A lipid-lowering pharmaceutical composition and a preparation method thereof

Abstract

The application provides a lipid-lowering pharmaceutical composition and a preparation method thereof, and belongs to the technical field of medicines.The lipid-lowering pharmaceutical composition comprises the following components: 10-20 parts of spirulina extract, 5-15 parts of grape seed proanthocyanidin, 3-10 parts of conjugated linoleic acid, 2-8 parts of phytosterol and 1-5 parts of coenzyme Q10.Based on the lipid-lowering pharmaceutical composition, liposomes of spirulina extract and coenzyme Q10, microspheres of grape seed proanthocyanidin and phytosterol, and inclusion compounds of conjugated linoleic acid are prepared in sequence, and the liposomes, the microspheres and the inclusion compounds are mixed to obtain the lipid-lowering pharmaceutical composition.Through multi-level embedding and gradient release technology, the active ingredients can play roles in different parts of the body and at different time periods, the lipid-lowering effect is significantly improved, the side effect risk of high concentration exposure of single component is reduced, and the synergistic effect and precise release of the pharmaceutical composition are realized.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a lipid-lowering drug composition and its preparation method. Background Technology

[0002] Hyperlipidemia, a common metabolic disease, is characterized by abnormally elevated levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C), or decreased levels of high-density lipoprotein cholesterol (HDL-C) in the blood. In recent years, with the shift towards a high-sugar, high-fat diet and the prevalence of sedentary lifestyles, the incidence of hyperlipidemia has exploded, and the patient population is gradually becoming younger, leading to numerous health problems.

[0003] Currently used lipid-lowering drugs have significant limitations: statins, while effectively inhibiting cholesterol synthesis, may cause myopathy and elevated liver enzymes with long-term use, and their effect on triglyceride regulation is limited; fibrates primarily lower triglycerides but may increase the risk of gallstones, and their combination with statins increases the incidence of myopathy; niacin, while comprehensively regulating blood lipids, suffers from side effects such as facial flushing and gastrointestinal discomfort, leading to extremely low patient compliance. Furthermore, existing drugs often target single targets, making it difficult to address the complex multifactorial pathological mechanisms of hyperlipidemia, and their treatment efficacy for mixed hyperlipidemia is often unsatisfactory. While lipid-lowering components derived from natural products have become a research hotspot due to their high safety and multi-target regulation advantages, single components suffer from insufficient efficacy and low bioavailability, thus failing to exert significant therapeutic effects. Therefore, developing drug combinations based on natural active ingredients, achieving highly effective lipid-lowering through multi-target synergistic effects, and possessing high safety and few side effects, has become a key direction for overcoming current treatment bottlenecks. Summary of the Invention

[0004] To address the problems existing in the prior art, the first objective of this invention is to provide a lipid-lowering drug composition.

[0005] A second objective of this invention is to provide a method for preparing the above-mentioned lipid-lowering drug composition.

[0006] A third objective of this invention is to provide a lipid-lowering drug.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a lipid-lowering drug composition, comprising the following components in parts by weight:

[0009] Spirulina extract 10-20 parts, grape seed proanthocyanidins 5-15 parts, conjugated linoleic acid 3-10 parts, phytosterols 2-8 parts, and coenzyme Q10 1-5 parts.

[0010] The present invention also provides a method for preparing the above-mentioned lipid-lowering drug composition, comprising the following steps:

[0011] (1) Preparation of liposomes of spirulina extract and coenzyme Q10: Soybean lecithin and cholesterol were mixed at a mass ratio of 3:1 and dissolved in anhydrous ethanol to prepare an organic phase with a concentration of 20 mg / mL; Spirulina extract and coenzyme Q10 were mixed and dissolved in PBS to prepare an aqueous phase; Under a water bath at 55-65℃, the organic phase was slowly added dropwise to the aqueous phase and stirred to form a primary emulsion; The ethanol in the primary emulsion was removed under reduced pressure to form a liposome suspension; The liposome suspension was homogenized under high pressure to obtain liposomes;

[0012] (2) Preparation of grape seed proanthocyanidins and phytosterol microspheres: Grape seed proanthocyanidins and phytosterols were mixed, and an anhydrous ethanol solution of chitosan was added. The mixture was ultrasonically dispersed to form a drug-chitosan mixture. Under stirring conditions of 50℃ and 200r / min, the drug-chitosan mixture was slowly added dropwise to a medium-chain triglyceride containing 3% Span80 and stirred for 20-30 min to form a W / O type emulsion. Genipin solution was added dropwise to the W / O type emulsion and stirring was continued for crosslinking for 5-7 h. Anhydrous ethanol was added to break the emulsion, and the mixture was washed with 50% ethanol solution, then washed with purified water and dried to obtain microspheres.

[0013] (3) Inclusion complex of conjugated linoleic acid: Conjugated linoleic acid was added dropwise to an aqueous solution of β-cyclodextrin while stirring magnetically at 400 r / min for 2 h. Then the mixture was cooled to room temperature and refrigerated at 4 °C for 12 h. The precipitate was collected by filtration, washed with anhydrous ethanol, and dried to obtain the inclusion complex.

