Berberine liposome and preparation method thereof

Abstract

The invention discloses berberine liposome and a preparation method thereof. The preparation method comprises the following steps: (1) dissolving soybean lecithin, cholesterol and DSPE-PEG2000-COOH in an organic solvent according to a molar ratio of (8-12): (4-8): (0.5-1); (2) carrying out rotary evaporation to dryness on the mixed solution, and forming a phospholipid-cholesterol film on the bottle wall after the organic solvent is evaporated to dryness; (3) adding a PBS (Phosphate Buffer Solution) into the bottle, dissolving the phospholipid-cholesterol membrane, and hydrating; (4) adding a berberine solution into the blank liposome suspension, carrying out ultrasonic treatment, adjusting the pH value of the suspension to 6.5-7.2, incubating at 35-45 DEG C overnight, carrying out suction filtration, and homogenizing by using a high-pressure homogenizer; and (5) quickly freezing the obtained lipidosome with liquid nitrogen, and freeze-drying to obtain the berberine lipidosome. According to the invention, the encapsulation efficiency of the berberine liposome can be obviously improved (greater than 80%); the method is simple in process, easy in large-scale production and beneficial to industrialization.

Description

Technical Field

[0001] The invention relates to the technical field of drug carrier preparation, and in particular to a berberine liposome and a preparation method thereof. Background Art

[0002] Coptis chinensis is a medicinal plant of the Ranunculaceae family. Berberine, also known as berberine, is the active ingredient in the traditional Chinese medicine Coptis chinensis. It is an isoquinoline alkaloid that can inhibit or kill a variety of Gram-negative bacteria, Gram-positive bacteria, and Mycobacterium tuberculosis. Its clinical application is mainly to treat diarrhea caused by intestinal bacterial infection. Berberine has a hypoglycemic effect by resisting hyperglycemic hormones and promoting insulin cell regeneration and functional recovery. In addition, berberine has many new clinical uses. It has a definite effect on metabolic diseases such as hyperlipidemia. It also has anti-inflammatory, anti-tumor and immunomodulatory effects, and can be used as an adjuvant treatment for tumors and inflammatory bowel disease.

[0003] Liposome is a new drug formulation technology with a structure similar to that of cell membrane. As a drug carrier that has been widely studied in recent years, its main features are that it can protect the encapsulated drug, increase drug stability, change the distribution behavior of the drug in the body, and carry the drug to the lesion site passively or actively. It is a closed vesicle formed by lipid materials (such as phospholipids) dispersed in an aqueous medium, which has targeting and sustained release effects, is suitable for degradation in the body, and can reduce the dosage of the drug and reduce toxic side effects. Therefore, the application of liposomes has become more and more extensive.

[0004] Berberine has a high polarity and is difficult to be absorbed through the gastrointestinal tract to reach an effective therapeutic concentration; however, berberine liposomes have a strong affinity with tissue cells and can improve drug absorption and distribution. At present, the main methods for preparing berberine liposomes include thin film dispersion method, reverse evaporation method, solvent injection method, pH gradient method, ammonium sulfate gradient method, thin film dispersion-pH gradient method, supercritical CO2 method, etc.

[0005] The berberine liposomes currently prepared have the disadvantages of residual organic solvents in the product, resulting in reduced safety of the liposomes, low encapsulation efficiency, and long time consumption. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the technical defects of the background technology and provide a berberine liposome and a preparation method thereof. The present invention can significantly improve the encapsulation rate of the berberine liposome (>80%); the present invention has a simple process, does not require complex instruments and equipment, is easy to scale up production, and is conducive to industrialization; the present invention can reduce the use of solvents and reduce costs.

