Oral medicine composition containing kidney bean kinase and application of oral medicine composition

Abstract

The invention provides an oral pharmaceutical composition containing kidney bean kinase and application thereof.The pharmaceutical composition comprises kidney bean kinase serving as an active component and a pharmaceutically acceptable carrier, the activity of kidney bean kinase is not lower than 2000 U / mg, and the kidney bean kinase up-regulates the LDL-R expression level and promotes LDL uptake and degradation by hydrolyzing PCSK9 protein in liver cells, so that the activity of the kidney bean kinase is improved, and the activity of the kidney bean kinase is improved. The lipid-lowering effect is achieved. The pharmaceutical composition containing the kidney bean kinase can be taken orally, is natural in source and high in safety, can effectively reduce expression of PCSK9 protein, and is used for treating or preventing hypercholesteremia, atherosclerosis and complications thereof.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a kind of oral drug composition containing kidney bean kinase and application thereof. BACKGROUND

[0002] Hypercholesterolemia and atherosclerosis are the main risk factors of cardiovascular and cerebrovascular diseases, and their occurrence is closely related to the increase of plasma low-density lipoprotein cholesterol (LDL-C). Proprotein convertase subtilisin / kexin type 9 (PCSK9) is a key protein that regulates the degradation of hepatocyte low-density lipoprotein receptor (LDL-R), and by promoting the degradation of LDL-R in lysosomes, it reduces the clearance of LDL by the liver, thereby causing an increase in blood cholesterol levels. Therefore, targeting PCSK9 has become an important direction for current lipid-lowering therapy. Existing PCSK9 inhibitors are mainly monoclonal antibody drugs, such as Alirocumab and Evinoloumonab, which block the interaction of PCSK9 protein with LDL-R by binding to it, thereby reducing plasma LDL-C. Although this type of drug has significant efficacy, it generally has high cost, requires cold chain storage and transportation, is inconvenient for injection administration, has poor long-term compliance, and may induce immunogenicity. In addition, in recent years, siRNA-based PCSK9 inhibitors such as Inclisiran have appeared, which can reduce the frequency of administration, but still rely on injection administration, and their long-term safety and tissue targeting need to be verified.

[0003] All currently marketed or in development PCSK9-targeted drugs mainly focus on blocking the binding of PCSK9 to LDL-R or interfering with PCSK9 gene expression, and there is no drug that can directly act on specific hydrolysis sites of PCSK9 (such as key amino acid sequences necessary for its maturation processing) to affect the structure, processing or activity of PCSK9. This mechanism is considered a potential key link in PCSK9 regulation, but no drug has achieved a breakthrough in targeting. Therefore, there is still a huge technical gap and clinical need in the field of lipid-lowering therapy, i.e. to develop an orally available, naturally derived, highly safe and moderately priced drug formulation that can regulate PCSK9 function through a novel mechanism.

[0004] Therefore, there is an urgent need to explore naturally active substances that can be taken orally, have long-term safety, and can inhibit the activity of PCSK9 through multiple mechanisms, to provide high accessibility and more in line with the needs of chronic disease management for patients with hypercholesterolemia and atherosclerosis. SUMMARY

[0005] Kidney bean enzyme (KBE) is a serine protease secreted by Bacillus subtilis var. niger during fermentation, which has been widely studied and applied for its thrombolytic, antithrombotic and hemorheological properties. Recent studies have found that KBE can regulate liver lipid metabolism and improve vascular endothelial function in addition to its fibrinolytic activity. The applicant has found that KBE can directly act on PCSK9-LDL-R to lower lipid levels.

[0006] Therefore, the present application provides an oral pharmaceutical composition containing KBE and its application to solve the technical problem that there is a lack of orally administrable, naturally derived, safe and effective drug preparation for inhibiting PCSK9 expression. The present application discloses for the first time that KBE has the ability to hydrolyze PCSK9 protein and can promote the uptake and metabolism of low-density lipoprotein cholesterol (LDL-C) in hepatocytes by regulating the PCSK9-LDL-R signaling pathway, thereby achieving an oral lipid-lowering effect.

[0007] To achieve the above object, the present application proposes the following technical solutions:

[0008] An oral pharmaceutical composition comprises:

[0009] KBE as an active ingredient and a pharmaceutically acceptable carrier;

[0010] The pharmaceutical composition is formulated as:

[0011] After oral administration, the KBE can lower lipid levels by down-regulating the expression level of PCSK9 protein and up-regulating the expression of low-density lipoprotein receptor.

[0012] As a preferred technical solution of the present application, the pharmaceutical composition is formulated as:

[0013] After oral administration, the KBE can lower lipid levels by hydrolyzing the YSPASA and / or FAPGED sites of PCSK9 protein.

[0014] As a preferred technical solution of the present application, the specific activity of the KBE is not less than 2000 U / mg.

[0015] As a preferred technical solution of the present application, the pharmaceutical composition further comprises one or more additional lipid-lowering active ingredients, and the weight ratio of the KBE to the additional lipid-lowering active ingredients is 1: (0.1-10).

[0016] As a preferred technical solution of the present application, the additional lipid-lowering active ingredients are statins, ezetimibe, phytosterols and probiotics.

[0017] As a preferred technical solution of the present application, the pharmaceutical composition is formulated into a capsule, a tablet, a granule, an oral liquid, a microcapsule or a liposome.

[0018] As a preferred technical solution of the present application, the microcapsule is prepared by sodium alginate and chitosan embedding technology.

[0019] As a preferred technical solution of the present application, the pharmaceutical composition is a soft capsule, and the content thereof is composed of at least the following components in addition to the Phaseolus vulgaris kinase:

[0020] Fish oil, the mass ratio of the fish oil to the Phaseolus vulgaris kinase being 1:4-1:6;

[0021] An antioxidant, the content of the antioxidant being 0.1%-0.3% based on the total weight of the content;

[0022] A suspending agent, the content of the suspending agent being 0.2%-0.5% based on the total weight of the content.

[0023] As a preferred technical solution of the present application, the liposome is formed by phospholipid and cholesterol to form a double membrane to encapsulate the Phaseolus vulgaris kinase, wherein the weight ratio of the phospholipid to the cholesterol is 3:1, and the encapsulation rate of the Phaseolus vulgaris kinase is not less than 80%.

[0024] The present application also provides an application of the oral pharmaceutical composition, which is applied to the preparation of an oral lipid-lowering drug, and / or;

[0025] The application is applied to the preparation of a drug for preventing and / or treating hypercholesterolemia and atherosclerosis.

[0026] The present application also provides an application of the oral pharmaceutical composition, which is applied to the preparation of a functional food or a nutritional supplement.

[0027] The present application discloses the following mechanism of the Phaseolus vulgaris kinase in lowering lipid:

[0028] The Phaseolus vulgaris kinase can significantly inhibit the protein expression of a proprotein convertase subtilisin / kexin type 9 (PCSK9) in a liver cell. In vitro test proves that the Phaseolus vulgaris kinase can reduce the PCSK9 protein level in a time-dependent and concentration-dependent manner at a concentration of 20-100 U / mL, and the maximum inhibition rate can reach 65%, p<0.01.