[0014] (4) The above liposomes, microspheres and inclusion complexes are mixed to obtain a lipid-lowering drug composition.

[0015] Preferably, in step (1), the pH of PBS is 7.4; and the concentration of spirulina extract in the aqueous phase is 15 mg / mL.

[0016] Preferably, in step (1), the dropping rate of the organic phase is 1 mL / min, and the stirring is magnetic stirring at 300 r / min.

[0017] Preferably, in step (1), the primary emulsion is subjected to rotary evaporation under reduced pressure at 40°C and 0.08 MPa to remove ethanol; the liposome suspension is homogenized three times under 100 MPa pressure to obtain liposomes.

[0018] Preferably, in step (2), the concentration of the chitosan solution is 2%; the volume ratio of the drug-chitosan mixture and the medium-chain triglyceride containing 3% Span80 is 6:1.

[0019] Preferably, in step (2), the concentration of the genipin solution is 10%, the solvent is anhydrous ethanol, the amount of genipin used is 10-15% of the mass of chitosan, and the crosslinking temperature is 60°C.

[0020] Preferably, in step (2), three times the volume of anhydrous ethanol is added to break the emulsion.

[0021] Preferably, in step (3), the concentration of the β-cyclodextrin aqueous solution is 5%, the mass ratio of conjugated linoleic acid to β-cyclodextrin is 1:8, and the magnetic stirring temperature is 55°C.

[0022] The present invention also provides a lipid-lowering drug, wherein the active ingredient of the drug is the above-mentioned lipid-lowering drug composition or the lipid-lowering drug composition prepared by the above-mentioned preparation method; the drug further includes a pharmaceutically acceptable carrier or excipient.

[0023] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows:

[0024] The lipid-lowering drug composition of the present invention is made of a variety of natural ingredients, which have synergistic effects and can significantly reduce the levels of TC, TG and LDL-C in serum, while increasing the level of HDL-C. It has a significant lipid-lowering effect, few side effects, and high safety.

[0025] The preparation method of the lipid-lowering drug composition in this invention utilizes multi-level encapsulation and gradient release technology to enable each active ingredient to exert its effects at different sites and time periods in the body, significantly improving the lipid-lowering effect while reducing the risk of side effects from high-concentration exposure of a single ingredient, thus achieving synergistic enhancement and precise release of the drug composition. Detailed Implementation

[0026] This invention provides a lipid-lowering drug composition, comprising, by weight parts: 10-20 parts of spirulina extract, 5-15 parts of grape seed proanthocyanidins, 3-10 parts of conjugated linoleic acid, 2-8 parts of phytosterols, and 1-5 parts of coenzyme Q10; preferably comprising 12-16 parts of spirulina extract, 8-12 parts of grape seed proanthocyanidins, 5-6 parts of conjugated linoleic acid, 4-5 parts of phytosterols, and 2-4 parts of coenzyme Q10. The phytosterols used in this invention are preferably any one or more of stigmasterol, campesterol, sitosterol, or alfalfa.

[0027] The present invention also provides a method for preparing the above-mentioned lipid-lowering drug composition, comprising the following steps:

[0028] (1) Preparation of liposomes of spirulina extract and coenzyme Q10: Soybean lecithin and cholesterol were mixed at a mass ratio of 3:1 and dissolved in anhydrous ethanol to prepare an organic phase with a concentration of 20 mg / mL; Spirulina extract and coenzyme Q10 were mixed and dissolved in PBS to prepare an aqueous phase; Under a water bath at 55-65℃, the organic phase was slowly added dropwise to the aqueous phase and stirred to form a primary emulsion; The ethanol in the primary emulsion was removed under reduced pressure to form a liposome suspension; The liposome suspension was homogenized under high pressure to obtain liposomes;

[0029] (2) Preparation of grape seed proanthocyanidins and phytosterol microspheres: Grape seed proanthocyanidins and phytosterols were mixed, and an anhydrous ethanol solution of chitosan was added. The mixture was ultrasonically dispersed to form a drug-chitosan mixture. Under stirring conditions of 50℃ and 200r / min, the drug-chitosan mixture was slowly added dropwise to a medium-chain triglyceride containing 3% Span80 and stirred for 20-30 min to form a W / O type emulsion. Genipin solution was added dropwise and stirring was continued for cross-linking for 5-7 h. Anhydrous ethanol was added to break the emulsion, and the mixture was washed with 50% ethanol solution, then washed with purified water and dried to obtain microspheres.

[0030] (3) Inclusion complex of conjugated linoleic acid: Conjugated linoleic acid was added dropwise to an aqueous solution of β-cyclodextrin while stirring magnetically at 400 r / min for 2 h. Then the mixture was cooled to room temperature and refrigerated at 4 °C for 12 h. The precipitate was collected by filtration, washed with anhydrous ethanol, and dried to obtain the inclusion complex.

[0031] (4) The above liposomes, microspheres and inclusion complexes are mixed to obtain a lipid-lowering drug composition.

