Pharmaceutical composition containing lactoferrin and application of pharmaceutical composition in blood fat reduction

Abstract

The invention provides a pharmaceutical composition containing lactoferrin and application of the pharmaceutical composition in blood fat reduction. Specifically, the KGT-C polypeptide provided by the invention has a very strong effect of reducing the synthesis amount of cholesterol in cells, and after the KGT-C polypeptide is combined with lactoferrin, the generation of cholesterol and LDL can be remarkably reduced, so that the KGT-C polypeptide has a relatively good effect of treating hyperlipidemia.

Description

Technical Field

[0001] The present application relates to the field of biological therapy, and specifically to a pharmaceutical composition comprising lactoferrin and its application in lowering blood lipids. Background Art

[0002] Hyperlipidemia is one of the main diseases that plague human health. The complexity of the causes of this disease and the difficulty of curing this disease have always been a difficult problem in the medical community. Coupled with the rapid changes in people's environment and the diversity of diet, various potential factors may cause people to suffer from hyperlipidemia. Data show that more and more people around the world are suffering from hyperlipidemia, and the population is getting wider and wider. Hyperlipidemia is a disease that may manifest in various parts of the human body. This disease can induce more complex diseases such as cardiovascular and cerebrovascular diseases, diabetes, and coronary heart disease. These diseases generally have many complications, slow treatment, and are difficult to cure. How to reduce the incidence and increase the cure rate has become the focus of current medical research. Hyperlipidemia is mostly 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 lowering blood lipids in clinical practice. They are highly selective and effective, and a large number of clinical results have shown that they have few side effects and extremely small side effects. It is precisely this characteristic and advantage of statins that makes them the most ideal drugs for treating hypercholesterolemia. They can significantly treat atherosclerosis and coronary heart disease caused by high total cholesterol, and have now become a popular drug in the medical community. Statins mainly include lovastatin, simvastatin and other drugs.

[0004] Lactoferrin (Lf) is an 80kDa non-heme iron-binding glycoprotein. The isolated protein structure is similar to serum transferrin, with 60% sequence homology. Therefore, Lf is classified as a member of the transferrin family together with serum transferrin, melanotransferrin and ovotransferrin. Lf also has a variety of biological functions, including anti-inflammatory, antibacterial, antioxidant, anticancer and immunomodulatory. Its wide application prospects make it one of the hot spots for current research and development. Lf shows 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 pregnant women with anemia from various diseases were intervened and 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. Combined oral and intravaginal administration of bLf has been shown to be an excellent therapy for preventing PTD by reducing cervicovaginal IL-6 and prostaglandin F2a (PGF2a) without any side effects, indicating the anti-inflammatory activity of bLf. The chronic inflammatory process in brain tissue of Alzheimer's disease (AD) mainly occurs around amyloid plaques in activated microglia. These microglia promote the synthesis of different proinflammatory cytokines, such as IL-1p, IL-6, TNF, and IFN. In addition, an increase in serum IL-6 and a decrease in anti-inflammatory IL-10 were observed in AD patients. Inflammation is one of the pathogenic mechanisms of different neurodegenerative diseases. The detection of serum inflammatory mediators is helpful for preclinical diagnosis of AD and monitoring the progression of the disease. Studies have shown that intranasal administration of recombinant human lactoferrin (rhLf) can reduce Ap deposition and alleviate cognitive decline in AD model mice. rhLf treatment significantly reduced TNF and IL-6 levels in the brain. In a clinical trial, oral administration of 250 mg / d of commercial bovine lactoferrin (cbLf) to AD patients for three months showed that cbLf showed significant effects 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 shown that lactoferrin has a certain regulatory effect on obesity and diabetes in mice induced by a high-fat diet. Lactoferrin can reduce the body's insulin resistance by regulating lipid metabolism and insulin-involved signaling pathways, thereby slowing down the occurrence and progression of obesity and diabetes. However, current studies have shown that the effect of using lactoferrin alone to treat hyperlipidemia is not significant enough, and further combined use 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 separated from momordica charantia, which is named KGT-C and has an amino acid sequence as shown in SEQ ID NO:1.

[0007] The lipid-lowering polypeptide separated from bitter melon is prepared by crushing fresh bitter melon into pulp, enzymolyzing it with cellulase and neutral protease respectively, ultrasonically treating it, then inactivating the enzymes, centrifuging it, and ultrafiltration. The low-molecular-weight polypeptide is first purified by S-8 macroporous adsorption resin, and then further separated and purified by Sephadex G-15 gel column. The component with better effect on lowering blood lipids is further analyzed and identified by Nano-HPLC-MS / MS technology for C component, and the lipid-lowering polypeptide KGT-C is identified by database comparison.

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

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

[0010] Therefore, another aspect of the present invention provides a use of a lipid-lowering polypeptide KGT-C and lactoferrin in preparing a drug kit for treating hyperlipidemia.

