Drug composition containing lactoferrin and its application in reducing blood lipid

Abstract

The present invention provides a pharmaceutical composition containing lactoferrin and its application in reducing blood lipid. Specifically, the present invention provides that the KGT-C polypeptide has a strong effect of reducing the synthesis amount of cholesterol in cells. After combining it with lactoferrin, it can significantly reduce the production of cholesterol and LDL, and has a good effect in treating hyperlipidemia.

Description

Technical Field

[0001] The present application relates to the field of biotherapy, and specifically to a pharmaceutical composition containing lactoferrin and its application in reducing blood lipid levels. Background Art

[0002] Hyperlipidemia is one of the major diseases that plague human health. The complexity of the causes of this disease and its intractability have always been a difficult problem in the medical field. Coupled with the rapid changes in the environment and the diversity of diets that people are in now, various potential factors may lead to hyperlipidemia in people. Data shows that the number of people suffering from hyperlipidemia globally is increasing, and the population is becoming wider. Hyperlipidemia is a disease that can manifest in various parts of the human body. This disease can induce relatively complex diseases such as cardiovascular and cerebrovascular diseases, diabetes, and coronary heart disease. These diseases generally have the characteristics of multiple complications, slow treatment, and difficulty in complete recovery. How to reduce the incidence rate and improve the cure rate has become the key research direction in the current medical field. Most cases of hyperlipidemia are caused by excessive lipids in the blood. How to reduce the lipid content in the blood has become an important research direction for the prevention and treatment of this type of disease.

[0003] Statins are currently the most widely used drugs for reducing blood lipid levels in clinical practice. These drugs have the characteristics of strong selectivity and good efficacy. Moreover, a large number of clinical results have shown that these drugs also have the advantages of few side effects and extremely minor accompanying side effects. It is precisely these characteristics and advantages of statins that make them the most ideal drugs for treating hypercholesterolemia, and they can significantly treat atherosclerosis and coronary heart disease caused by high total cholesterol. They have now become a much-favored drug in the medical field. The main statin lipid-lowering drugs include lovastatin, simvastatin, and other drugs.

[0004] Lactoferrin (Lf) is an 80 kDa non-heme iron-binding glycoprotein. The isolated protein structure is similar to that of serum transferrin, with 60% sequence homology. Therefore, Lf, together with serum transferrin, melanotransferrin, and ovotransferrin, is classified as a member of the transferrin family. Lf also has a variety of biological functions, including anti-inflammatory, antibacterial, antioxidant, anti-cancer, and immunomodulatory effects. Its broad application prospects have made it one of the current research and development hotspots. Lf exhibits good anti-inflammatory activity in various diseases, mainly in iron deficiency anemia, Alzheimer's disease, obesity, diabetes, and inflammatory bowel disease. In an intervention study, 29 women with mild thalassemia, 149 women with hereditary thrombotic anemia, and 20 anemic pregnant women with various diseases were intervened. They were treated with 100 mg of 20-30% iron-saturated bLf orally twice a day, showing that bLf can effectively reduce serum IL-6 and hepcidin levels. The combined administration of oral and intravaginal bLf has been proven to be an excellent therapy for preventing PTD by reducing cervical-vaginal IL-6 and prostaglandin F2α (PGF2α) without any side effects, indicating the anti-inflammatory activity of bLf. In Alzheimer's disease (AD), the chronic inflammatory process in the brain tissue mainly occurs around the amyloid plaques of activated microglia. These microglia promote the synthesis of different pro-inflammatory cytokines, such as IL-1β, IL-6, TNF, and IFN. In addition, an increase in serum IL-6 and a decrease in anti-inflammatory IL-10 have been observed in AD patients. Inflammation is one of the pathogenic mechanisms of different neurodegenerative diseases. The detection of serum inflammatory mediators helps in the preclinical diagnosis of AD and monitoring the disease progression. Studies have shown that intranasal administration of recombinant human lactoferrin (rhLf) can reduce Aβ deposition in AD model mice and alleviate the decline in their cognitive ability. rhLf treatment significantly reduced the levels of TNF and IL-6 in the brain. In a clinical trial, treatment of AD patients with 250 mg / d of commercial bovine lactoferrin (cbLf) for three months showed that cbLf had a significant effect in reducing serum IL-6 and increasing serum IL-10, also confirming the anti-inflammatory activity of Lf in this pathology.