[0032] This invention first prepares liposomes of spirulina extract and coenzyme Q10. In this invention, the pH of the PBS is 7.4, and the concentration of the spirulina extract in the prepared aqueous phase is 15 mg / mL. The preferred water bath temperature is 60°C. The organic phase is added at a dropping rate of 1 mL / min with stirring, preferably using magnetic stirring at 300 r / min. The primary emulsion is preferably prepared by removing ethanol under reduced pressure via rotary evaporation at 40°C and 0.08 MPa to obtain a liposome suspension; the liposome suspension is preferably homogenized three times under 100 MPa pressure to obtain liposomes. This invention also produces encapsulated liposomes with an average particle size of 150–200 nm through encapsulation.

[0033] The present invention further prepares microspheres of grape seed proanthocyanidins and phytosterols: In this invention, the concentration of the anhydrous ethanol solution of chitosan is preferably 2% (w / v, i.e., 2g chitosan dissolved in 100mL anhydrous ethanol), and the ultrasonic dispersion conditions are preferably 250-300W ultrasonic treatment for 8-10min (using intermittent mode, ultrasonic treatment for 20 seconds, with a 10-second interval to prevent local overheating and drug degradation). The medium-chain triglyceride containing 3% Span80 refers to Span80 added to the medium-chain triglyceride (MCT) at a mass ratio of 3%. The volume ratio of the drug-chitosan mixture to the medium-chain triglyceride containing 3% Span80 is 6:1. The concentration of the genipin solution is 10% (w / v, i.e., 10g of genipin dissolved in 100mL of anhydrous ethanol); the amount of genipin used is 10-15% of the chitosan mass, preferably 12-13%; the stirring speed is preferably 100-300r / min; the crosslinking temperature is preferably 60℃; the crosslinking time is preferably 6h; after crosslinking, preferably 3 times the volume of anhydrous ethanol is added to break the emulsion, and after standing for 10min, the microspheres are collected by vacuum filtration. They are first washed 3 times with 50% ethanol solution (after each wash, vacuum filtration is performed to remove residual MCT and Span80), then washed 2 times with purified water, and then vacuum dried at 45℃ to constant weight to obtain microspheres with an average particle size of 6-12μm.

[0034] This invention further prepares the inclusion complex of conjugated linoleic acid: In this invention, the concentration of the β-cyclodextrin aqueous solution is 5% (w / v, i.e., 5g of β-cyclodextrin dissolved in 100mL of water), the mass ratio of conjugated linoleic acid to β-cyclodextrin is 1:8, the magnetic stirring temperature is 55℃, and the speed is 300-400r / min. After refrigeration at 4℃ for 12h, the inclusion complex is allowed to fully precipitate. The precipitate is collected by filtration, washed three times with anhydrous ethanol, and vacuum dried at 45℃ to constant weight to obtain the conjugated linoleic acid inclusion complex.

[0035] The present invention mixes the above-mentioned liposomes, microspheres and inclusion complexes to obtain a lipid-lowering drug composition. Preferably, the mixture is mixed at a speed of 50 r / min for 5 to 15 min under the conditions of relative humidity ≤30% and temperature 25°C to ensure that the components are fully dispersed and mixed.

[0036] In this invention, hydrophilic spirulina extract and lipid-soluble coenzyme Q10 are encapsulated in liposomes, utilizing the lipid bilayer structure to achieve synergistic delivery of both and improve transmembrane transport efficiency. The liposomes rapidly release spirulina extract and coenzyme Q10 in the stomach, initiating early lipid-lowering effects. Chitosan microspheres encapsulate grape seed proanthocyanidins and phytosterols, utilizing the pH-responsiveness of chitosan to allow for slow drug release in the alkaline intestinal environment, prolonging the duration of action and continuously regulating lipid metabolism. β-cyclodextrin encapsulates conjugated linoleic acid, improving its stability and water solubility, reducing oxidative loss and odor, and gradually releasing conjugated linoleic acid in the distal small intestine, enhancing fatty acid oxidation and decomposition. This invention, through stepwise encapsulation of each component, allows each component to exert its effect at different sites and time points in the body, significantly improving lipid-lowering efficacy while reducing the risk of side effects from high-concentration exposure to a single component, achieving synergistic enhancement and precise release of the drug composition.

[0037] This invention also provides a lipid-lowering drug, wherein the active ingredient of the drug is the above-described lipid-lowering drug composition or the lipid-lowering drug composition prepared by the above-described preparation method; the drug further includes a pharmaceutically acceptable carrier or excipient. This invention does not limit the dosage form of the prepared drug; corresponding dosage forms can be prepared by adding corresponding carriers or excipients according to the preparation methods of pharmaceutical formulations in the art. More preferably, the pharmaceutical excipients of this invention include, but are not limited to, at least one of diluents, wetting agents, binders, lubricants, colorants, and coating agents. The dosage form of the drug of this invention is selected from at least one of tablets, capsules, granules, pills, injections, suspensions, dispersants, and syrups.

[0038] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0039] Unless otherwise specified, the following embodiments are all conventional methods.

