Preparation method of quercetin liposome

Abstract

The invention relates to a preparation method of quercetin liposome, which comprises the following steps: (1) adding phospholipid, cholesterol, phospholipid polyethylene glycol and quercetin into ethanol, and dissolving to obtain an ethanol solution containing quercetin; (2) dropwise adding the quercetin-containing ethanol solution prepared in the step (1) into water to obtain a quercetin liposome-containing solution; (3) removing ethanol in the quercetin-containing liposome solution obtained in the step (2) in an aqueous medium to obtain a quercetin-containing liposome aqueous solution; and (4) performing ultrasonic treatment on the aqueous solution containing the quercetin liposome prepared in the step (3) to obtain the quercetin liposome. According to the invention, toxic and harmful organic solvents used in traditional methods are avoided, and ethanol is used as a solvent, so that a safer and more environment-friendly preparation process is realized. The prepared liposome has high encapsulation efficiency and good solubility, and the market application prospect of the quercetin liposome is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a method for preparing quercetin liposomes. Background Art

[0002] Quercetin is a flavonoid compound widely found in nature, with multiple biological activities such as antioxidant, anti-inflammatory, anti-tumor, and cardiovascular protection. These activities make quercetin have a wide range of application potential in clinical and health care fields. However, quercetin has poor stability and low water solubility, resulting in low bioavailability of pure quercetin, thus limiting its application in medicine and scientific research.

[0003] Liposome technology provides an effective way to solve the problems of quercetin stability and water solubility. By encapsulating quercetin in liposomes, its stability and bioavailability in vivo can be significantly enhanced. Therefore, developing a new method for preparing quercetin liposomes can not only overcome the limitations of traditional methods, but also broaden the application range of quercetin and give it greater potential in application.

[0004] In the prior art, the preparation method of quercetin liposomes is mainly "thin film dispersion method". For example, in Chinese patent application CN108478534A, lipid components and quercetin are dissolved in a mixed solvent of chloroform and methanol, and an organic film is formed by rotary evaporation. After hydration, a liposome solution is obtained, which is further processed for use. The preparation method of this patent application requires the use of organic solvents chloroform and methanol to form a film. However, the use of chloroform and methanol has the following problems:

[0005] (1) Toxicity and safety issues: Organic solvents chloroform and methanol are toxic to a certain extent and pose potential hazards to operators and the environment. In addition, the chloroform and methanol solvent vapors in the working environment may cause damage to the human body.

[0006] (2) Environmental pollution: The use of organic solvents may cause environmental pollution. They may release volatile organic compounds (VOCs) during the production process, causing pollution to the atmosphere and water resources. In addition, improper disposal of waste organic solvents may cause environmental pollution, further increasing the environmental burden.

[0007] (3) High cost: Organic solvents are expensive to purchase and require additional safety facilities and operating procedures during handling, which increases the cost and complexity of the preparation process.

[0008] The "thin film dispersion method" adopted by Chinese patent application CN116440081A requires the use of an organic reagent containing chloroform as a solvent, and an organic film is formed after vacuum rotary evaporation, and then it needs to be hydrated and emulsified at a speed of 6000-8000r / min for 30min and stabilized for 2-3h. Its operation is cumbersome, time-consuming, costly, and toxic, and requires additional safety facilities and operating procedures, which increases production costs and complexity. Therefore, a method for preparing environmentally friendly and safe quercetin liposomes is needed. Summary of the invention

[0009] In order to overcome the deficiencies of the prior art, the present invention provides an innovative method for preparing quercetin liposomes, which makes the preparation of quercetin liposomes safer and more environmentally friendly and reduces costs, thereby improving the application of quercetin in biomedicine and expanding its application in medicine and health products.

[0010] The purpose of the present invention is achieved through the following technical solutions.