[0005] Previous studies have also shown that lactoferrin has a certain regulatory effect on high-fat diet-induced obesity and diabetes in mice. Lactoferrin can reduce the body's insulin resistance and then slow down the occurrence and progression of obesity and diabetes by regulating multiple pathways such as lipid metabolism and the signal pathways involved in insulin. However, current studies show that the effect of using lactoferrin alone to treat hyperlipidemia is not significant enough, and further combination with other drugs to improve the treatment effect is the future research direction of traditional Chinese medicine. Summary of the Invention

[0006] The present invention provides a lipid-lowering polypeptide isolated from Momordica charantia, named KGT-C, and its amino acid sequence is shown in SEQ ID NO: 1.

[0007] The lipid-lowering polypeptide isolated from Momordica charantia is prepared by crushing fresh Momordica charantia into pulp, enzymatically hydrolyzing it with cellulase and neutral protease respectively, followed by ultrasonic treatment, then inactivating the enzyme, centrifuging, and then performing ultrafiltration. The low-molecular-weight polypeptide is first purified by S-8 macroporous adsorption resin, and further separated and purified by SephadexG-15 gel column. The component with better lipid-lowering effect is further analyzed and identified by Nano-HPLC-MS / MS technology for component C. Through database comparison, the lipid-lowering polypeptide KGT-C is identified.

[0008] On the one hand, the present invention provides the use of the lipid-lowering polypeptide KGT-C in the preparation of a pharmaceutical composition for treating hyperlipidemia.

[0009] The inventors also found in the research that lactoferrin regulates fat accumulation by modulating the intestinal flora structure and improves the glycolipid metabolism disorder in high-fat diet mice. Therefore, the present invention combines the lipid-lowering polypeptide KGT-C with lactoferrin for better treatment of hyperlipidemia.

[0010] Therefore, on the other hand, the present invention provides the use of the lipid-lowering polypeptide KGT-C and lactoferrin in the preparation of a kit for treating hyperlipidemia.

[0011] The pharmaceutical composition of the present invention can be used for treating, preventing or controlling cholesterol, dyslipidemia and related disorders, including but not limited to: cardiovascular diseases; atherosclerosis; stroke; peripheral vascular diseases; dyslipidemia; abnormal lipoproteinemia; restenosis; glucose metabolism disorders; Alzheimer's disease; syndrome X; peroxisome proliferator-activated receptor-related disorders; sepsis; thrombotic disorders; obesity; pancreatitis; hypertension; kidney diseases; cancer; inflammation; inflammatory muscle diseases, such as rheumatoid polymyositis, polymyositis and fibrosis; impotence; gastrointestinal diseases; irritable bowel syndrome; inflammatory bowel disease; inflammatory disorders, such as asthma, vasculitis, ulcerative colitis, Crohn's disease, Kawasaki disease, Wegener's granulomatosis, (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS) and autoimmune chronic hepatitis; impotence; arthritis, such as rheumatoid arthritis, juvenile rheumatoid arthritis and osteoarthritis; osteoporosis, soft tissue rheumatism, such as tendinitis; bursitis; autoimmune diseases, such as systemic lupus erythematosus and lupus erythematosus; scleroderma; ankylosing spondylitis; gout; pseudogout; non-insulin-dependent diabetes mellitus (NTDDM); septic shock; polycystic ovary disease; hyperlipidemia, such as familial hypercholesterolemia (FH), familial combined hyperlipidemia (FCH); lipoprotein lipase deficiency, such as hypertriglyceridemia, hypolipoproteinemia and hypercholesterolemia; lipoprotein abnormalities associated with diabetes; lipoprotein abnormalities associated with obesity; and lipoprotein abnormalities associated with Alzheimer's disease.