[0040] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0041] Example 1

[0042] A lipid-lowering drug composition (by mass parts): 10 parts of spirulina extract, 5 parts of grape seed proanthocyanidins, 3 parts of conjugated linoleic acid, 2 parts of phytosterols and 1 part of coenzyme Q10; the phytosterol is alfalfa sterol.

[0043] Preparation method of lipid-lowering drug composition:

[0044] (1) Preparation of liposomes of spirulina extract and coenzyme Q10: Soybean lecithin and cholesterol were mixed at a mass ratio of 3:1 and dissolved in anhydrous ethanol to prepare an organic phase with a concentration of 20 mg / mL; Spirulina extract and coenzyme Q10 were mixed and dissolved in PBS (pH 7.4) to prepare an aqueous phase (water was added until the concentration of spirulina extract was 15 mg / mL); Under the condition of 55℃ water bath, the organic phase was slowly added dropwise to the aqueous phase at a drop rate of 1 mL / min and magnetically stirred at 300 r / min to form a primary emulsion; The primary emulsion was subjected to rotary evaporation at 40℃ and 0.08 MPa to remove ethanol and obtain a liposome suspension; The liposome suspension was homogenized by cycling at 100 MPa pressure 3 times to obtain liposomes.

[0045] (2) Preparation of grape seed proanthocyanidins and phytosterol microspheres: Grape seed proanthocyanidins and phytosterols were mixed, and an anhydrous ethanol solution of chitosan with a concentration of 2% was added. The mixture was then ultrasonically dispersed (ultrasonic treatment at 300W for 9 min, using intermittent mode, 20 seconds of sonication with a 10-second interval) to form a drug-chitosan mixture. Under stirring conditions of 50℃ and 200r / min, the drug-chitosan mixture was slowly added dropwise at a volume ratio of 6:1 to medium-chain triglycerides containing 3% Span80. In the triglyceride, the mixture was stirred for 20 min to form a W / O emulsion. A 10% anhydrous ethanol solution of genipin was added dropwise (the amount of genipin was 10% of the mass of chitosan). The mixture was stirred at 300 r / min and 60 °C for 5 h to crosslink. Three times the volume of anhydrous ethanol was added to break the emulsion. After standing for 10 min, the microspheres were collected by vacuum filtration. The microspheres were washed three times with 50% ethanol solution and then twice with purified water (filtered after each wash). Finally, the microspheres were dried under vacuum at 45 °C to constant weight to obtain the microspheres.

[0046] (3) Inclusion complex of conjugated linoleic acid: Conjugated linoleic acid was added dropwise to a 5% aqueous solution of β-cyclodextrin at a mass ratio of 1:8. The mixture was magnetically stirred at 400 r / min for 2 h while being added dropwise at 55 °C. The mixture was then cooled to room temperature and refrigerated at 4 °C for 12 h. The precipitate was collected by vacuum filtration, washed three times with anhydrous ethanol (after each wash by vacuum filtration), and dried under vacuum at 45 °C to constant weight to obtain the inclusion complex of conjugated linoleic acid.

[0047] (4) The above liposomes, microspheres and inclusion complexes are mixed at a speed of 50 r / min for 15 min under the conditions of relative humidity ≤30% and temperature 25℃ to obtain a lipid-lowering drug composition.

[0048] Example 2

[0049] A lipid-lowering drug composition (by mass parts): 20 parts of spirulina extract, 15 parts of grape seed proanthocyanidins, 10 parts of conjugated linoleic acid, 8 parts of phytosterols and 5 parts of coenzyme Q10; the phytosterol is phytosterol.

[0050] Preparation method of lipid-lowering drug composition:

[0051] (1) Preparation of liposomes of spirulina extract and coenzyme Q10: Soybean lecithin and cholesterol were mixed at a mass ratio of 3:1 and dissolved in anhydrous ethanol to prepare an organic phase with a concentration of 20 mg / mL; Spirulina extract and coenzyme Q10 were mixed and dissolved in PBS (pH 7.4) to prepare an aqueous phase (water was added until the concentration of spirulina extract was 15 mg / mL); Under a water bath at 65℃, the organic phase was slowly added dropwise to the aqueous phase at a drop rate of 1 mL / min and magnetically stirred at 300 r / min to form a primary emulsion; The primary emulsion was subjected to rotary evaporation at 40℃ and 0.08 MPa to remove ethanol and obtain a liposome suspension; The liposome suspension was homogenized three times under a pressure of 100 MPa to obtain liposomes.

[0052] (2) Preparation of grape seed proanthocyanidins and phytosterol microspheres: Grape seed proanthocyanidins and phytosterols were mixed, and an anhydrous ethanol solution of chitosan with a concentration of 2% was added. The mixture was then ultrasonically dispersed (300W ultrasonic treatment for 8 min, using intermittent mode, ultrasonic treatment for 20 seconds, with a 10-second interval) to form a drug-chitosan mixture. Under stirring conditions of 50℃ and 200r / min, the drug-chitosan mixture was slowly added dropwise at a volume ratio of 6:1 to medium-chain triglycerides containing 3% Span80. In the triglyceride, the mixture was stirred for 30 min to form a W / O emulsion. A 10% anhydrous ethanol solution of genipin (15% of the chitosan mass) was added dropwise, and the mixture was stirred at 300 r / min and 60 °C for 7 h to crosslink. Three times the volume of anhydrous ethanol was added to break the emulsion. After standing for 10 min, the microspheres were collected by vacuum filtration. The microspheres were washed three times with 50% ethanol solution and then twice with purified water (filtered after each wash). Finally, the microspheres were dried under vacuum at 45 °C to constant weight to obtain the microspheres.