[0011] The pharmaceutical compositions of the present invention can be used to treat, prevent or control cholesterol, dyslipidemia and related disorders, including but not limited to: cardiovascular disease; atherosclerosis; stroke; peripheral vascular disease; dyslipidemia; dyslipoproteinemia; restenosis; glucose metabolism disorders; Alzheimer's disease; syndrome X; peroxisome proliferator-activated receptor-related disorders; sepsis; thrombotic disorders; obesity; pancreatitis; hypertension; kidney disease; cancer; inflammation; inflammatory muscle diseases, such as rheumatic polymyositis, polymyositis and fibrosis; impotence; gastrointestinal diseases; irritable bowel syndrome; inflammatory bowel disease; inflammatory disorders, such as asthma, vasculitis, ulcerative colitis, Crohn's disease, Toosendan disease, Wegen's granulomatosis, (RA), systemic lupus erythematosus (SLE) E), multiple sclerosis (MS) and autoimmune chronic hepatitis; impotence; arthritis, such as rheumatoid arthritis, juvenile rheumatoid arthritis and osteoarthritis; osteoporosis, soft tissue rheumatic diseases, such as tendinitis; capsulitis; 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; dyslipidemia associated with diabetes; dyslipidemia associated with obesity; and dyslipidemia associated with Alzheimer's disease.

[0012] As used herein, the term "dyslipidemia" refers to a condition 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, 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 website of the American Heart Association and the website of the National Cholesterol Education Program of the National Heart, Lung and Blood Association. 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 less than 130 mg / dl; the recommended LDL:HDL cholesterol ratio in the blood is less than 5:1, ideally 3.5:1; and the recommended level of free triglycerides in the blood is less than 200 mg / dl.

[0013] Unless otherwise indicated, the term "pharmaceutically acceptable" as used herein means approved by a regulatory agency of a federal or state government or listed in the U.S. Pharmacopeia or other recognized pharmacopoeia for use in animals, particularly humans. The term "carrier" refers to a diluent, adjuvant, excipient or vehicle for administering the compounds of the present invention. Such pharmaceutical carriers can be liquids, such as water and oils, including liquids of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Pharmaceutical carriers can be saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. In addition, adjuvants, stabilizers, thickeners, lubricants and colorants can be used. When administered to a patient, the compounds and compositions of the present invention and pharmaceutically acceptable carriers are preferably sterile. When the compounds of the present invention are administered intravenously, water is a preferred excipient. Saline solutions and aqueous solutions of dextrose and glycerol 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 gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk, glycerol, propylene glycol, water, ethanol, etc. If necessary, the composition of the present invention may also contain a small amount of a wetting agent or emulsifier, or a pH buffering agent.

[0014] Unless otherwise indicated, the term "pharmaceutically acceptable salts" as used herein include, but are not limited to, salts of acidic or basic groups which may be present in the compounds of the present invention. Compounds that are basic in nature are capable of forming a variety of salts with various inorganic and organic acids. The acids that can be used to prepare the 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, gentiooxalate, fumarate, gluconate, gluconate, 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 various amino acids. The compounds of the present invention, which are acidic in nature, are able to form basic salts with various 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 indicated, the term "pharmaceutically acceptable solvate" as used herein refers to a compound of the present invention or a salt thereof, 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, and are administered to humans in trace amounts. The term solvate includes hydrates, refers to a compound of the present invention or a salt thereof, which further includes a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces, and includes monohydrates, dihydrates, trihydrates, tetrahydrates, and the like.

[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, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oils (e.g., 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, mucosal (e.g., nasal, sublingual, vaginal, buccal or rectal), parenteral (e.g., subcutaneous, intravenous, bolus injection, intramuscular or intraarterial) 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; tablets; lozenges; dispersions; suppositories; ointments; cataplasms (poultry); pastes; powders; dressings; creams; plasters; solutions; pouches; 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 sterile solids that can be reconstituted 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 lipids. Specifically, the present invention provides a KGT-C polypeptide which has a strong ability to reduce the amount of cholesterol synthesis in cells. When used in combination with lactoferrin, it can significantly reduce the generation of cholesterol and LDL, and has a good effect in treating hyperlipidemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Effects of peptides on cholesterol synthesis in a hypercholesterolemia cell model

[0020] Figure 2 Effects of each group on the liver, peritesticular and perirenal fat content of mice. DETAILED DESCRIPTION