[0012] As used herein, the term "dyslipidemia" refers to a disorder that causes or manifests as abnormal levels of circulating lipids. To the extent that lipid levels in the blood are too high, the methods of the present invention can be used to restore normal levels. Normal levels of lipids are reported in medical articles known to those skilled in the art. For example, the recommended blood levels of low-density lipoprotein (LDL), high-density lipoprotein, free triglycerides and other parameters related to lipid metabolism can be found on the websites of the American Heart Association and the National Cholesterol Education Program of the National Heart, Lung, and Blood Institute of the United States. Currently, the recommended level of HDL cholesterol in the blood is above 35 mg / dl; the recommended level of low-density lipoprotein cholesterol in the blood is below 130 mg / dl; the recommended LDL:HDL cholesterol ratio in the blood is below 5:1, ideally 3.5:1; and the recommended level of free triglycerides in the blood is below 200 mg / dl.

[0013] Unless otherwise indicated, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or State government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals and, particularly, in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound of the invention is administered. Such pharmaceutical carriers can be liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. The pharmaceutical carrier can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. Additionally, adjuvants, stabilizers, thickening agents, lubricants, and coloring agents can be used. The compounds and compositions of the invention and the pharmaceutically acceptable carriers are preferably sterile when administered to a patient. When the compounds of the invention are administered intravenously, water is the preferred excipient. Aqueous solutions of saline and dextrose and aqueous glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like. If desired, the compositions of the invention can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0014] Unless otherwise specified, the term "pharmaceutically acceptable salt" as used herein includes, but is not limited to, salts of acidic or basic groups that may be present in the compounds of the present invention. Compounds that are basic in nature are capable of forming various salts with a variety of inorganic and organic acids. The acids that can be used to prepare pharmaceutically acceptable acid addition salts of these basic compounds are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, including but not limited to sulfuric acid, citric acid, maleic acid, acetic acid, oxalate, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, oleate, tannate, pantothenate, tartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., L,L'-methylene-bis-(2-hydroxy-3-naphthoate)). In addition to the above acids, the compounds of the present invention containing an amino moiety can also form pharmaceutically acceptable salts with a variety of amino acids. Compounds of the present invention that are acidic in nature are capable of forming base salts with a variety of pharmacologically acceptable cations. Examples of such salts include alkali metal or alkaline earth metal salts, particularly calcium, magnesium, sodium, lithium, zinc, potassium, and iron salts. Unless otherwise specified, the term "pharmaceutically acceptable solvate" as used herein refers to a compound or a salt thereof of the present invention, which further includes a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. Preferred solvents are volatile, non-toxic, and / or acceptable when administered to humans in trace amounts. The term solvate includes hydrates, which refers to a compound or a salt thereof of the present invention, which further includes a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces, and includes monohydrates, dihydrates, trihydrates, tetrahydrates, etc.

[0015] Lubricants that can be used in the pharmaceutical compositions and dosage forms of the present invention include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerol, sorbitol, mannitol, polyethylene glycol, other diols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oils (such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laurate, agar, and mixtures thereof.

[0016] The single unit dosage form of the present invention is suitable for oral administration, mucosal (e.g., nasal, sublingual, vaginal, buccal or rectal), parenteral (e.g., subcutaneous, intravenous, bolus injection, intramuscular or intra-arterial) or transdermal administration to a patient. Examples of dosage forms include, but are not limited to: tablets; cores; capsules, such as soft elastic gelatin capsules; caches; pills; lozenges; dispersions; suppositories; ointments; cataplasms (poultry); pastes; powders; dressings; creams; plasters; solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage forms suitable for oral or mucosal administration to a patient, including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions or water-in-oil liquid emulsions), solutions and elixirs; liquid dosage forms suitable for parenteral administration to a patient; and reconstitutable sterile solids to provide liquid dosage forms suitable for parenteral administration to a patient.