[0007] The technical solution adopted by the present invention to solve the above technical problems is as follows:

[0008] A method for preparing berberine liposomes comprises the following steps:

[0009] (1) dissolving soybean lecithin, cholesterol and DSPE-PEG2000-COOH in an organic solvent at a molar ratio of 8-12:4-8:0.5-1 to obtain a mixed solution;

[0010] (2) rotating the mixed solution to dryness, and forming a phospholipid-cholesterol membrane on the bottle wall after the organic solvent is evaporated;

[0011] (3) Add PBS buffer to the bottle to dissolve the phospholipid-cholesterol membrane and hydrate it to obtain a blank liposome suspension;

[0012] (4) adding berberine solution to the blank liposome suspension, ultrasonically adjusting the pH of the suspension to 6.5-7.2, incubating at 35-45° C. overnight, filtering, and homogenizing with a high-pressure homogenizer to obtain liposomes;

[0013] (5) The obtained liposomes are quickly frozen with liquid nitrogen and freeze-dried to obtain berberine liposomes.

[0014] Preferably, in step (1), the dissolving temperature is 35-40°C.

[0015] Preferably, in step (1), the organic solvent is any one or both of chloroform and methanol.

[0016] Preferably, in step (2), the evaporation temperature is 40-45°C.

[0017] Preferably, in step (3), the pH of the PBS buffer is 5.5-7.

[0018] Preferably, in step (3), the hydration time is 1 to 4 hours.

[0019] Preferably, in step (4), an equal volume of 1 mg / ml berberine solution is added to the blank liposome suspension.

[0020] Preferably, in step (4), the power of the ultrasound is 400 W, the time is 8 min, and the ultrasound is performed once every 2 s.

[0021] Preferably, in step (4), the pore size of the filter membrane used in the filtration is 0.22 μm.

[0022] Preferably, in step (4), the homogenization pressure is 20 to 100 MPa and the time is 1 to 3 hours.

[0023] A berberine liposome is prepared by using the above-mentioned method for preparing the berberine liposome.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The present invention can shorten the preparation time of berberine liposomes and significantly improve the encapsulation efficiency of berberine liposomes (>80%);

[0026] (2) The process of the present invention is simple, does not require complicated instruments and equipment, is easy to scale up for production, and is conducive to industrialization;

[0027] (3) The present invention can reduce the use of solvents and reduce costs. DETAILED DESCRIPTION

[0028] In order to better understand the content of the present invention, further description is given below in conjunction with specific embodiments. It should be understood that these embodiments are only used to further illustrate the present invention, and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content described in the present invention, those skilled in the art make some non-essential changes or adjustments to the present invention, which still belong to the protection scope of the present invention.

[0029] Example 1

[0030] A method for preparing berberine liposomes, comprising the following steps:

[0031] (1) Dissolve soybean lecithin, cholesterol and DSPE-PEG2000-COOH in methanol at a molar ratio of 10:6:1 at 35° C. to obtain a mixed solution;

[0032] (2) The mixed solution is subjected to rotary evaporation at 40°C to evaporate methanol, and a phospholipid-cholesterol film is formed on the bottle wall after the methanol is evaporated;

[0033] (3) Add PBS buffer at pH 6.5 to the bottle to dissolve the phospholipid-cholesterol membrane and hydrate for 3 h to obtain a blank liposome suspension;

[0034] (4) Add an equal volume of 1 mg / ml berberine solution to the blank liposome suspension, sonicate at 400 W for 8 min, sonicate once every 2 s, adjust the pH of the suspension to 6.8, incubate at 42°C overnight, filter through a 0.22 μm filter membrane with a suction filter, and homogenize with a high-pressure homogenizer for 2 h at a pressure of 50 MPa to obtain liposomes;

[0035] (5) The obtained liposomes were quickly frozen with liquid nitrogen and freeze-dried to obtain berberine liposomes; after testing, the encapsulation efficiency of the berberine liposomes was 85.7%.

