Water-soluble cholesterol solution as well as freeze-dried solid and preparation method thereof

Abstract

The invention relates to the technical field of cholesterol preparation, in particular to a water-soluble cholesterol solution, a freeze-dried solid of the water-soluble cholesterol solution and a preparation method of the freeze-dried solid of the water-soluble cholesterol solution. Not only is the accuracy of cell biological research related to cholesterol improved, but also the problem of cell apoptosis caused by precipitation after the cholesterol is diluted to 10-17mg / mL in the prior art in the field of cell incubation is effectively solved.

Description

Technical Field

[0001] The present invention relates to the technical field of cholesterol preparation, and particularly relates to a water-soluble cholesterol solution, its freeze-dried solid and a preparation method thereof. Background Art

[0002] Cholesterol is a derivative of cyclopentane polyhydrophenanthrene, existing inside and outside cells. It interacts with adjacent lipids on the cell membrane to regulate the fluidity and permeability of the membrane, etc. It is the main steroid compound in mammals and plays an important role in basic cell life activities.

[0003] Cholesterol is an organic compound with extremely low water solubility. The solubility of pure cholesterol in water is only about 0.095 mg / L (30 °C), which is 105 - 158 times different from the water solubility of cholesterol required for cell culture (10 - 17 mg / L). However, during cell culture and biological experimental research, either too high or too low cholesterol will lead to cell apoptosis. Therefore, it is particularly important to ensure that the cholesterol aqueous solution does not precipitate at 10 - 17 mg / L.

[0004] In the prior art, cholesterol is mainly dissolved in organic reagents such as ethanol and chloroform and then added to the cell aqueous solution. Not only does it have the problem that cholesterol precipitates after dilution, resulting in cell apoptosis due to uncontrolled cholesterol intake, but also the organic reagents are toxic to cells.

[0005] Currently, commercially available HyClone TM LS1000 and MERCK's C4951 products, although they improve the water solubility of cholesterol on the basis of organic reagent dissolution, do not solve the problem of complete non-precipitation under the application concentration conditions of cholesterol required for cell culture. Cholesterol-PEG series compounds, although they improve the water solubility of cholesterol, but cholesterol and PEG are combined in a chemical bonding manner, which is not only not conducive to cell absorption, but also the preparation process is complex and not easy to mass-produce industrially.

[0006] The water-soluble cholesterol preparation process described in Patent CN102645358A is different from the present invention. Its preparation process features extracting water-soluble cholesterol from serum, but there are problems with serum sources and defects that it cannot be mass-produced industrially. Therefore, it is of great significance to develop a product that can be mass-produced industrially and meets the requirement that cholesterol does not precipitate during cell culture, etc. Summary of the Invention

[0007] The first object of the present invention is to develop a water-soluble cholesterol solution. By encapsulating cholesterol with β-cyclodextrin, the solubility of cholesterol in water is improved, cholesterol precipitation is avoided, and a water-soluble cholesterol solution with stable properties that can be directly applied to cell research, especially a water-soluble cholesterol solution for cell culture, is obtained.

[0008] The above technical object of the present invention is achieved by the following technical solutions:

[0009] A water-soluble cholesterol solution provided by the present invention comprises plant-derived cholesterol, β-cyclodextrin derivatives and water, wherein the molar ratio of plant-derived cholesterol to β-cyclodextrin derivatives is 1:7 to 1:30.

[0010] The present invention uses plant-derived cholesterol as the raw material for the cholesterol aqueous solution, which has the advantage of less interference factors compared with the cholesterol aqueous solution of animal origin. The present invention uses β-cyclodextrin derivatives as solubilizers. By utilizing the special molecular structure of β-cyclodextrin derivatives, cholesterol is included inside the molecular structure, and at the same time, the hydrophilicity of β-cyclodextrin derivatives is used to improve the solubility of cholesterol in water.

[0011] Furthermore, the concentration of cyclodextrin derivatives is 0.5 to 200 g / L.