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

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

[0023] When the HepG2 liver cancer cells covered 80%-90% of the bottom of the cell bottle, they were digested with trypsin, suspended, and counted. The final concentration was 2x10 5 / mL were inoculated in a 96-well plate and cultured statically in a carbon dioxide incubator. When the cell monolayer covered the bottom of the well plate, it was replaced with serum-free culture medium, and an optimized cholesterol solution with a mass concentration of 20μg / mL was added to the culture medium. After 24 hours of action, a high cholesterol cell model was prepared. Subsequently, KGT-C polypeptide (SEQ ID NO: 1) samples with different final mass concentrations (10μg / mL, 50μg / mL, 100μg / mL, 200μg / mL, 500μg / mL) were added to the culture medium and cultured for 24 hours. The supernatant was then removed, digested with trypsin, centrifuged, washed three times with PBS, and 250μL of 1% TritonX-100 was added. Then, the cells were 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 It can be seen that the TC content of the model group (83.56±3.87) μmol / g was significantly higher than that of each experimental group (P<0.01). After the addition of the peptide, the TC content of the model decreased in a dose-dependent manner. When the concentration of the peptide was 200 μg / mL, the TC content decreased to (1.82±0.62) μmol / g, which was basically close to the TC content of 500 μg / ml, indicating that the optimal peptide concentration was 200 μg / mL.

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

[0026] The formation of foam cells is a typical pathological feature of early atherosclerosis (As). Macrophages are the main source of foam cells. The main reason for their formation is that the intracellular cholesterol homeostasis is broken. Macrophages take up a large amount of oxidized low-density lipoprotein and accumulate a large amount of cholesterol esters and triglycerides in the cells.

[0027] RAW264.7 cells were cultured in RPMI1640 complete medium containing 10% inactivated fetal bovine serum in a 37°C, 5% CO2 saturated humidity incubator. The experimental cells were divided into three groups, namely the control group, the oxidized low-density lipoprotein group, and the polypeptide + oxidized low-density lipoprotein group (the concentration of KGT-C polypeptide was 200μg / mL). The latter two groups were treated with oxidized low-density lipoprotein (final concentration of 30μg / mL) for 24h to induce macrophage foaming; the control group was not treated. After the cell treatment was completed, the culture medium was discarded, PBS was washed 3 times, 1mL of normal saline was added to dilute the cells, and the cells were repeatedly frozen and thawed, and ultrasonically lysed 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 ]]> Peptide+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] From the results in Table 1, it can be seen that oxidized low-density lipoprotein can significantly increase the intracellular TC content, which is consistent with the biological characteristics of foam cells. After the polypeptide treated cells treated with oxidized low-density lipoprotein, 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 a good cholesterol-lowering property.

[0032] Example 3 Experiment on hyperlipidemia animal model

[0033] Healthy male C57BL / 6J mice, 4 weeks old, were purchased from AlphaGo. They were kept in an SPF animal room with a room temperature of 22°C and a relative humidity of 50-60%. The mice were free to eat and drink during the 12-hour light-dark alternation experiment. After 1 week of adaptive feeding, they were randomly divided into 5 groups according to body weight, with 10 mice in each group. The specific grouping and intervention were as follows:

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

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

[0036] (3) Peptide group: The mice were fed a high-fat diet and given KGT-C peptide orally at a dose of 200 mg / kg / d.

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

[0038] (5) Peptide combined with lactoferrin group: The group was fed a high-fat diet, lactoferrin was added to the drinking water, and KGT-C peptide was gavaged at the same dosage as above.

[0039] (6) Positive control group: fed with high-fat diet and gavaged with Zhibituo capsule 200 mg / kg / d. The amount of high-fat diet was the same in each group.

[0040] The intervention period was 12 weeks. After the end of the intervention period, the tail blood of the mice was collected and the corresponding TG, TC and LDL data in the serum were measured using 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 were all increased, and there was a significant difference (P<0.05). Compared with the high-fat diet group, the serum TG, TC and LDL levels of the four intervention treatment groups were significantly reduced. In particular, the serum TC, TG and LDL levels of the peptide combined with lactoferrin group were significantly lower than those of the high-fat diet group (P<0.05), and the treatment effect was better than that of the positive control group. This fully shows that peptide combined with lactoferrin can be effectively used to treat hyperlipidemia.

[0044] The mice in each group were then killed by cervical dislocation, and the liver, peritesticular and perirenal fat were quickly removed and weighed. Figure 2 shown.

[0045] like Figure 2 As shown in the results, the high-fat diet group significantly increased the weight of visceral fat in mice and was significantly higher than the normal diet group (P<0.05), and each intervention group was significantly lower than the high-fat diet group (P<0.05). The peptide group or the peptide combined with lactoferrin group was able to significantly reduce the weight of visceral fat, and the therapeutic effect was significant. At the same time, the visceral organs of mice in each peptide group were tested, and there were no obvious pathological characteristics, which also showed that the peptide and lactoferrin had no obvious toxic 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 it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, 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 bitter melon, characterized in that The amino acid sequence is shown in SEQ ID NO:

1.

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

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

4. Use of the lipid-lowering polypeptide KGT-C and lactoferrin as claimed in claim 1 in preparing a medicine kit for treating hyperlipidemia.

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