[0036] Example 2

[0037] A method for preparing berberine liposomes, comprising the following steps:

[0038] (1) Dissolve soybean lecithin, cholesterol and DSPE-PEG2000-COOH in methanol at a molar ratio of 8:4:0.5 at 40° C. to obtain a mixed solution;

[0039] (2) The mixed solution is subjected to rotary evaporation at 45°C to evaporate methanol, and a phospholipid-cholesterol film is formed on the bottle wall after the methanol is evaporated;

[0040] (3) Add PBS buffer at pH 5.5 to the bottle to dissolve the phospholipid-cholesterol membrane and hydrate for 1 h to obtain a blank liposome suspension;

[0041] (4) Add an equal volume of 1 mg / ml berberine solution to the blank liposome suspension, sonicate at 400 W for 8 min, sonicate once every 2 s, adjust the pH of the suspension to 6.5, incubate at 35°C overnight, filter through a 0.22 μm filter membrane with a suction filter, and homogenize with a high-pressure homogenizer for 2 h at a pressure of 20 MPa to obtain liposomes;

[0042] (5) The obtained liposomes were quickly frozen with liquid nitrogen and freeze-dried to obtain berberine liposomes; and the encapsulation efficiency of the berberine liposomes was found to be 83.3%.

[0043] Example 3

[0044] A method for preparing berberine liposomes, comprising the following steps:

[0045] (1) Dissolve soybean lecithin, cholesterol and DSPE-PEG2000-COOH in chloroform at a molar ratio of 12:8:1 at 40° C. to obtain a mixed solution;

[0046] (2) The mixed solution is subjected to rotary evaporation at 45°C to evaporate the chloroform, and a phospholipid-cholesterol film is formed on the bottle wall after the chloroform is evaporated;

[0047] (3) Add PBS buffer at pH 7.0 to the bottle to dissolve the phospholipid-cholesterol membrane and hydrate for 4 h to obtain a blank liposome suspension;

[0048] (4) Add an equal volume of 1 mg / ml berberine solution to the blank liposome suspension, sonicate at 400 W for 8 min, sonicate once every 2 s, adjust the pH of the suspension to 7.2, incubate at 45°C overnight, filter through a 0.22 μm filter membrane with a suction filter, and homogenize with a high-pressure homogenizer for 3 h at a pressure of 100 MPa to obtain liposomes;

[0049] (5) The obtained liposomes were quickly frozen with liquid nitrogen and freeze-dried to obtain berberine liposomes. After testing, the encapsulation efficiency of the berberine liposomes was 82.6%.

[0050] Comparative Example 1

[0051] Compared with Example 1, the only difference is that in step (1), the molar ratio of soybean lecithin, cholesterol and DSPE-PEG2000-COOH is 7:6:1; and the encapsulation efficiency of the prepared berberine liposome is 70.2%.

[0052] Comparative Example 2

[0053] Compared with Example 1, the only difference is that in step (4), ultrasound is applied and the pH of the suspension is adjusted to 6.0; the encapsulation efficiency of the prepared berberine liposome is 27.4%.

[0054] Comparative Example 3

[0055] Compared with Example 1, the only difference is that in step (4), ultrasound is applied and the pH of the suspension is adjusted to 7.5; the encapsulation efficiency of the prepared berberine liposome is 20.5%.

[0056] Comparative Example 4

[0057] Compared with Example 1, the only difference is that in step (4), the incubation is carried out at 30° C. overnight; the encapsulation efficiency of the prepared berberine liposomes is 28.1%.

[0058] Comparative Example 5

[0059] Compared with Example 1, the only difference is that in step (4), the incubation is carried out at 50° C. overnight; the encapsulation efficiency of the prepared berberine liposomes is 31.7%.

[0060] The detection method of the berberine liposomes prepared in Examples 1 to 3 and Comparative Examples 1 to 5 is as follows:

[0061] 1. Berberine content determination conditions

[0062] 1.1 Column conditions

[0063] Chromatographic column: Diamonsil C18 (200mm×4.6mm, 5μm); mobile phase: acetonitrile-0.05mol / ml KH2PO4 (35:65), adjusted to pH3.2 with H3PO4; flow rate: 1.0ml / min; column temperature: 30℃; detection wavelength: 345nm, injection 10μl.