[0053] (3) Inclusion complex of conjugated linoleic acid: Conjugated linoleic acid was added dropwise to a 5% aqueous solution of β-cyclodextrin at a mass ratio of 1:8. The mixture was magnetically stirred at 400 r / min for 2 h while being added dropwise at 55 °C. The mixture was then cooled to room temperature and refrigerated at 4 °C for 12 h. The precipitate was collected by vacuum filtration, washed three times with anhydrous ethanol (after each wash by vacuum filtration), and dried under vacuum at 45 °C to constant weight to obtain the inclusion complex of conjugated linoleic acid.

[0054] (4) The above liposomes, microspheres and inclusion complexes are mixed at a speed of 50 r / min for 5 min under the conditions of relative humidity ≤30% and temperature 25℃ to obtain a lipid-lowering drug composition.

[0055] Example 3

[0056] A lipid-lowering drug composition (by mass parts): 15 parts of spirulina extract, 10 parts of grape seed proanthocyanidins, 5 parts of conjugated linoleic acid, 6 parts of phytosterols and 3 parts of coenzyme Q10; the phytosterol is stigmasterol.

[0057] Preparation method of lipid-lowering drug composition:

[0058] (1) Preparation of liposomes of spirulina extract and coenzyme Q10: Soybean lecithin and cholesterol were mixed at a mass ratio of 3:1 and dissolved in anhydrous ethanol to prepare an organic phase with a concentration of 20 mg / mL; Spirulina extract and coenzyme Q10 were mixed and dissolved in PBS (pH 7.4) to prepare an aqueous phase (water was added until the concentration of spirulina extract was 15 mg / mL); Under a water bath at 60℃, the organic phase was slowly added dropwise to the aqueous phase at a drop rate of 1 mL / min and magnetically stirred at 300 r / min to form a primary emulsion; The primary emulsion was subjected to rotary evaporation at 40℃ and 0.08 MPa to remove ethanol and obtain a liposome suspension; The liposome suspension was homogenized three times under a pressure of 100 MPa to obtain liposomes.

[0059] (2) Preparation of grape seed proanthocyanidins and phytosterol microspheres: Grape seed proanthocyanidins and phytosterols were mixed, and an anhydrous ethanol solution of chitosan with a concentration of 2% was added. The mixture was then ultrasonically dispersed (ultrasonic treatment at 300W for 10 min, using intermittent mode, 20 seconds of sonication with a 10-second interval) to form a drug-chitosan mixture. Under stirring conditions of 50℃ and 200r / min, the drug-chitosan mixture was slowly added dropwise at a volume ratio of 6:1 to medium-chain triglycerides containing 3% Span80. In the triglyceride, the mixture was stirred for 25 min to form a W / O emulsion. A 10% anhydrous ethanol solution of genipin (15% of the chitosan mass) was added dropwise, and the mixture was stirred at 300 r / min and 60 °C for 6 h to crosslink. Three times the volume of anhydrous ethanol was added to break the emulsion. After standing for 10 min, the microspheres were collected by vacuum filtration. The microspheres were washed three times with 50% ethanol solution and then twice with purified water (filtered after each wash). Finally, the microspheres were dried under vacuum at 45 °C to constant weight to obtain the microspheres.

[0060] (3) Inclusion complex of conjugated linoleic acid: Conjugated linoleic acid was added dropwise to a 5% aqueous solution of β-cyclodextrin at a mass ratio of 1:8. The mixture was magnetically stirred at 400 r / min for 2 h while being added dropwise at 55 °C. The mixture was then cooled to room temperature and refrigerated at 4 °C for 12 h. The precipitate was collected by vacuum filtration, washed three times with anhydrous ethanol (after each wash by vacuum filtration), and dried under vacuum at 45 °C to constant weight to obtain the inclusion complex of conjugated linoleic acid.

[0061] (4) The above liposomes, microspheres and inclusion complexes are mixed at a speed of 50 r / min for 10 min under the conditions of relative humidity ≤30% and temperature 25℃ to obtain a lipid-lowering drug composition.

[0062] Comparative Example 1

[0063] The difference between this comparative example and Example 3 is that it does not contain coenzyme Q10.

[0064] The lipid-lowering drug composition (by mass parts) consists of: 15 parts of spirulina extract, 10 parts of grape seed proanthocyanidins, 5 parts of conjugated linoleic acid, and 6 parts of phytosterols; the phytosterols are stigmasterol.