[0017] Beneficial effects

[0018] The present invention provides a pharmaceutical composition containing lactoferrin and its application in reducing blood lipid. Specifically, the present invention provides that the KGT-C polypeptide has a strong effect on reducing the synthesis amount of cholesterol in cells. After combining it with lactoferrin, it can significantly reduce the generation of cholesterol and LDL, and has a good effect on treating hyperlipidemia. Description of the drawings

[0019] Figure 1 Results graph of the effect of polypeptide on cholesterol synthesis in a high-cholesterol cell model

[0020] Figure 2 Effects of each group on the fat contents in the liver, peritesticular and perirenal regions of mice. Detailed implementation manners

[0021] The specific embodiments of the present invention will be described in more detail below with reference to the drawings. Although the specific embodiments of the present invention are shown in the drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0022] Example 1 Effect of KGT-C polypeptide on a high-cholesterol cell model

[0023] When the hepatoma cells HepG2 cover 80%-90% of the bottom of the cell culture flask, they are digested with trypsin, suspended and counted, and according to a final concentration of 2x10 5Cells were seeded at 100 μg / mL in a 96-well plate and cultured statically in a carbon dioxide incubator. When the cell monolayer covered the bottom of the well plate, the medium was replaced with serum-free medium, and an optimized cholesterol solution with a mass concentration of 20 μg / mL was added to the medium. After 24 hours, a high cholesterol cell model was prepared. Subsequently, KGT-C polypeptide (SEQ ID NO: 1) samples with different final mass concentrations (10 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 500 μg / mL) were added to the culture medium and cultured for 24 hours. The supernatant was then removed, the cells were digested with trypsin, centrifuged, washed three times with PBS, and 250 μL of 1% TritonX-100 was added. The cells were then lysed in an ice bath at 4°C, and the cholesterol (TC) concentration in each group of cells was measured. The results are as follows. Figure 1 shown.

[0024] from Figure 1 As can be seen, the TC content of the model group (83.56±3.87) μmol / g was significantly higher than that of all experimental groups (P<0.01). After the addition of the peptide, the TC content of the model decreased in a dose-dependent manner. At a peptide concentration of 200 μg / mL, the TC content decreased to (1.82±0.62) μmol / g, which is basically close to the TC content of 500 μg / mL, indicating that the optimal peptide concentration is 200 μg / mL.

[0025] Example 2 Effect of KGT-C polypeptide on cholesterol metabolism in foam cells

[0026] Foam cell formation is a typical pathological feature of the early stages of atherosclerosis (AAS). Macrophages are the primary source of foam cells. Their formation is primarily due to a disruption in intracellular cholesterol homeostasis. Macrophages ingest large amounts of oxidized low-density lipoprotein, leading to the accumulation of large amounts of cholesterol esters and triglycerides within the cells.

[0027] RAW264.7 cells were cultured in RPMI1640 complete medium supplemented with 10% inactivated fetal bovine serum in a 37°C, 5% CO2 saturated humidity incubator. The experimental cells were divided into three groups: a control group, an oxidized low-density lipoprotein group, and a peptide + oxidized low-density lipoprotein group (KGT-C peptide concentration was 200 μg / mL). The latter two groups were treated with oxidized low-density lipoprotein (final concentration of 30 μg / mL) for 24 hours to induce macrophage foaming; the control group was not treated. After the cells were treated, the culture medium was discarded, the cells were washed three times with PBS, and 1 mL of saline was added to dilute the cells. The cells were repeatedly frozen and thawed, and then sonicated in an ice bath. High-performance liquid chromatography (HPLC) was used to analyze the intracellular cholesterol content, and the results are shown in Table 1.

[0028] Table 1 Effect of each group on cholesterol content (mg / g protein)

[0029] Group Total cholesterol (TC) Control group 160.82±9.57 Oxidized low density lipoprotein group <![CDATA[471.59±29.54 a > Polypeptide + oxidized low density lipoprotein group <![CDATA[198.75±13.54 b >

[0030] Compared with the control group, a: P < 0.05; compared with the oxidized low-density lipoprotein group, b: P < 0.05;

[0031] As can be seen from the results in Table 1, oxidized low-density lipoprotein can significantly increase the intracellular TC content, which is consistent with the biological characteristics of foam cells. After treating the cells treated with oxidized low-density lipoprotein with the polypeptide, the T content decreased compared with the control group, and the difference was significant. This fully shows that the KGT-C polypeptide of the present invention has good cholesterol-lowering properties.