[0029] As a serine protease, kidney bean kinase can specifically recognize and hydrolyze the conserved amino acid sequence site on PCSK9 protein. Through site mutation experiment, it is confirmed that YSPASA and FAPGED sites are the key hydrolysis target sites of kidney bean kinase. Kidney bean kinase directly enzymatically cuts to destroy the structural integrity and stability of PCSK9 protein.

[0030] After kidney bean kinase hydrolyzes PCSK9 at the level through the above mechanism, the degradation promotion of PCSK9 to LDL-R on the membrane surface of liver cells is released, so that the expression amount of LDL-R is significantly up-regulated. Both in vivo and in vitro experiments have confirmed that after the intervention of kidney bean kinase, the PCSK9 protein in liver cells can be hydrolyzed, the level of LDL-R is up-regulated, thereby promoting the uptake and metabolism of LDL, the expression level of liver cell LDL-R protein can be increased by about 2.1 times, and the uptake and clearance capacity of plasma low-density lipoprotein (LDL) is enhanced, which indicates that it has the biological activity of directly regulating the PCSK9-LDL-R pathway.

[0031] In animal models, kidney bean kinase ultimately shows significant lipid-lowering effect through the above-mentioned pathway, including reducing the levels of serum total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C), and effectively reducing liver steatosis, inflammatory reaction and aortic plaque formation induced by high-fat diet.

[0032] The kidney bean kinase described in the application is suitable for intervention and adjuvant therapy of dyslipidemia in mammalian individuals (including humans); and in animal models (ApoE - / - Mice, high-fat diet C57BL / 6J mice) can significantly reduce the plasma PCSK9 level, increase the expression of LDL-R protein and reduce the formation of aortic plaques.

[0033] Therefore, the kidney bean kinase is applied to prepare an oral lipid-lowering drug for inhibiting PCSK9 expression. After oral administration, kidney bean kinase can intervene in the PCSK9 protein secretion process, improve the lipid metabolism homeostasis, and be used for preventing and / or treating hypercholesterolemia, atherosclerosis and its complications.

[0034] The kidney bean kinase can be used alone or in combination with statins, ezetimibe, phytosterols, probiotics and the like to prepare a pharmaceutical composition or a compound composition. When combined with statins, kidney bean kinase can inhibit the up-regulation of PCSK9 induced by statins, thereby enhancing the recycling of LDL receptors, synergistically reducing the levels of serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C), and significantly improving the overall lipid-lowering efficacy.

[0035] The pharmaceutical composition can be prepared into capsule, tablet, granule, oral liquid, microcapsule or liposome, etc. to meet different clinical and consumer needs. The microencapsulation, liposome encapsulation or nanometer delivery system can significantly improve the stability and bioavailability of the kidney bean kinase in the gastrointestinal tract, so that the PCSK9 inhibitory activity is maintained after oral administration.

[0036] The kidney bean kinase can be derived from the fermentation product of Bacillus subtilis var. niger, and the purity is x≥2000 U / mg. Preferably, a biological fermentation purification process is used to obtain an enzyme preparation with high specific activity and high stability, so as to ensure the consistency of drug efficacy and long-term storage stability.

[0037] In order to ensure that the kidney bean kinase still maintains biological activity after oral administration, the application provides various advanced dosage form strategies.

[0038] The microcapsule prepared by sodium alginate and chitosan embedding technology can effectively protect the kidney bean kinase from passing through the gastric acid environment, so that it is released in the intestinal tract, and the active retention rate in the gastric juice is more than 85%.

[0039] The kidney bean kinase is encapsulated by using phospholipid and cholesterol to form a bilayer membrane structure, and the encapsulation rate is not less than 80%. The liposome system can improve the stability and intestinal absorption rate of the drug.

[0040] In summary, the application provides an oral pharmaceutical composition and its application in the preparation of lipid-lowering drugs. Compared with the prior art, the application has the following beneficial effects:

[0041] 1. Novel and clear mechanism of action. It is first discovered and confirmed that the kidney bean kinase can play a role through the dual mechanisms of down-regulating PCSK9 expression and directly cutting PCSK9 protein, and the molecular target of the kidney bean kinase acting on the PCSK9-YSPASA / FAPGED site is clear, which opens up a new action path for natural enzyme preparations in the field of lipid-lowering.

[0042] 2. Oral effectiveness and high bioavailability. Through microcapsule, liposome and other delivery technologies, the problem of protein inactivation after oral administration is successfully solved, so that the kidney bean kinase can maintain high biological activity after oral delivery, and reach the target to play a curative effect.

[0043] 3. In animal models, the kidney bean kinase can significantly reduce serum total cholesterol and low-density lipoprotein cholesterol, and can effectively improve liver steatosis and other pathological changes, and the effect is equivalent to that of existing PCSK9 antibody drugs.

[0044] 4. Natural source and excellent safety. The kidney bean kinase is derived from food-grade microbial fermentation products, and has good long-term use safety record. Animal experiments show that long-term administration has no significant effect on liver function indicators and no hepatotoxicity, and is suitable for long-term use.

[0045] 5. Low cost, good patient compliance. The mature microbial fermentation process is used for production, the raw material is renewable, and the preparation cost is much lower than that of antibody drugs. Oral administration avoids the inconvenience and pain caused by injection, greatly improves the treatment compliance of patients, and is suitable for long-term management of chronic patients.

[0046] 6. Various application forms, broad industrialization prospects. Kidney bean kinase can be used not only for preparing prescription drugs, but also as an active ingredient for functional food or nutritional supplements, meeting the prevention and health care needs of different populations, and having huge market application space.

[0047] In summary, the application uses the inhibitory effect of kidney bean kinase on PCSK9 expression to construct a natural, safe and orally administrable new lipid-lowering strategy, which has significant scientific innovation and industrial application value.

[0048] It should be understood that any combination of the foregoing concepts and additional concepts described in more detail below falls within the scope of the present application, provided that there is no contradiction between them.

[0049] The technical solutions taught by the application and the embodiments and features thereof can be more fully understood in conjunction with the accompanying drawings. Other aspects, preferred embodiments, and beneficial effects of the application will be further described in the following description or can be embodied in the implementation of the application. BRIEF DESCRIPTION OF DRAWINGS

[0050] The drawings do not represent true proportions according to the real reference. In the drawings, each same or approximately same component shown in each figure can be indicated by the same reference numeral. For the sake of clarity, not every component is labeled in each figure. Now, embodiments of various aspects of the application will be described by example and with reference to the accompanying drawings, in which:

[0051] Figure 1 Figure 1 is a graph of the test detection results in Example 2 of the application; wherein A is the expression change of PCSK9 protein at different time points; B is the expression change of PCSK9 protein in HepG2 cells at different concentrations; C is the expression change of PCSK9 protein in Hepa1-6 cells at different concentrations; D is the expression change of LDL-R protein in HepG2 cells at different concentrations; E is the expression change of LDL-R protein in Hepa1-6 cells at different concentrations;

[0052] Figure 2The following are the experimental results from Example 3 of this invention: A shows morphological photographs of mice in the normal diet group, high-fat diet control group, KBE7.5K group, and KBE15K group; B shows morphological photographs of liver tissue from mice in the normal diet group, high-fat diet control group, KBE7.5K group, and KBE15K group; and C shows a statistical chart of liver tissue weight from mice in the normal diet group, high-fat diet control group, KBE7.5K group, and KBE15K group.