[0065] Preparation method of lipid-lowering drug composition:

[0066] (1) Preparation of liposomes from spirulina extract: Soybean lecithin and cholesterol were mixed at a mass ratio of 3:1 and dissolved in anhydrous ethanol to prepare an organic phase with a concentration of 20 mg / mL; Spirulina extract was added to PBS (pH 7.4) and dissolved to prepare an aqueous phase (water was added until the concentration of spirulina extract was 15 mg / mL); Under a water bath at 60℃, the organic phase was slowly added dropwise to the aqueous phase at a drop rate of 1 mL / min and magnetically stirred at 300 r / min to form a primary emulsion; The primary emulsion was subjected to rotary evaporation at 40℃ and 0.08 MPa to remove ethanol and obtain a liposome suspension; The liposome suspension was homogenized three times under a pressure of 100 MPa to obtain liposomes.

[0067] (2) Preparation of grape seed proanthocyanidins and phytosterol microspheres: Same as in Example 3.

[0068] (3) Inclusion complex of conjugated linoleic acid: Same as in Example 3.

[0069] (4) Same as Example 3.

[0070] Comparative Example 2

[0071] The difference between this comparative example and Example 3 is that it does not contain phytosterols.

[0072] The lipid-lowering drug composition (by mass parts) consists of: 15 parts of spirulina extract, 10 parts of grape seed proanthocyanidins, 5 parts of conjugated linoleic acid, and 3 parts of coenzyme Q10.

[0073] Preparation method of lipid-lowering drug composition:

[0074] (1) Same as Example 3.

[0075] (2) Preparation of grape seed proanthocyanidin microspheres: Grape seed proanthocyanidins were mixed with an anhydrous ethanol solution of chitosan at a concentration of 2%, and ultrasonically dispersed evenly (ultrasonic treatment at 300W for 10 min, using intermittent mode, 20 seconds of sonication with a 10-second interval) to form a drug-chitosan mixture; under stirring conditions of 50℃ and 200r / min, the drug-chitosan mixture was slowly added dropwise at a volume ratio of 6:1 to a medium-chain triglyceride containing 3% Span80, and stirred. Stir for 25 min to form a W / O type emulsion; add dropwise a 10% anhydrous ethanol solution of genipin (genipin amount is 15% of chitosan mass), and continue stirring at 300 r / min and 60℃ for 6 h for crosslinking; add 3 times the volume of anhydrous ethanol to break the emulsion, let stand for 10 min, and then filter to collect microspheres. Wash the microspheres 3 times with 50% ethanol solution, then wash them 2 times with purified water (filter after each wash), and then vacuum dry at 45℃ to constant weight to obtain microspheres.

[0076] (3) Same as Example 3.

[0077] (4) Same as Example 3.

[0078] Comparative Example 3

[0079] The difference between this comparative example and Example 3 is that: liposomes of spirulina extract and coenzyme Q10, microspheres of grape seed proanthocyanidins and phytosterols, and inclusion complexes of conjugated linoleic acid are not prepared.

[0080] The lipid-lowering drug composition (by mass parts) consists of: 15 parts of spirulina extract, 10 parts of grape seed proanthocyanidins, 5 parts of conjugated linoleic acid, 6 parts of phytosterols and 3 parts of coenzyme Q10; the phytosterol is stigmasterol.

[0081] Preparation method of lipid-lowering drug composition: Mix each component at a speed of 50 r / min for 10 min under conditions of relative humidity ≤30% and temperature 25℃ to obtain lipid-lowering drug composition.

[0082] Experimental Example 1

[0083] 1. Cellular verification experiments were conducted on the components of the pharmaceutical composition of the present invention:

[0084] Experimental materials and methods: Human hepatocellular carcinoma cell line HepG2; HepG2 cells in logarithmic growth phase were harvested and cultured at a concentration of 5 × 10⁻⁶ cells / cells. 4 Cells were seeded per well in 96-well plates and cultured at 37°C and 5% CO2 for 24 h. Then, different culture media were used for each group, and the cells were cultured for another 48 h (n=10). Intracellular total cholesterol (TC) and triglyceride (TG) levels were detected using enzyme-linked immunosorbent assay (ELISA).

[0085] Experimental grouping and drug administration: The blank control group was replaced with ordinary culture medium without drugs; the model group and each drug administration group were replaced with high lipid induction medium (high glucose DMEM medium containing 0.2 mmol / L cholesterol + 0.1 mmol / L oleic acid) to construct a lipid accumulation model (the modeling success criteria were: the total cholesterol (TC) content in the model group cells increased by ≥60% and the triglyceride (TG) content increased by ≥50% compared with the blank control group).

[0086] Blank control group: cultured in ordinary culture medium, without any drug intervention, for 72 hours.

[0087] Model group: After culturing in high-fat induction medium for 48 hours, the medium was replaced with ordinary medium without drugs and cultured for another 24 hours.

[0088] Positive drug group: After culturing in high-fat induction medium for 48 h, the medium was replaced with ordinary medium containing atorvastatin (concentration 10 μmol / L) and cultured for 24 h.