[0064] 1.2 Preparation of reference solution and sample solution

[0065] Accurately weigh an appropriate amount of berberine reference substance dried to constant weight at 105°C, add methanol to make a 8μg / ml solution, filter with a 0.45μm microporous filter membrane, and take the filtrate to obtain. Accurately measure 0.2ml of berberine liposomes in a 10ml volumetric bottle, dissolve in methanol and dilute to the scale, filter with a 0.45μm microporous filter membrane, and take the filtrate to obtain.

[0066] 1.3 Determination of the maximum absorption wavelength of berberine

[0067] Accurately measure an appropriate amount of berberine standard into a 10 ml volumetric flask, dissolve it with methanol solution and make up to the scale. Using methanol as a blank, perform UV scanning in the wavelength range of 200-400 nm and determine that 345 nm is selected as the maximum absorption wavelength.

[0068] 2. Establishment of Encapsulation Efficiency Determination Method

[0069] The encapsulation efficiency of berberine was determined by Sephadex-G50 column (1cm×20cm) method. 0.2ml of berberine liposome suspension was accurately measured and loaded onto the column, eluted with PBS, and the yellow milky band (4-10ml) was collected with a 10ml volumetric bottle. When it completely passed, stop collecting, add methanol to the scale, ultrasonically break the emulsion for 5min, and obtain the liposome sample solution; 0.2ml of berberine liposome suspension was accurately measured in a 10ml volumetric bottle, and methanol was adjusted to the scale, ultrasonically break the emulsion for 5min, and obtain the total sample solution; 10μl of the liposome and total sample solutions were injected, and the peak area was recorded to calculate the encapsulation efficiency of berberine: EE%=W package / W total×100%.

[0070] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A method for preparing berberine liposomes, characterized in that: The steps include: (1) dissolving soybean lecithin, cholesterol and DSPE-PEG2000-COOH in an organic solvent at a molar ratio of 8-12:4-8:0.5-1 to obtain a mixed solution; (2) rotating the mixed solution to dryness, and forming a phospholipid-cholesterol membrane on the bottle wall after the organic solvent is evaporated; (3) Add PBS buffer to the bottle to dissolve the phospholipid-cholesterol membrane and hydrate it to obtain a blank liposome suspension; (4) adding berberine solution to the blank liposome suspension, ultrasonically adjusting the pH of the suspension to 6.5-7.2, incubating at 35-45° C. overnight, filtering, and homogenizing with a high-pressure homogenizer to obtain liposomes; (5) The obtained liposomes are quickly frozen with liquid nitrogen and freeze-dried to obtain berberine liposomes.

2. The method for preparing berberine liposomes according to claim 1, characterized in that: In step (1), the dissolving temperature is 35-40°C.

3. The method for preparing berberine liposomes according to claim 1, characterized in that: In step (1), the organic solvent is any one or both of chloroform and methanol.

4. The method for preparing berberine liposomes according to claim 1, characterized in that: In step (2), the evaporation temperature is 40-45°C.

5. The method for preparing berberine liposomes according to claim 1, characterized in that: In step (3), the pH of the PBS buffer is 5.5-7.

6. The method for preparing berberine liposomes according to claim 1, characterized in that: In step (3), the hydration time is 1 to 4 hours.

7. The method for preparing berberine liposomes according to claim 1, characterized in that: In step (4), the power of the ultrasound is 400 W, and the time is 8 min, with ultrasound being performed once every 2 s.

8. The method for preparing berberine liposomes according to claim 1, characterized in that: In step (4), the pore size of the filter membrane used in the filtration is 0.22 μm.

9. The method for preparing berberine liposomes according to claim 1, characterized in that: In step (4), the homogenization pressure is 20 to 100 MPa and the time is 1 to 3 hours.

10. A berberine liposome, characterized in that: The berberine liposome is prepared by the preparation method of any one of claims 1 to 9.