[0011] The present invention provides a method for preparing quercetin liposomes, which comprises the following steps:

[0012] (1) adding phospholipids, cholesterol, phospholipid polyethylene glycol and quercetin into ethanol and dissolving them to obtain an ethanol solution containing quercetin;

[0013] (2) adding the quercetin-containing ethanol solution prepared in step (1) dropwise into water to obtain a solution containing quercetin liposomes;

[0014] (3) removing ethanol from the quercetin-containing liposome solution obtained in step (2) in an aqueous medium to obtain an aqueous solution of quercetin-containing liposomes;

[0015] (4) Ultrasonicate (e.g., probe ultrasonicator) the aqueous solution containing quercetin liposomes prepared in step (3) to obtain quercetin liposomes.

[0016] Preferably, in step (1), the phospholipid is selected from one or more of egg yolk lecithin, soybean lecithin, sodium dioleoylphosphatidylserine (DOPS), dioleoyl L-α-phosphatidylethanolamine (DOPE), 1,2-dioleoyl phosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine (DSPC), preferably egg yolk lecithin, soybean lecithin, DOPC and DPPC.

[0017] Preferably, in step (1), the phospholipid polyethylene glycol is selected from one or more of phospholipid polyethylene glycols having a number average molecular weight of 500, 1000 and 2000, preferably phospholipid polyethylene glycol having a molecular weight of 2000.

[0018] Preferably, in step (1), the molar ratio of the phospholipid, the cholesterol, the phospholipid polyethylene glycol and the quercetin is 1:0.2-1:0-0.05:0.1-0.3, preferably 1:0.5-1:0.015-0.05:0.1-0.2.

[0019] Preferably, in step (2), the volume ratio between the quercetin-containing ethanol solution prepared in step (1) and water is 1:3-12, preferably 1:3-10.

[0020] Preferably, in step (2), the molar concentration of phospholipids in the solution containing quercetin liposomes is 4-12 mmol / L, preferably 5-10 mmol / L.

[0021] Preferably, in step (3), ethanol in the solution containing quercetin liposomes obtained in step (2) is removed by dialysis or ultrafiltration.

[0022] Preferably, the dialysis is performed using a dialysis bag or an ultrafiltration tube with a molecular weight cut-off of 1000-10000Da.

[0023] Preferably, in step (3), the aqueous medium is water, a glucose aqueous solution with a mass fraction of 5-10%, a sodium chloride aqueous solution with a mass fraction of 0.9%, or a phosphate buffer solution with a pH value of 7.4.

[0024] Preferably, in step (4), the power of the ultrasound is 100-300W, preferably 100-200W.

[0025] Preferably, in step (4), the ultrasonication time is 1-10 minutes, preferably 1-5 minutes.

[0026] Compared with the existing preparation methods of similar products, the method of the present invention does not need to use highly toxic and costly organic solvents such as chloroform and methanol, thereby reducing potential hazards to operators and the environment during the production process, and has the characteristics of safety, environmental protection and low preparation cost.

[0027] The present invention simplifies the preparation process by using ethanol as a solvent and combining it with an aqueous medium, avoids complicated emulsification and high-speed rotation steps, and reduces the complexity and time cost of the operation.

[0028] The present invention ensures the particle size and quality of the liposomes through operations such as dialysis or ultrafiltration, ultrasound, etc., overcomes the shortcomings of the traditional method, reduces the preparation cost of the quercetin liposomes, and improves the production practicability of the quercetin liposomes.

[0029] The method of the present invention overcomes the safety and cost issues in the traditional film dispersion method, improves the application of quercetin in the field of biomedicine, ensures safety and environmental protection in the production process, and provides a new direction for industry production. The preparation method provides a more optimized liposome preparation scheme, has high practical value in research and production, and is helpful to further exert and study the biological activity of quercetin.

[0030] The quercetin liposomes obtained by the method of the present invention have high encapsulation rate, for example, the encapsulation rate can reach more than 90%, uniform particle size distribution, for example, the particle size distribution index is below 0.3, good solubility, and great market application prospects.