[0089] Low-dose group of the composition: After culturing in high-lipid induction medium for 48 h, the medium was replaced with ordinary medium containing the drug composition (15 parts of spirulina extract, 10 parts of grape seed proanthocyanidins, 5 parts of conjugated linoleic acid, 6 parts of stigmasterol and 3 parts of coenzyme Q10, concentration 50 μg / mL), and cultured for 24 h.

[0090] High-dose group of the composition: After culturing in high-lipid induction medium for 48 h, the medium was replaced with ordinary medium containing the drug composition (15 parts of spirulina extract, 10 parts of grape seed proanthocyanidins, 5 parts of conjugated linoleic acid, 6 parts of stigmasterol and 3 parts of coenzyme Q10, concentration 200 μg / mL), and cultured for 24 h.

[0091] High-dose control composition 1: After culturing in high-lipid induction medium for 48 h, the medium was replaced with ordinary medium containing the drug composition (15 parts of spirulina extract, 10 parts of grape seed proanthocyanidins, 5 parts of conjugated linoleic acid and 6 parts of stigmasterol, at a concentration of 200 μg / mL), and cultured for 24 h.

[0092] High-dose control composition 2: After culturing in high-lipid induction medium for 48 h, the medium was replaced with ordinary medium containing the drug composition (15 parts of spirulina extract, 10 parts of grape seed proanthocyanidins, 5 parts of conjugated linoleic acid and 3 parts of coenzyme Q10, with a concentration of 200 μg / mL), and cultured for 24 h.

[0093] The lipid content per milligram of cellular protein was calculated using an enzymatic assay kit, and the results are shown in Table 1.

[0094] Table 1. Total cholesterol (TC) and triglyceride (TG) content in each group

[0095] Group Name Intracellular TC (mmol / g protein) Intracellular TG (mmol / g protein) Blank control group 1.20±0.10 0.81±0.06 Model group 3.94±0.26 2.73±0.37 Positive drug group 1.98±0.23 1.36±0.16 Low-dose group of the composition 2.26±0.19 1.54±0.21 High-dose group of the composition 1.62±0.15 1.01±0.09 High-dose control composition 1 2.37±0.21 1.89±0.16 High-dose control composition 2 2.46±0.17 2.01±0.34

[0096] The results showed that the pharmaceutical composition of the present invention can significantly reduce the content of TC and TG, and the reduction effect of the high-dose group is better than that of the positive drug group.

[0097] 2. Mouse experiments were conducted on the pharmaceutical composition prepared according to the present invention:

[0098] Experimental animals: Male C57BL / 6 mice, weighing 20–25g.

[0099] Experimental groups (n=10) were formed for the pharmaceutical compositions and positive control drugs prepared in Examples 1-3, Comparative Examples 1-3, and respectively:

[0100] Normal control group: fed with ordinary feed (5% fat content).

[0101] Model control group: fed a high-fat diet (basal diet + 18% lard + 1.5% cholesterol + 0.3% sodium cholate).

[0102] Experimental group 1: fed with a high-fat diet and simultaneously administered the drug composition prepared in Example 1 by gavage (dosage 100 mg / kg).

[0103] Experimental group 2: fed with a high-fat diet and simultaneously administered the drug composition prepared in Example 2 by gavage (dosage: 100 mg / kg).

[0104] Experimental group 3: fed with a high-fat diet and simultaneously administered the drug composition prepared in Example 3 by gavage (dosage: 100 mg / kg).

[0105] Experimental group 4: fed with a high-fat diet and simultaneously administered the drug composition prepared in Example 3 by gavage (dose of 200 mg / kg).

[0106] Experimental group 5: fed with a high-fat diet and simultaneously administered the drug composition prepared in Comparative Example 1 by gavage (dose of 200 mg / kg).

[0107] Experimental group 6: fed with a high-fat diet and simultaneously administered the drug composition prepared in Comparative Example 2 by gavage (dose of 200 mg / kg).

[0108] Experimental group 7: fed with a high-fat diet and simultaneously administered the drug composition prepared in Comparative Example 3 by gavage (dose of 200 mg / kg).

[0109] Positive control group: fed a high-fat diet and administered atorvastatin by gavage (prepared as a 1 mg / mL suspension with physiological saline before use, dose 10 mg / kg).

[0110] Experimental methods: After a one-week acclimatization period, mice in each group were treated according to the above groupings for four consecutive weeks. Following the last administration, mice were fasted for 12 hours, and blood was collected from the orbital sinus. Serum was separated, and the levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) in the serum were detected using an automated biochemical analyzer. The results are shown in Table 2.

[0111] Table 2 shows the levels of total cholesterol, triglycerides, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol in each group.