[0053] Figure 3 The figure shows the experimental results in Example 4 of this invention; AC represents the changes in the levels of TC, TG, and LDL-C in the serum of mice in the normal feed group, high-fat feed control group, KBE7.5K group, and KBE15K group.

[0054] Figure 4 The changes in PCSK9 content in the serum of mice in the normal feed group, high-fat feed control group, KBE7.5K group, and KBE15K group in Example 4 of this invention;

[0055] Figure 5 The figure shows the experimental results in Example 4 of this invention; AB represents the changes in ALT and AST levels in the serum of mice in the normal feed group, high-fat feed control group, KBE7.5K group, and KBE15K group.

[0056] Figure 6 The images show the HE staining results of mouse liver tissues from the normal feed group, high-fat feed control group, KBE7.5K group, and KBE15K group in Example 5 of this invention.

[0057] Figure 7 The following is a graph showing the experimental results in Example 5 of this invention; where A represents the immunohistochemical expression of LDL-R in the liver tissue of mice in the normal diet group, high-fat diet control group, KBE7.5K group, and KBE15K group; and B represents the statistical graph of LDL-R expression in the liver tissue of mice in the normal diet group, high-fat diet group, KBE7.5K group, and KBE15K group.

[0058] Figure 8 The figures shown are the experimental results of Example 5 of the present invention; where A is the Oil Red staining result of liver tissue of mice in the normal diet group, high-fat diet control group, KBE7.5K group, and KBE15K group; and B is the statistical graph of lipid accumulation in liver tissue of mice in the normal diet group, high-fat diet control group, KBE7.5K group, and KBE15K group.

[0059] Figure 9Figure A is the test result chart of the embodiment 6 of the present application; wherein A is the sequence of three potential hydrolysis sites YSPASA, FAPGED and WFPEDQ of PCSK9; B is the conservative sequence of human and mouse PCSK9 protein; C-D are the empty, PCSK9 and three potential site mutation plasmids of human and mouse PCSK9 mut1 , PCSK9 mut2 , PCSK9 mut3 Expression of PCSK9 protein in 293 cells

[0060] Figure 10 Figure A is the test result chart of the embodiment 7 of the present application; wherein A is the morphological photo of normal feed group, high-fat feed control group, KBE7.5K group, KBE15K group, PCSK9 mut1 +KBE, PCSK9 mut2 +KBE mice; B is the morphological photo of the mice after 4 weeks of modeling, PCSK9 mut1 , PCSK9 mut2 Liver injection, and the change of body weight of the mice after 4 weeks of KBE administration; C is the morphological photo and weight statistical chart of liver tissue of normal feed group, high-fat feed control group, KBE7.5K group, KBE15K group, PCSK9 mut1 +KBE, PCSK9 mut2 +KBE mice; D is the expression of PCSK9 and LDL-R protein in the liver tissue of normal feed group, high-fat feed control group, KBE7.5K group, KBE15K group, PCSK9 mut1 +KBE, PCSK9 mut2 +KBE mice. DETAILED DESCRIPTION

[0061] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme of the embodiments of the present application will be described clearly and completely below in combination with the drawings of the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the described embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without any creative effort belong to the scope of protection of the present application. Unless otherwise defined, the technical terms or scientific terms used herein should be understood as the usual meaning by those of ordinary skill in the art to which the present application belongs.

[0062] The terms "first", "second", and similar terms used in the specification and claims of the present patent application do not denote any order, quantity, or importance, but are used to distinguish different components. Similarly, the singular forms "a", "an", and "the" do not denote the quantity limitation, but denote the existence of at least one, unless the context clearly indicates otherwise. The terms "comprise", "comprising", and similar terms mean that the elements or objects before "comprise" or "comprising" encompass the features, integers, steps, operations, elements, and / or components listed after "comprise" or "comprising", and do not exclude one or more other features, integers, steps, operations, elements, components, and / or groups thereof. The terms "upper", "lower", "left", "right", and the like are used only to indicate relative positional relationships, and when the absolute positions of the described objects change, the relative positional relationships may also change accordingly.

[0063] Example 1

[0064] Preparation of novel oral PCSK9 inhibitor oral soft capsules.

[0065] (I) Preparation, fermentation and purification of kidney bean kinase

[0066] (1) Preparation and fermentation of kidney beans

[0067] a. Selecting kidney beans: high-quality, small-grained kidney beans with plump particles are selected because of their large surface area, which is more conducive to fermentation.

[0068] b. Washing and soaking: after washing, the kidney beans are soaked in warm water for about 12 hours to allow them to fully absorb water and swell.

[0069] c. Steaming: the soaked kidney beans are steamed in a high-pressure steam pot at high temperature until they are softened.

[0070] d. Inoculation and fermentation: the steamed kidney beans are cooled to 40-45℃, and then inoculated with purified Bacillus subtilis.

[0071] e. Constant-temperature fermentation: fermentation is carried out in a fermentation chamber with strictly controlled temperature (about 40℃) and humidity for 16-24 hours. After fermentation is completed, the surface of the kidney beans is covered with a layer of white bacterial film, and a unique sticky substance (containing kidney bean kinase) is produced. Then, an appropriate amount of purified water or buffer is added to the fermentation product for extraction, pressure filtration or centrifugation, and the obtained supernatant is collected as the fermentation broth containing kidney bean kinase.

[0072] (2) Extraction and purification of kidney bean kinase

[0073] The fermentation broth is centrifuged to remove the bacterial cells, and the supernatant is subjected to ammonium sulfate fractionation precipitation (40% to 80% saturation) to obtain a crude enzyme solution, which is then purified by DEAE-Sepharose anion exchange chromatography to collect the protein fraction having fibrinolytic activity. Further purification is performed by gel filtration chromatography (Sephadex G-75). The purified product is frozen at -80℃ and then lyophilized to obtain a high-purity kidney bean kinase lyophilized powder with an enzyme activity of 490000 U / g.

[0074] The specific activity (indicating the enzyme activity per milligram of protein, reflecting the purity of the enzyme) is greater than 2000 U / mg as determined by the agarose-fibrin plate method, which meets the requirements of the enzyme activity of the pharmaceutical composition and is a key parameter of the pharmaceutical grade enzyme.

[0075] (3) Enzymatic properties of kidney bean kinase

[0076] The optimal action pH of the kidney bean kinase is 7.5-8.5, and the optimal temperature is 37℃. After being protected by enteric coating in simulated gastric juice, the activity retention rate is >85%, wherein the simulated gastric juice condition is pH=2.0, the temperature is 37℃, and the time is 30 minutes.