[0031] The quercetin liposomes prepared by the present invention can exert biological activities such as anti-oxidation, anti-inflammatory, anti-tumor and cardiovascular protection in the field of medicine and health care, improve the practical value of quercetin in related fields, and have good market application prospects. The quercetin liposomes of the present invention can be applied to the pharmaceutical industry to develop quercetin drug products, including various therapeutic dosage forms such as anti-inflammatory, anti-oxidative and anti-tumor. The quercetin liposomes of the present invention can be used for scientific research experiments to study the biological effects of quercetin in cells and animal models, and improve the accuracy and practicality of the research. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The particle size characterization results of quercetin liposomes in Example 1;

[0033] Figure 2 Electron microscopic characterization results of quercetin liposomes in Example 2;

[0034] Figure 3 The stability test results of quercetin liposomes in Example 3. DETAILED DESCRIPTION

[0035] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies realized based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0036] In the following examples:

[0037] Particle size characterization: The particle size and particle size distribution index of quercetin liposomes were determined using a laser particle size analyzer.

[0038] Transmission electron microscopy was used to observe the size and shape of quercetin liposomes.

[0039] Stability test: The stability of quercetin liposomes was determined using a laser particle size analyzer, and the particle size was measured once a day for one week.

[0040] Example 1

[0041] Soy lecithin (0.06 mmol), cholesterol (0.03 mmol), phospholipid PEG (0.003 mmol) and quercetin (0.006 mmol) were added into an eggplant-shaped bottle containing 2 mL of ethanol, and all the raw materials were dissolved under magnetic stirring to obtain an ethanol solution containing quercetin;

[0042] The quercetin-containing ethanol solution was added dropwise into 8 mL of water and stirred at room temperature for half an hour to obtain a solution containing quercetin liposomes;

[0043] In a PBS solution with a concentration of 0.01 M, the liposome solution containing quercetin was dialyzed through a dialysis bag with a molecular weight cutoff of 3000 Da to remove ethanol, thereby obtaining an aqueous solution containing quercetin liposomes;

[0044] The aqueous solution containing quercetin liposomes was transferred to a 50 mL centrifuge tube and ultrasonicated for 5 minutes using a probe ultrasonicator (ultrasonic power: 100 W) to obtain a quercetin liposome aqueous solution with a particle size of 95 nm, a particle size distribution index of 0.19, and a quercetin encapsulation efficiency of 95%. Figure 1 The particle size characterization results of quercetin liposomes.

[0045] Example 2

[0046] 1,2-dioleoylphosphatidylcholine (DOPC) (0.06 mmol), cholesterol (0.03 mmol), phospholipid polyethylene glycol with a number average molecular weight of 2000 (0.003 mmol) and quercetin (0.012 mmol) were added into an eggplant-shaped bottle containing 2 mL of ethanol, and all the raw materials were dissolved under magnetic stirring to obtain an ethanol solution containing quercetin;

[0047] The quercetin-containing ethanol solution was added dropwise into 10 mL of water under stirring, and stirring was continued at room temperature for half an hour to obtain a solution containing quercetin liposomes;

[0048] In a glucose aqueous solution with a mass concentration of 5%, the solution containing quercetin liposomes is filtered through an ultrafiltration tube with a molecular weight cutoff of 10000Da to remove ethanol, thereby obtaining an aqueous solution containing quercetin liposomes;

[0049] The aqueous solution containing quercetin liposomes was transferred to a 50 mL centrifuge tube and ultrasonicated for 3 minutes by a probe ultrasonicator (ultrasonic power: 150 W) to obtain a quercetin liposome aqueous solution with a particle size of 92 nanometers, a particle size distribution index of 0.22, and a quercetin encapsulation efficiency of 93%.