[0112] Group Name TC (mmol / L) TG (mmol / L) LDL-C (mmol / L) HDL-C (mmol / L) normal control group 3.12±0.21 1.24±0.19 1.54±0.16 1.44±0.10 Model control group 6.95±0.25 3.61±0.46 4.32±0.21 0.81±0.16 Experimental group 1 4.02±0.25 1.62±0.16 2.10±0.19 1.20±0.22 Experimental group 2 4.01±0.16 1.68±0.30 2.05±0.26 1.20±0.18 Experimental group 3 3.94±0.26 1.51±0.42 2.07±0.43 1.19±0.20 Experimental group 4 3.46±0.30 1.37±0.24 1.84±0.14 1.36±0.19 Experimental group 5 4.95±0.45 2.09±0.41 2.91±0.43 0.98±0.24 Experimental group 6 5.06±0.37 2.26±0.34 2.89±0.15 0.92±0.13 Experimental group 7 5.87±0.42 2.45±0.27 3.12±0.04 0.90±0.21 Positive control group 3.81±0.24 1.49±0.24 1.99±0.20 1.30±0.15

[0113] The results showed that the pharmaceutical composition prepared in the embodiments of the present invention could significantly reduce the serum total cholesterol, triglycerides, and low-density lipoprotein cholesterol levels in mice induced by a high-fat diet, and increase the high-density lipoprotein cholesterol level. The effect was more significant with increasing dosage. Meanwhile, no obvious adverse reactions such as diarrhea, vomiting, or abnormal weight loss were observed in the experimental group mice. Their diet and activity were good, indicating that the pharmaceutical composition of the present invention has good safety.

[0114] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A lipid-lowering drug composition, characterized in that, By mass, it includes the following components: Spirulina extract 10-20 parts, grape seed proanthocyanidins 5-15 parts, conjugated linoleic acid 3-10 parts, phytosterols 2-8 parts and coenzyme Q10 1-5 parts; The preparation method of the lipid-lowering drug composition includes the following steps: (1) Preparation of liposomes of spirulina extract and coenzyme Q10: Soybean lecithin and cholesterol were mixed at a mass ratio of 3:1 and dissolved in anhydrous ethanol to prepare an organic phase with a concentration of 20 mg / mL; Spirulina extract and coenzyme Q10 were mixed and dissolved in PBS to prepare an aqueous phase; Under a water bath at 55-65℃, the organic phase was slowly added dropwise to the aqueous phase and stirred to form a primary emulsion; The ethanol in the primary emulsion was removed under reduced pressure to form a liposome suspension; The liposome suspension was homogenized under high pressure to obtain liposomes; (2) Preparation of grape seed proanthocyanidins and phytosterol microspheres: Grape seed proanthocyanidins and phytosterols were mixed, and an anhydrous ethanol solution of chitosan was added. The mixture was ultrasonically dispersed to form a drug-chitosan mixture. Under stirring conditions of 50℃ and 200r / min, the drug-chitosan mixture was slowly added dropwise to a medium-chain triglyceride containing 3% Span80 and stirred for 20-30 min to form a W / O type emulsion. Genipin solution was added dropwise to the W / O type emulsion and stirring was continued for crosslinking for 5-7 h. Anhydrous ethanol was added to break the emulsion, and the mixture was washed with 50% ethanol solution, then washed with purified water and dried to obtain microspheres. (3) Inclusion complex of conjugated linoleic acid: Conjugated linoleic acid was added dropwise to an aqueous solution of β-cyclodextrin while stirring magnetically at 400 r / min for 2 h. Then the mixture was cooled to room temperature and refrigerated at 4 °C for 12 h. The precipitate was collected by filtration, washed with anhydrous ethanol, and dried to obtain the inclusion complex. (4) The above liposomes, microspheres and inclusion complexes are mixed to obtain a lipid-lowering drug composition.

2. The lipid-lowering drug composition according to claim 1, characterized in that, In step (1), the pH of PBS is 7.4; the concentration of spirulina extract in the aqueous phase is 15 mg / mL.

3. The lipid-lowering drug composition according to claim 1, characterized in that, In step (1), the dropping rate of the organic phase is 1 mL / min, and the stirring is magnetic stirring at 300 r / min.

4. The lipid-lowering drug composition according to claim 1, characterized in that, In step (1), the primary emulsion is subjected to rotary evaporation at 40°C and 0.08 MPa to remove ethanol; the liposome suspension is homogenized three times under 100 MPa pressure to obtain liposomes.

5. The lipid-lowering drug composition according to claim 1, characterized in that, In step (2), the concentration of the chitosan solution is 2%; the volume ratio of the drug-chitosan mixture and the medium-chain triglyceride containing 3% Span80 is 6:

1.

6. The lipid-lowering drug composition according to claim 1, characterized in that, In step (2), the concentration of the genipin solution is 10%, the solvent is anhydrous ethanol, the amount of genipin used is 10-15% of the mass of chitosan, and the crosslinking temperature is 60℃.

7. The lipid-lowering drug composition according to claim 1, characterized in that, In step (2), three times the volume of anhydrous ethanol is added to break the emulsion.

8. The lipid-lowering drug composition according to claim 1, characterized in that, In step (3), the concentration of the β-cyclodextrin aqueous solution is 5%, the mass ratio of conjugated linoleic acid to β-cyclodextrin is 1:8, and the magnetic stirring temperature is 55℃.

9. A lipid-lowering drug, characterized in that, The active ingredient of the drug is the lipid-lowering drug composition according to any one of claims 1 to 8; the drug also includes a pharmaceutically acceptable carrier or excipient.