[0077] Based on the high-activity and high-stability kidney bean kinase raw material obtained above, the present application provides various preparation methods of oral pharmaceutical compositions, including but not limited to capsule, tablet, granule, oral liquid, microcapsule, liposome, etc. and ensures that it still maintains PCSK9 inhibitory activity after oral administration, which can treat or prevent cholesteremia, atherosclerosis, metabolic syndrome and related cardiovascular diseases by adjusting the PCSK9-LDLR signal pathway.

[0078] The kidney bean kinase can be used alone to prepare a PCSK9 inhibitor, and can also be used in combination with other lipid-lowering components. Specifically, when used in combination, at least one additional lipid-lowering active component is mixed in a mass ratio of 1:(0.1-10), preferably a pharmaceutically acceptable carrier is added to prepare a compound composition, so as to synergistically reduce the plasma cholesterol level. Among them, the additional lipid-lowering active component is preferably a statin, ezetimibe, phytosterol, probiotic bacteria or other lipid-lowering component; the pharmaceutically acceptable carrier or excipient is preferably microcrystalline cellulose, lactose, magnesium stearate.

[0079] (II) Preparation of specific pharmaceutical dosage forms

[0080] 1. Preparation of soft capsules

[0081] (1) Preparation of soft capsule contents

[0082] As preferred, the soft capsule content of the present embodiment, in addition to the lentiviral kinase, further comprises fish oil, antioxidant, and suspending agent, wherein the fish oil can be a commercially available product meeting the quality standards for fish oil in the pharmacopoeia, the antioxidant is vitamin E or an equivalent allowed by the pharmacopoeia, and the suspending agent is preferably silicon dioxide.

[0083] The lentiviral kinase concentrate is mixed with refined fish oil at a mass ratio of 1:4 to 1:6 (w / w) as the carrier oil, so that the water-soluble and fat-soluble components of the lentiviral kinase can be fully dispersed and remain active. On this basis, 0.1% to 0.3% of the total content of antioxidant (preferably vitamin E) is added to prevent oil oxidation and spoilage, and if necessary, 0.2% to 0.5% of the total content of suspending agent is added to avoid sedimentation and stratification of the content after long-term storage. Then, a high-speed shearing device or a homogenizer is used to homogenize at 3000-8000 rpm for 5-10 min, to obtain a uniform and stable oil suspension, which provides a content with good consistency for soft capsule filling.

[0084] (2) Compression molding of soft capsules

[0085] The soft capsule shell is prepared by mixing gelatin, plasticizer, and purified water at a mass ratio of 45% to 55%: 25% to 35%: 15% to 25% (w / w), wherein gelatin is used as the main film-forming material, and the plasticizer can be glycerol or sorbitol to make the shell have appropriate softness and elasticity. To improve stability, 0.2% to 0.5% of titanium dioxide can be added as a light shielding agent, and 0.01% to 0.1% of a coloring agent or flavoring agent can be added according to product requirements. After the raw materials are heated, melted, mixed, and degassed, a gel liquid with good flowability is obtained. During the pill pressing process, two pieces of heated gelatin are supplied from left and right rollers on the soft capsule press, and a pre-prepared lentiviral kinase inner material (the conventional filling amount is 30% to 45% of the total mass of a single particle) is injected with high precision by a metering pump. Under the conditions of set pressure and vacuum, the two rollers are closed synchronously, so that the gelatin wraps the inner material to form a sealed soft capsule and completes the cutting and molding. The freshly pressed capsules are still relatively soft and need to be immediately put into a shaping and drying machine for preliminary drying to fix the shape under controlled conditions of 20-24°C and relative humidity of 20%-35%.

[0086] (3) Post-processing and packaging

[0087] The soft capsules after preliminary sizing need to be post-processed to ensure quality and stability. First, the capsules are transferred into a large drying rotating cage and slowly dried at 20-30℃ and relative humidity of 20%-35% for 24-48 h to fully remove the excess water in the capsule skin, so that the capsule reaches the appropriate hardness and long-term storage stability. After drying, the capsule is surface cleaned with an appropriate amount of ethanol (usually 70%-95%) or a special cleaner, and then polished by a rotary polisher to make it smooth and clean in appearance. Then, quality inspection is carried out according to the regulations, including appearance, weight difference, disintegration time limit, microbial limit and kidney bean kinase activity unit determination to ensure that the product meets the quality standards. The qualified capsules are packaged according to the plastic bottle or aluminum-plastic blister (PVC / Al) and 1-3 g of desiccant is added to maintain a low-humidity environment; the packaging link completes the boxing and labeling, clearly marking the product name, specification, kidney bean kinase activity unit, production date and shelf life, etc.

[0088] Through this embodiment, high-activity and high-stability kidney bean kinase raw materials are obtained to ensure the consistency, repeatability and pharmacological reliability of subsequent experiments and preparations.

[0089] 2. Preparation of compositions containing additional active ingredients and microcapsules, liposomes

[0090] Alternatively, the specific preparation method of the pharmaceutical composition containing kidney bean kinase is as follows:

[0091] Take kidney bean kinase with specific activity ≥ 2000 U / mg and at least one additional lipid-lowering active ingredient with a mass ratio of 1:(0.1-10), add a pharmaceutically acceptable carrier, and prepare oral preparations such as capsules, tablets, granules, oral solutions, microcapsules, and liposomes. The specific ratio is preferably exemplified as follows:

[0092] The weight ratio of kidney bean kinase to statins is 1:1, and the statins can be selected from atorvastatin.

[0093] The weight ratio of kidney bean kinase to ezetimibe is 1:0.5.

[0094] The weight ratio of kidney bean kinase to phytosterol is 1:5.

[0095] The weight ratio of kidney bean kinase to probiotics is 1:2, and the probiotics can be selected from lactobacillus.

[0096] The above mixture is prepared into tablets by wet granulation or dry compression or filled into capsules, each unit dose containing 50-200 mg of kidney bean kinase.

[0097] (1) Preparation of microcapsules

[0098] As preferred, the microcapsule is prepared by using sodium alginate and chitosan embedding technology to improve the stability and bioavailability of kidney bean kinase in gastrointestinal environment. The specific steps are as follows:

[0099] The kidney bean kinase is dissolved in phosphate buffer saline with pH 7.4, and the concentration is 50 mg / mL; and the sodium alginate (2% w / v) solution is mixed with the kidney bean kinase solution, and is added dropwise into the calcium chloride solution (2% w / v) by using a spraying device to form gel microspheres. The microspheres are collected and washed with deionized water, and then are immersed in a chitosan solution (0.5% w / v, pH 5.5) for stirring for 10 minutes; after being washed again, the kidney bean kinase microcapsule is obtained by freeze-drying.

[0100] The in-vitro release experiment simulates the gastrointestinal environment, and the results show that the enzyme activity retention rate of the microcapsule is greater than 90% in the simulated gastric juice (pH 2.0) for 2 hours, and the cumulative release rate is more than 85% in the simulated intestinal juice (pH 6.8) for 4 hours, indicating that the dosage form can effectively protect the activity of kidney bean kinase and achieve controlled release.