[0050] Example 3

[0051] Dipalmitoylphosphatidylcholine (DPPC) (0.06 mmol), cholesterol (0.06 mmol), phospholipid polyethylene glycol with a number average molecular weight of 2000 (0.0009 mmol) and quercetin (0.012 mmol) were added into an eggplant-shaped bottle containing 2 mL of ethanol, and all the raw materials were dissolved under magnetic stirring to obtain an ethanol solution containing quercetin;

[0052] The quercetin-containing ethanol solution was added dropwise into 10 mL of water under stirring, and stirring was continued at room temperature for half an hour to obtain a solution containing quercetin liposomes;

[0053] In a glucose aqueous solution with a mass concentration of 5%, the solution containing quercetin liposomes is filtered through an ultrafiltration tube with a molecular weight cutoff of 5000Da to remove ethanol, thereby obtaining an aqueous solution containing quercetin liposomes;

[0054] The aqueous solution containing quercetin liposomes was transferred to a 50 mL centrifuge tube and ultrasonicated for 1 minute using a probe ultrasonicator (ultrasonic power: 200 W) to obtain an aqueous solution of quercetin liposomes with a particle size of 90 nanometers, a particle size distribution index of 0.20, and a quercetin encapsulation rate of 95%.

[0055] Example 4

[0056] Egg yolk lecithin (0.06 mmol), cholesterol (0.06 mmol), phospholipid polyethylene glycol with a number average molecular weight of 2000 (0.003 mmol) and quercetin (0.006 mmol) were added into an eggplant-shaped bottle containing 2 mL of ethanol, and all the raw materials were dissolved under magnetic stirring to obtain an ethanol solution containing quercetin;

[0057] The quercetin-containing ethanol solution was added dropwise into 10 mL of water under stirring, and stirring was continued at room temperature for half an hour to obtain a solution containing quercetin liposomes;

[0058] In a phosphate buffer solution having a pH value of 7.4, the solution containing quercetin liposomes is filtered through an ultrafiltration tube having a molecular weight cut-off of 3000 Da to remove ethanol, thereby obtaining an aqueous solution containing quercetin liposomes;

[0059] The aqueous solution containing quercetin liposomes was transferred to a 50 mL centrifuge tube and ultrasonicated for 2 minutes (ultrasonic power: 150 W) by a probe ultrasonicator to obtain a quercetin liposome aqueous solution with a particle size of 98 nanometers, a particle size distribution index of 0.30, and a quercetin encapsulation rate of 90%.

[0060] Comparative Example 1

[0061] 1,2-dioleoylphosphatidylcholine (DOPC) (0.06 mmol), cholesterol (0.03 mmol), phospholipid polyethylene glycol with a number average molecular weight of 2000 (0.003 mmol) and quercetin (0.012 mmol) were added into an eggplant-shaped bottle filled with 2 mL of dichloromethane, and all the raw materials were dissolved under magnetic stirring to obtain a dichloromethane solution containing quercetin;

[0062] The dichloromethane solution containing quercetin was added dropwise into 6 mL of 5% glucose aqueous solution and stirred at room temperature for half an hour. The solution was emulsified and separated into upper and lower layers after standing for a period of time, and no quercetin liposome solution was formed.

[0063] Comparative Example 2

[0064] 1,2-dioleoylphosphatidylcholine (DOPC) (0.06 mmol), cholesterol (0.03 mmol), phospholipid polyethylene glycol with a number average molecular weight of 2000 (0.003 mmol) and quercetin (0.012 mmol) were added into an eggplant-shaped bottle containing 2 mL of ethanol, and all the raw materials were dissolved under magnetic stirring to obtain an ethanol solution containing quercetin;

[0065] The quercetin-containing ethanol solution was added dropwise into 1 mL of water and stirred at room temperature for half an hour, but no solution containing quercetin liposomes was obtained.

