth muscle cells were visible in the media; the adventitia was loose connective tissue. In the model group, the arrangement of the aortic intima of rats was disordered, the endothelium was thickened, and obvious plaque formation was observed; the smooth muscle cells in the media proliferated, irregularly arranged smooth muscle cells were visible, local inflammatory cell infiltration was observed, and lipid plaques were formed, showing the pathological morphological changes of atherosclerosis combined with hyperlipidemia; the arterial structure of rats in the low-dose and high-dose donkey-hide gelatin groups was clearer than that in the model group, with intimal thickening, smooth muscle cell proliferation, and less obvious plaques. This study suggests that the aortic vascular wall of rats in the model group showed the endothelial injury changes of atherosclerosis combined with hyperlipidemia, and the pathological changes after intervention with donkey-hide gelatin were alleviated compared with those in the model group, indicating that donkey-hide gelatin can improve the injury of vascular endothelial cells and protect blood vessels.

Claims

1. A new use of donkey-hide gelatin, characterized in that: For the preparation of pharmaceutical preparations and / or health products and / or foods for improving atherosclerosis combined with hyperlipidemia.

2. The new use of donkey-hide gelatin according to claim 1, characterized in that: The weight-average molecular weight Mw of the donkey-hide gelatin is 16,001 - 16,654 Da, wherein the proportion of molecules > 50 kDa is 8.34 - 8.38%, the proportion of molecules 10 - 50 kDa is 47.74 - 49.68%, the proportion of molecules 5 - 10 kDa is 13.21 - 13.75%, and the proportion of molecules < 5 kDa is 28.71 - 29.89%.

3. The new use according to claim 1, characterized in that, The pharmaceutical preparations include: tablets, infusions, pills, emulsions, granules, soft capsules, hard capsules or microcapsules.

4. The new use according to claim 1, characterized in that, The health products include: tablets, infusions, pills, emulsions, granules, soft capsules, hard capsules or microcapsules.

5. The new use according to claim 1, characterized in that, The foods include one or more of: beverages, paste foods, preserved fruit foods and pastry foods.

6. The new use according to claim 1, wherein The pharmaceutical preparations include donkey-hide gelatin and pharmaceutically acceptable excipients or carriers.

7. The new use according to claim 1, characterized in that, The health products include donkey-hide gelatin and excipients or carriers acceptable in the field of health products.

8. The new use according to claim 1, characterized in that, The foods include donkey-hide gelatin and excipients or food additives acceptable in the field of foods.

9. The preparation method of the donkey-hide gelatin powder according to any one of claims 1 - 8, comprising the following steps: a. Raw material treatment: Select high-quality donkey skins, after rinsing to remove impurities, dehairing and defatting, cut them into 5×5 cm squares, soak for 12 - 24 h, and change water 2 - 3 times during soaking to remove blood and odor; b. Decoction: Add water in a ratio of 1:5, and decoct the donkey skins at a low temperature of 40 - 60 °C and under a vacuum condition of -0.08 ~ -0.10 Mpa for 12 - 24 h; c. Filtration and concentration: After the decoction, filter with gauze or a filter screen to remove residues; use an ultrafiltration membrane or a nanofiltration membrane to concentrate the filtrate, remove small molecule impurities, and retain macromolecular active ingredients; d. Freeze-drying: Freeze the concentrated solution and perform sublimation drying under vacuum conditions to obtain freeze-dried donkey-hide gelatin powder with high active ingredients.

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