[0101] (2) Preparation of liposome

[0102] As preferred, the kidney bean kinase liposome is prepared, and the specific steps are as follows:

[0103] The soybean phospholipid and cholesterol are dissolved in chloroform according to the weight ratio of 3:1 to form a film by rotary evaporation; the kidney bean kinase is dissolved in neutral PBS with a concentration of 20 mg / mL; then the kidney bean kinase solution is added into the film, and is hydrated and shaken, and then the liposome suspension is obtained by high-pressure homogenization or ultrasonic treatment; the liposome suspension is purified by ultracentrifugation, and the encapsulation efficiency is determined by using the BCA protein method, and the encapsulation efficiency of not less than 80% is qualified.

[0104] By transmission electron microscopy observation, the liposome has a double-membrane structure, and the particle size distribution is 100-200 nm. The in-vitro release experiment shows that the kidney bean kinase can be continuously released in the simulated intestinal juice, and the cumulative release rate is more than 75% for 24 hours.

[0105] (Three) Application of the pharmaceutical composition

[0106] (1) The kidney bean kinase is used for preparing an oral lipid-lowering drug

[0107] The embodiments of the present application also provide the application of the kidney bean kinase and the pharmaceutical composition thereof. Specifically, the kidney bean kinase and the pharmaceutical composition thereof can be applied to the preparation of an oral lipid-lowering drug, and can be applied to the preparation of drugs for preventing and / or treating hypercholesterolemia, atherosclerosis and the like, so as to prevent and / or treat hypercholesterolemia, atherosclerosis and complications thereof. For example, the kidney bean kinase and the pharmaceutical composition thereof are prepared into a capsule, and 50-200 mg of the kidney bean kinase is taken per day, which can effectively reduce the serum LDL-C level of a patient.

[0108] (2) Kidney bean kinase for preparing functional food or nutritional supplement

[0109] In addition, the kidney bean kinase can be used as an active ingredient of functional food or nutritional supplement. Preferably, the kidney bean kinase microcapsule or liposome is added to a food matrix to prepare granules or oral liquid, and 50-200 mg of kidney bean kinase is ingested daily. After oral administration of kidney bean kinase, the kidney bean kinase is absorbed through the intestinal tract, can inhibit the up-regulated expression of PCSK9 by directly acting on hepatocytes or regulating the intestinal-liver axis signal pathway, and can safely and long-term maintain normal blood lipid level.

[0110] Example 2

[0111] This Example 2 studies the effect of kidney bean kinase on the expression of PCSK9 in hepatocytes. The specific test method is as follows:

[0112] (1) The human hepatoma cell line HepG2 and the murine hepatoma cell Hepa1-6 are used as models. The cells are cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin. The cells are treated with 0, 20, 40, 60, 80, and 100 U / mL kidney bean kinase for 4 hours.

[0113] (2) Western blot detection and analysis

[0114] To detect the PCSK9 and LDL-R protein levels in the cells, Western blot method is used for semi-quantitative analysis. The main analysis process is as follows: the protein samples are extracted and quantified, subjected to molecular weight separation, electroblotting, and antibody incubation on the membrane, followed by chemiluminescence or other suitable detection means for development and photographing. In the experiment, the internal reference protein, which can be selected as β-Tubulin, is used to correct the protein loading difference.

[0115] To investigate the time and dose-dependent relationship of kidney bean kinase on the expression of PCSK9, HepG2 and Hepa1-6 cells are detected at different concentrations and treatment times.

[0116] Reference Figure 1 A. Under the action of 80 U / mL kidney bean kinase, the PCSK9 protein level shows a significant downward trend with the extension of the treatment time. Specifically, a slight down-regulation occurs at 1 hour of treatment, the decrease is about 40% at 3 hours, and the decrease is about 65% at 5 hours (p<0.01), indicating that the kidney bean kinase has a time-dependent inhibitory effect on PCSK9.

[0117] Reference Figure 1B and 1C, in different concentration treatment groups, kidney bean kinase hydrolysis of PCSK9 showed concentration-dependent changes. With the concentration from 20 U / mL to 100 U / mL, PCSK9 / β-Tubulin relative gray value gradually decreased.

[0118] Reference Figure 1 D and 1E, LDL-R / β-Tubulin ratio increased significantly, and the change was particularly significant in 80 U / mL and 100 U / mL groups.

[0119] HepG2 and Hepa1-6 cell lines showed consistent results, indicating that kidney bean kinase can promote LDL receptor-mediated cholesterol clearance by down-regulating PCSK9 and up-regulating LDL-R.

[0120] Example 3

[0121] This example 3 studies the effect of kidney bean kinase on obese animal models. The specific test method is as follows:

[0122] (1) Preparation of mice

[0123] 5-6 week old C57B / 6 male mice weighing (18±20) g were selected from Changzhou Cavens Experimental Animal Co., Ltd. The mice were raised in SPF level laminar flow feeding cabinet, with 12 hours: 12 hours cycle rhythm, environmental temperature of 23-25℃, environmental humidity of 40%-60%, and adaptive feeding for 1 week before formal experiment.

[0124] Twelve mice were randomly divided into four groups, three in each group, namely normal diet group, high-fat diet control group, low-dose kidney bean kinase group (kidney bean kinase dosage 7500 U / kg, recorded as KBE7.5K group), and high-dose kidney bean kinase group (kidney bean kinase dosage 15000 U / kg, recorded as KBE15K group).

[0125] (2) Administration method and period

[0126] After the model animals were induced by high-fat diet, they were given intragastric administration at the 4th week, once every other day, and the administration lasted for 4 weeks. During the experiment, the high-fat diet was maintained to evaluate the effect of continuous oral kidney bean kinase on blood lipid level and PCSK9 expression.

[0127] Reference Figure 2 A and 2B, the normal diet group of mice was healthy and had bright fur; the high-fat diet control group of mice had significantly increased body weight, dull fur, and obvious characteristics of liver enlargement and fat deposition.

[0128] The KBE7.5K group and the KBE15K group after intervention by kidney bean kinase, the growth rate of the mice was significantly slowed down, the liver volume was reduced, the color was restored from light yellow to reddish brown, and the overall state was close to the normal group, suggesting that kidney bean kinase can effectively improve obesity and liver steatosis caused by high-fat diet.

[0129] Reference Figure 2 C, the liver weight statistical structure showed that the high-fat feed control group significantly increased the liver weight, while the kidney bean kinase intervention group significantly reduced the liver weight, which had statistically significant difference compared with the high-fat feed control group (p<0.05), and the improvement effect of the high-dose kidney bean kinase group was more significant. The results show that kidney bean kinase can effectively reduce liver hypertrophy and fat deposition induced by high-fat diet, and has a protective effect on maintaining liver structure and function.