[0066] Comparative Example 3

[0067] Soy lecithin (0.06 mmol), cholesterol (0.12 mmol), phospholipid PEG (0.003 mmol) and quercetin (0.001 mmol) were added into an eggplant-shaped bottle containing 1 mL of ethanol, and all the raw materials were dissolved under magnetic stirring to obtain an ethanol solution containing quercetin;

[0068] The quercetin-containing ethanol solution was added dropwise into 4 mL of water and stirred at room temperature for half an hour to obtain a solution containing quercetin liposomes;

[0069] In a PBS solution with a concentration of 0.01 M, the liposome solution containing quercetin was dialyzed through a dialysis bag with a molecular weight cutoff of 3000 Da to remove ethanol, thereby obtaining an aqueous solution containing quercetin liposomes;

[0070] The aqueous solution containing quercetin liposomes was transferred to a 50 mL centrifuge tube and ultrasonicated for 5 minutes using a probe ultrasonicator (ultrasonic power: 100 W) to obtain a quercetin liposome aqueous solution with a particle size of 232 nm, a particle size distribution index of 0.35, and a quercetin encapsulation efficiency of 77%.

Claims

1. A method for preparing quercetin liposomes, the method comprising the following steps: (1) adding phospholipids, cholesterol, phospholipid polyethylene glycol and quercetin into ethanol and dissolving them to obtain an ethanol solution containing quercetin; (2) adding the quercetin-containing ethanol solution prepared in step (1) dropwise into water to obtain a solution containing quercetin liposomes; (3) removing ethanol from the quercetin-containing liposome solution obtained in step (2) in an aqueous medium to obtain an aqueous solution of quercetin-containing liposomes; (4) Ultrasonicate (e.g., probe ultrasonicator) the aqueous solution containing quercetin liposomes prepared in step (3) to obtain quercetin liposomes.

2. The preparation method according to claim 1, wherein In step (1), the phospholipid is selected from one or more of egg yolk lecithin, soybean lecithin, sodium dioleoylphosphatidylserine (DOPS), dioleoyl L-α-phosphatidylethanolamine (DOPE), 1,2-dioleoyl phosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine (DSPC), preferably egg yolk lecithin, soybean lecithin, DOPC, and DPPC.

3. The preparation method according to claim 1 or 2, wherein In step (1), the phospholipid polyethylene glycol is selected from one or more of phospholipid polyethylene glycols having a number average molecular weight of 500, 1000 and 2000, preferably phospholipid polyethylene glycol having a molecular weight of 2000.

4. The preparation method according to any one of claims 1 to 3, wherein In step (1), the molar ratio of the phospholipid, the cholesterol, the phospholipid polyethylene glycol and the quercetin is 1:0.2-1:0-0.05:0.1-0.3, preferably 1:0.5-1:0.015-0.05:0.1-0.

2.

5. The preparation method according to any one of claims 1 to 4, wherein In step (2), the volume ratio between the quercetin-containing ethanol solution prepared in step (1) and water is 1:3-12, preferably 1:3-10.

6. The preparation method according to any one of claims 1 to 5, wherein In step (2), the molar concentration of phospholipids in the solution containing quercetin liposomes is 4-12 mmol / L, preferably 5-10 mmol / L.

7. The preparation method according to any one of claims 1 to 6, wherein In step (3), ethanol in the solution containing quercetin liposomes obtained in step (2) is removed by dialysis or ultrafiltration.

8. The preparation method according to any one of claims 1 to 7, wherein The dialysis is performed using a dialysis bag or an ultrafiltration tube with a molecular weight cutoff of 1000-10000Da.

9. The preparation method according to any one of claims 1 to 8, wherein In step (3), the aqueous medium is water, a glucose aqueous solution with a mass fraction of 5-10%, a sodium chloride aqueous solution with a mass fraction of 0.9%, or a phosphate buffer solution with a pH value of 7.

4.

10. The preparation method according to any one of claims 1 to 9, wherein In step (4), the power of the ultrasound is 100-300 W, preferably 100-200 W; Preferably, in step (4), the ultrasonication time is 1-10 minutes, preferably 1-5 minutes.

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