[0032] Example 3 Experiment on Hyperlipidemia Animal Model

[0033] Healthy male C57BL / 6J mice, 4 weeks old, were purchased from Mouse Treasure Biotechnology. They were housed in an SPF-class animal room at a room temperature of 22 °C and a relative humidity of 50 - 60%. During the experiment, the mice had free access to food and water under a 12-hour light-dark cycle. After 1 week of adaptive feeding, they were randomly divided into 5 groups of 10 mice each according to body weight. The specific grouping and interventions were as follows:

[0034] (1) Normal diet group: Fed with normal feed with a fat content of 14% and drank sterile water.

[0035] (2) High-fat diet group: Fed with high-fat feed with a fat content of 60% and drank sterile water.

[0036] (3) Polypeptide group: Fed with high-fat feed and intragastrically administered KGT-C polypeptide at a dose of 200 mg / kg / d.

[0037] (4) Lactoferrin group: Fed with high-fat feed and added lactoferrin to the drinking water at a dose of 2 g / 100 ml.

[0038] (5) Polypeptide combined with lactoferrin group: Fed with high-fat feed, added lactoferrin to the drinking water, and at the same time intragastrically administered KGT-C polypeptide at the same dose as above.

[0039] (6) Positive control group: Fed with high-fat feed and intragastrically administered Zhibituo capsules at a dose of 200 mg / kg / d. The amount of high-fat feed used in each group was the same.

[0040] The intervention period was 12 weeks. After 12 weeks of intervention, mouse tail blood was taken, and the corresponding TG, TC, and LDL data in the serum were measured according to the corresponding kits. The results are shown in Table 2.

[0041] Table 2 Results of Serum Lipid Metabolism Indexes of Mice in Each Group

[0042]

[0043] As shown in Table 2, after the intervention, compared with the normal diet group, the blood lipid indexes (TG, TC, LDL) in the high-fat diet group increased, and there were significant differences (P<0.05). Compared with the high-fat diet group, the serum TG, TC and LDL levels in the four intervention treatment groups were significantly decreased. In particular, the serum TC, TG and LDL levels in the polypeptide combined with lactoferrin group were significantly lower than those in the high-fat diet group (P<0.05), and the treatment effect was also better than that of the positive control group. This fully shows that the polypeptide combined with lactoferrin can be effectively used to treat hyperlipidemia.

[0044] Subsequently, the mice in each group were sacrificed by cervical dislocation, and the liver, peritesticular and perirenal fat were quickly removed and the fat weight was weighed. The results are as Figure 2 shown.

[0045] As Figure 2 shown, the high-fat diet group significantly increased the weight of visceral fat in mice and was significantly higher than that in the normal diet group (P<0.05). Each intervention group was significantly lower than the high-fat diet group (P<0.05). The polypeptide group or the polypeptide combined with lactoferrin group could more significantly reduce the weight of visceral fat, and the treatment effect was significant. At the same time, the visceral organs of the mice in each polypeptide group were detected, and there were no obvious pathological characteristics, which also indicated that the polypeptide and lactoferrin had no obvious toxic and side effects.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A lipid-lowering polypeptide KGT-C isolated from Momordica charantia, characterized in that The amino acid sequence is shown as SEQ ID NO:

1.

2. Use of the lipid-lowering polypeptide KGT-C according to claim 1 in the preparation of a pharmaceutical composition for treating hyperlipidemia.

3. The use according to claim 2, characterized in that The pharmaceutical composition further contains a pharmaceutically acceptable carrier.

4. Use of the lipid-lowering polypeptide KGT-C and lactoferrin according to claim 1 in the preparation of a kit for treating hyperlipidemia.

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