[0130] Example 4

[0131] This Example 4 studies the lipid-lowering effect of kidney bean kinase on obese animal models. The specific test method is as follows:

[0132] (1) Changes in blood lipid biochemical indicators

[0133] At the end of the experiment, blood was collected after fasting, and serum was separated for detection of total cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C) levels. The detection method can be selected from existing commercial enzyme colorimetric reagent kits and operated according to the instructions.

[0134] The serum PCSK9 content can be determined by ELISA method, and standard curve and positive and negative controls are set to ensure quantitative accuracy. The detection results are expressed as mean ± standard deviation, and statistical analysis is used to compare the differences between groups.

[0135] (2) Safety evaluation

[0136] At the end of the experiment, blood was collected after fasting, and serum was separated for determination of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity by existing commercial enzyme colorimetric reagent kits or automatic analyzers. The reagent kit and instrument instructions were strictly followed for quality control and operation. Each batch of detection included standard and positive and negative controls, the results were reported in U / L units, and the group mean ± standard deviation was expressed, and statistical methods were used to compare the differences between groups to evaluate the hepatotoxicity risk of drug administration.

[0137] Reference Figure 3 A, Figure 3 B and Figure 3The animal experiment results of C showed that after continuous oral administration of kidney bean kinase for 4 weeks, the blood lipid level and PCSK9 expression of mice were significantly improved. Compared with the high-fat diet control group, the low-dose kidney bean kinase group and the high-dose kidney bean kinase group can significantly reduce the levels of serum total cholesterol and triglyceride, and low-density lipoprotein cholesterol. Among them, the decrease of the high-dose kidney bean kinase group is more significant, the serum total cholesterol decreases by about 32%, and the low-density lipoprotein cholesterol decreases by about 45%, p<0.01.

[0138] Reference Figure 4 The serum ELISA detection results showed that the PCSK9 content was significantly increased in the high-fat diet control group, and was significantly decreased after the intervention of kidney bean kinase, especially in the high-dose kidney bean kinase group, which decreased by about 55% (p<0.01), indicating that kidney bean kinase may improve lipid metabolism disorder by inhibiting PCSK9 secretion.

[0139] Reference Figure 5 A and Figure 5 B, the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity detection results showed that the liver function indexes of the kidney bean kinase intervention group were similar to those of the normal group, and no obvious increase or tissue damage was found, indicating that long-term oral administration of kidney bean kinase had good safety and tolerability. The overall results verified that kidney bean kinase could safely and effectively improve hyperlipidemia and lipid metabolism disorder by down-regulating PCSK9 expression and restoring LDL-R-mediated lipid clearance pathway.

[0140] Example 5

[0141] This Example 5 studies the effect of kidney bean kinase on the expression of LDL-R and lipid accumulation in the liver tissue of obese mice. The specific test method is as follows:

[0142] (1) Preparation of pathological sections

[0143] The normal diet group, high-fat diet control group, low-dose kidney bean kinase group (kidney bean kinase dose 7500 U / kg, recorded as KBE7.5K group), and high-dose kidney bean kinase group (kidney bean kinase dose 15000 U / kg, recorded as KBE15K group) mice were dissected. Immediately after, the colon tissue was taken out for length measurement and photography, and a part of the colon was taken after PBS rinsing for fixation. After the colon tissue was fixed in 4% paraformaldehyde for 24-48 hours, it was washed with water, dehydrated, transparent, immersed in wax, embedded, sectioned, spread, and baked to prepare tissue pathological sections with a thickness of about 5 µm.

[0144] (2) HE staining and analysis of mouse liver tissue

[0145] a. Baking the section: bake the paraffin section at 95°C for 15 minutes.

[0146] b. Dewaxing and hydration: after baking, put the slice into xylene for 10 minutes, repeat 3 times; put into 100% ethanol for 3 minutes, repeat 3 times; put into 95% ethanol for 3 minutes, repeat 2 times; put into 70% ethanol for 3 minutes, repeat 2 times; wash with distilled water.

[0147] c. Hematoxylin staining: put the slice into hematoxylin staining solution for 4-5 minutes, wash with distilled water.

[0148] d. Eosin staining: put the slice into eosin staining solution for 20-30 seconds, wash with distilled water.

[0149] e. Dehydration, transparency and mounting: put the slice into 70% ethanol for 2 minutes; put into 95% ethanol for 2 minutes; put into 100% ethanol for 2 minutes, repeat 2 times; put into xylene for 5 minutes, repeat 3 times; mount with neutral resin, and dry.

[0150] f. After microscopic examination, use a pathological slice scanner for scanning analysis.

[0151] (3) Mouse liver tissue immunostaining and analysis

[0152] a. Baking: bake the paraffin slice at 95°C for 15 minutes.

[0153] b. Dewaxing and hydration: after baking, put the slice into xylene for 10 minutes, repeat 3 times; put into 100% ethanol for 3 minutes, repeat 3 times; put into 95% ethanol for 3 minutes, repeat 2 times; put into 70% ethanol for 3 minutes, repeat 2 times; wash with distilled water.

[0154] c. Perform antigen repair, optionally heat or enzyme method, to restore the antigen epitope in the tissue.

[0155] d. Block endogenous peroxidase and non-specific binding sites with blocking solution.

[0156] e. Add specific primary antibody against target protein, preferably PCSK9 or LDL-R, for incubation, followed by detection with secondary antibody or labeled streptavidin-peroxidase complex.

[0157] f. Develop color with color developing reagent, optionally DAB color developing reagent, and re-stain with appropriate dye, optionally hematoxylin, for nuclear contrast.

[0158] g. Dehydrate, transparentize and mount, and observe and record photos under microscope.

[0159] (4) Mouse liver tissue oil red staining and analysis

[0160] After the liver tissue is taken out, it is fixed or frozen and prepared into sections, and the oil red 0 (oil Red 0) staining method is used to detect the lipid droplets and fatty degeneration in the liver. After staining, an optical microscope is used to take pictures and record, and the area ratio of lipid droplets in the liver tissue or the staining intensity is evaluated by image analysis software or semi-quantitative scoring method. The results are expressed as mean ± standard deviation, and statistical comparison is performed to evaluate the differences in liver fat deposition of different treatment groups. Each staining batch should include negative and positive controls to ensure staining specificity and comparability.

[0161] Reference Figure 6 The liver tissue structure of the normal group fed with ordinary feed is complete, the liver cord arrangement is regular, the hepatocyte morphology is normal, and no obvious fatty degeneration or inflammatory cell infiltration is observed. The cytoplasm of the hepatocytes in the high-fat feed control group is obviously vacuolated, and the liver cord structure is disordered, and a large number of fatty degeneration and cell swelling can be seen, indicating that high-fat diet leads to obvious liver injury. After intervention of kidney bean kinase, the liver cell structure of the low-dose kidney bean kinase group and the high-dose kidney bean kinase group gradually recovers, the liver cord arrangement is relatively neat, the degree of fatty degeneration and inflammation is obviously reduced, and there is a dose-dependent improvement trend, and the improvement of the high-dose kidney bean kinase group is more significant.

[0162] Reference Figure 7 A and Figure 7 B, the LDL-R immunohistochemical staining shows that the positive signal of LDL-R in the liver tissue of the ordinary feed group of mice is significantly reduced, indicating that high-fat diet inhibits the expression of LDL-R. In the kidney bean kinase intervention group, strong brownish yellow positive signal can be seen in the hepatocyte membrane and cytoplasm, indicating that the expression level of LDL-R is significantly up-regulated, especially in the high-dose kidney bean kinase group, the expression intensity of LDL-R is increased by about 2.1 times compared with the high-fat feed control group (p<0.01). It is indicated that kidney bean kinase may down-regulate PCSK9 to remove the inhibition of LDL-R degradation and promote the LDL-R-mediated cholesterol clearance.

[0163] Reference Figure 8 A and Figure 8 B, the oil red O staining results show that there are a large number of red lipid droplets deposited in the liver tissue of the high-fat feed control group, and the area ratio of lipid droplets is significantly higher than that of the normal diet group (p<0.001), indicating that the lipid accumulation is serious. After intervention of kidney bean kinase, the number and area of lipid droplets are obviously reduced, and the staining intensity is significantly weakened, such as the area ratio of lipid droplets in the high-dose kidney bean kinase group is decreased by about 60% (p<0.01), indicating that kidney bean kinase has a significant effect on inhibiting the accumulation of liver lipids.

[0164] The above results show that kidney bean kinase can effectively improve the lipid deposition and structural damage of the liver of obese mice, significantly up-regulate the level of LDL-R, thereby enhancing the uptake and metabolism of serum LDL-C by the liver, and reducing fatty degeneration and inflammatory reaction.

[0165] Example 6

[0166] This Example 6 investigates the protease domain of kidney bean kinase specifically binds to the key sites of PCSK9. The specific test method is as follows:

[0167] (1) Cell model

[0168] Human embryonic kidney cell line HEK293T is selected as a model. The cells are cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin.

[0169] (2) Construction of mutant plasmid

[0170] Site-specific mutants are designed for the predicted three candidate hydrolysis sites YSPASA, FAPGED, and WFPEDQ of PCSK9. For each candidate short peptide, an alanine scan and conservative / non-conservative substitution strategy is used to construct single-point and combined mutant, such as Y→A, Y→F, P→A, G→V, E→Q, etc. Two-site and three-site combined mutants are constructed to evaluate the redundancy or synergy between sites. By comparing the wild type and the mutant, the stability and the fragmentation product after treatment with kidney bean kinase can determine which site is the actual hydrolysis site, thereby verifying the direct hydrolysis mechanism mediated by kidney bean kinase KBE. The mutants are Human / Mouse PCSK9 mut1 , Human / Mouse PCSK9 mut2 , Human / Mouse PCSK9 mut3 .

[0171] (3) Treatment method

[0172] The expression plasmid of recombinant PCSK9 and its site mutant is transfected into the appropriate cell line, and the transfection / transduction process is performed according to the supplier's instructions. After treatment, the cells are collected at the predetermined time point, and immunological or mass spectrometric methods are used to detect the integrity and fragmentation product of PCSK9, in order to compare the stability difference between wild type and mutant before and after treatment with kidney bean kinase.

[0173] Reference Figure 9 A and Figure 9 B, Hydrolysis site characteristics of subtilisin serine protease substrate, further analysis of the protein sequence of PCSK9 found three potential hydrolysis sites YSPASA, FAPGED, WFPEDQ, and through sequence alignment found that the three sites are conserved sequences of human and mouse PCSK9 protein. Kidney bean kinase KBE can directly act on recombinant PCSK9 protein under in vitro conditions, producing a characteristic fragmentation band with a molecular weight of about 40-50 kDa, suggesting that it has specific proteolytic activity.

[0174] ReferenceFigure 9 C, PCSK9 mut1 (YSPASA mutation) and PCSK9 mut2 (FAPGED mutation) were significantly resistant to the cleavage mediated by kidney bean kinase KBE, and the broken bands were significantly weakened or completely disappeared, while PCSK9 mut3 (WFPEDQ mutation) showed partial protection, indicating that the first two sites are the main hydrolysis targets.

[0175] The results showed that kidney bean kinase can directly recognize and hydrolyze the conserved sequence YSPASA and FAPGED sites of PCSK9 protein, thereby destroying its structural stability and reducing its secretion level. This mechanism reveals the molecular basis of kidney bean kinase as a new type of PCSK9 protein hydrolysis inhibitor.

[0176] Example 7

[0177] This example 7 studies the effect of kidney bean kinase and its mutants in a high-fat diet mouse model. The specific test method is as follows:

[0178] (1) Experimental animal preparation

[0179] 5-6 week old C57BL / 6 male mice (body weight 18±2 g) were selected from Changzhou Cavens Experimental Animal Co., Ltd. The mice were raised in a SPF level barrier environment, with a 12 hour:12 hour day-night rhythm, temperature 23-25℃, and humidity 40%-60%. After 1 week of adaptive feeding, the mice were randomly divided into 6 groups (n=6): normal diet group, high-fat feed control group, kidney bean kinase low-dose group (kidney bean kinase given amount 7500U / kg, recorded as KBE7.5K group), kidney bean kinase high-dose group (kidney bean kinase given amount 15000U / kg, recorded as KBE15K group), high-fat+ PCSK9 mut1 plasmid group, high-fat+ PCSK9 mut2 plasmid group.

[0180] (2) Plasmid construction and liver in situ injection

[0181] Based on the kidney bean kinase gene, hydrolysis site mutant plasmids PCSK9 mut1 and PCSK9 mut2 were constructed, and the key amino acid sites were mutated to weaken or enhance the protease activity. After the plasmid was purified by EndoFree Plasmid Maxi, liver in situ injection was used for in vivo transfection, and each mouse was injected with 10% body weight of sterile PBS plasmid solution (plasmid concentration 1×10 8 PFU / mL), and the injection was done in one go to ensure that the plasmid was mainly distributed in the liver tissue. After injection, the mice were fed for 1 month to maintain a high-fat diet.

[0182] (3) Expression of PCSK9 and LDL-R in liver tissue protein

[0183] For detecting the protein level of PCSK9 and LDL-R in liver tissue or cells, Western blot method can be used for semi-quantitative analysis. The main process is as follows: extract and quantify the protein sample, perform molecular weight separation, electrotransfer membrane and antibody incubation on the membrane, and then develop and take pictures using chemiluminescence or other suitable detection methods. In the experiment, the internal reference protein, which can be β-Tubulin, is used to correct the protein loading difference, and positive and negative controls are set to verify the antibody specificity.

[0184] After 4 weeks of high-fat diet, the body weight of mice increased significantly compared with the normal diet group, indicating that the high-fat model was successfully constructed. Then, the mice were given kidney bean kinase or injected with PCSK9 mut1 mutant and PCSK9 mut2 mutant plasmid for liver in situ injection treatment for 4 weeks and maintained high-fat diet.

[0185] Reference Figure 10 A, the appearance of mice shows that the body size of high-fat diet control group mice increases significantly, while after kidney bean kinase intervention, especially in the high-dose kidney bean kinase group, the body size recovers close to normal; after PCSK9 mut1 mutant and PCSK9 mut2 mutant, the body size improvement effect of kidney bean kinase intervention is weakened.

[0186] Reference Figure 10 B, within the intervention period, the body weight of high-fat diet control group continues to rise, while the body weight of kidney bean kinase treatment group slows down gradually, and by the end of the 8th week, the body weight is significantly lower than that of high-fat diet control group. Among them, the high-dose kidney bean kinase (KBE15K) group has the most obvious body weight control effect, and the curve is flat; after PCSK9 mut1 and PCSK9 mut2 liver in situ injection, the body weight change of kidney bean kinase intervention is similar to that of high-fat diet control group. This result shows that kidney bean kinase significantly inhibits high-fat diet-induced body weight gain by regulating PCSK9 activity.

[0187] Reference Figure 10 C, the appearance of liver shows that the liver volume of high-fat diet control group increases and the color becomes lighter, while the liver volume of low-dose kidney bean kinase and high-dose kidney bean kinase decreases and the color returns to normal. After PCSK9 mut1 and PCSK9 mut2After in situ injection of the liver, kidney bean kinase intervention had no improvement effect on high-fat induced liver tissue. The liver weight statistical results showed that high-fat diet significantly increased the liver weight, and the kidney bean kinase intervention group significantly reduced, and the difference was statistically significant compared with the high-fat feed control group (p<0.05). After PCSK9 mut1 and PCSK9 mut2 After in situ injection of the liver, the kidney bean kinase intervention was basically the same as the high-fat feed control group.

[0188] Reference Figure 10 D, the Western blot results showed that high-fat diet significantly up-regulated the expression of PCSK9 protein in mouse liver tissue, accompanied by a significant decrease in LDL-R protein level, suggesting that high-fat intake led to imbalance of PCSK9-LDL-R regulatory axis. After kidney bean kinase intervention, the expression of PCSK9 protein decreased in a dose-dependent manner, while the expression of LDL-R gradually recovered. Especially in the high-dose kidney bean kinase group (KBE15K), the expression level of PCSK9 was significantly lower than that of the high-fat feed control group, and the level of LDL-R was significantly higher, indicating that kidney bean kinase could effectively improve the overexpression of PCSK9 and the degradation of LDL-R caused by high-fat diet.

[0189] Further, in the PCSK9 mut1 and PCSK9 mut2 After in situ injection of the liver, the changes of PCSK9 and LDL-R protein levels in the kidney bean kinase treatment group were no longer obvious, PCSK9 remained high expression and LDL-R remained low level, similar to the high-fat feed control group. The results suggest that kidney bean kinase plays a regulatory role by targeting the key hydrolysis site of PCSK9, and the mutation destroys the binding and degradation process of kidney bean kinase and PCSK9, thereby weakening its recovery effect on LDL-R.

[0190] In combination with Examples 1-7, the kidney bean kinase (Kidney bean enzyme, KBE) provided by the present application improves the mechanism of lipid metabolism disorder by regulating the PCSK9-LDL-R axis. In vitro experiments show that kidney bean kinase can significantly reduce PCSK9 protein level and up-regulate LDL-R expression in a time- and dose-dependent manner. Through mutation analysis of the predicted hydrolysis sites YSPASA, FAPGED and WFPEDQ of PCSK9, it is determined that the YSPASA and FAPGED sites are the key recognition and hydrolysis sites of kidney bean kinase.

[0191] Further verification in a high-fat diet mouse model found that kidney bean kinase significantly inhibited body weight gain and liver hypertrophy, reduced blood lipid levels and improved liver tissue morphology, and after mutation of the key sites of PCSK9, these protective effects disappeared.

[0192] The immunoblotting results show that kidney bean kinase treatment can down-regulate PCSK9 and up-regulate LDL-R expression, and the mutant group loses the regulation effect.

[0193] The comprehensive results show that kidney bean kinase specifically hydrolyzes the key site of PCSK9 through its protease activity, blocks the degradation of LDL-R, and thus improves the metabolic disorder induced by high fat, and provides a new molecular mechanism basis for the application of kidney bean kinase in the prevention and treatment of hyperlipidemia and atherosclerosis.

[0194] Although the present application has been disclosed with reference to the preferred embodiments, it is not intended to limit the present application. Those skilled in the art can make various modifications and improvements without departing from the spirit and scope of the present application. Therefore, the scope of protection of the present application shall be subject to the scope defined by the claims.

Claims

1. An oral pharmaceutical composition, characterized by, Comprise: kidney bean kinase as an active ingredient, and a pharmaceutically acceptable carrier; Wherein, the pharmaceutical composition is formulated to: After oral administration, the kidney bean kinase can exert lipid-lowering effect by down-regulating the expression level of PCSK9 protein and up-regulating the expression of low-density lipoprotein receptor.

2. The oral pharmaceutical composition according to claim 1, characterized in that, The pharmaceutical composition is formulated to: After oral administration, the kidney bean kinase can exert lipid-lowering effect by hydrolyzing YSPASA and / or FAPGED sites of PCSK9 protein.

3. The oral pharmaceutical composition according to claim 1, characterized in that, The specific activity of the kidney bean kinase is not less than 2000 U / mg.

4. The oral pharmaceutical composition according to claim 1, characterized by, Further comprising one or more additional lipid-lowering active ingredients, which are any one of statins, ezetimibe, phytosterols and probiotics, and the weight ratio of the kidney bean kinase to the additional lipid-lowering active ingredients is 1: (0.1-10).

5. The oral pharmaceutical composition according to claim 1, characterized in that, The pharmaceutical composition is formulated into capsules, tablets, granules, oral liquids, microcapsules or liposomes.

6. The oral pharmaceutical composition according to claim 5, characterized in that, The microcapsules are prepared by sodium alginate and chitosan embedding technology.

7. The oral pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition is a soft capsule, and the content thereof is composed of at least the following components in addition to the kidney bean kinase: Fish oil, and the mass ratio of the fish oil to the kidney bean kinase is 1:4-1:6; Antioxidants, and the content of the antioxidants is 0.1%-0.3% based on the total weight of the content; Suspending agents, and the content of the suspending agents is 0.2%-0.5% based on the total weight of the content.

8. The oral pharmaceutical composition according to claim 5, characterized in that, The liposomes are formed by phospholipids and cholesterol to form a double membrane to encapsulate the kidney bean kinase, wherein the weight ratio of the phospholipids to the cholesterol is 3:1, and the encapsulation rate of the kidney bean kinase is not less than 80%.

9. Use of an oral pharmaceutical composition according to any one of claims 1 to 8, characterized in that, Applied to the preparation of oral lipid-lowering drugs, and / or; Applied to the preparation of drugs for preventing and / or treating hypercholesterolemia and atherosclerosis.

10. Use of an oral pharmaceutical composition according to any one of claims 1 to 8, characterized in that, Applied to the preparation of functional foods or nutritional supplements. Applied to the preparation of functional foods or nutritional supplements.

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