[0012] Furthermore, the β-cyclodextrin derivative is any one or a mixture of more than one of methyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin or sulfobutyl-β-cyclodextrin.

[0013] The water-soluble cholesterol solution provided by the present invention uses β-cyclodextrin derivatives as solubilizers, which can improve the water solubility of cholesterol without adding any organic solvents, avoid the incorporation of organic solvents, and interfere with cell culture.

[0014] The second object of the present invention is to provide a preparation method of a water-soluble cholesterol solution, which effectively solves the problem that cholesterol precipitates when diluted to 10-17 mg / mL in the prior art in the field of cell culture, resulting in cell apoptosis. Moreover, the method proposed by the present invention is simple to operate, has good stability, and is easy to be mass-produced industrially.

[0015] The above technical object of the present invention is achieved by the following technical solutions:

[0016] A preparation method of a water-soluble cholesterol solution provided by the present invention specifically comprises:

[0017] Prepare an aqueous solution of β-cyclodextrin derivatives;

[0018] Under heating conditions, dissolve cholesterol in the aqueous solution of β-cyclodextrin derivatives and stir;

[0019] Cool to room temperature, filter, and obtain the water-soluble cholesterol solution.

[0020] Furthermore, the heating temperature is 60-100 °C, and the stirring time is 2-4 h.

[0021] Further, filtration is specifically carried out by filtering with a 0.45 μm filter membrane.

[0022] The third object of the present invention is to provide a method for preparing a freeze-dried solid based on the water-soluble cholesterol solution provided by the present invention. The obtained water-soluble cholesterol freeze-dried solid improves the solubility of cholesterol in water, avoids the precipitation of cholesterol during the application process, improves the accuracy of research results, and at the same time, the prepared water-soluble cholesterol has stable physical indicators, a high drug loading, is stable under the application temperature conditions, and there is no cholesterol precipitation.

[0023] The above technical object of the present invention is achieved by the following technical solutions:

[0024] A method for preparing a freeze-dried solid from a water-soluble cholesterol solution specifically includes the following operating steps:

[0025] Pre-freeze the water-soluble cholesterol solution;

[0026] After pre-freezing, carry out sublimation drying under vacuum;

[0027] After sublimation drying, carry out desorption drying under vacuum.

[0028] Further, pre-freezing includes reducing the temperature of the product to -35 to -45 °C and maintaining it for 2 h.

[0029] Further, sublimation drying specifically includes: after raising the temperature of the product to -5 to -10 °C, carry out low-temperature sublimation drying for 10 to 20 h, then raise the sample temperature to 0 °C, carry out low-temperature sublimation drying for 4 h, and evacuate to ≤0.3 mbar.

[0030] Further, desorption drying specifically includes: raising the temperature of the product to 25 to 40 °C, carry out desorption drying for 2 to 4 h, and evacuate to ≤0.1 mba.

[0031] Further, the preparation method of the water-soluble cholesterol solution specifically includes:

[0032] Prepare an aqueous solution of β-cyclodextrin derivative;

[0033] Under heating conditions, dissolve cholesterol in the aqueous solution of β-cyclodextrin derivative and stir;

[0034] Cool to room temperature, filter to obtain the water-soluble cholesterol solution.

[0035] Further, the β-cyclodextrin derivative is any one or a mixture of more than one of methyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, or sulfobutyl-β-cyclodextrin.

[0036] Further, in the aqueous solution of the β-cyclodextrin derivative, the concentration of the β-cyclodextrin derivative is 0.5 to 200 g / L.

[0037] Further, the heating temperature is 60 to 100 °C, and the stirring time is 2 to 4 h.

[0038] In summary, compared with the prior art, the beneficial effects of the present invention are as follows:

[0039] The present invention provides a water-soluble cholesterol, which not only increases the water solubility of cholesterol by 105 to 158 times, but also ensures that the cholesterol aqueous solution does not precipitate at 10 to 15 mg / L during cell culture and biological experimental research, eliminates the interference of cell apoptosis caused by nutrient deficiency or excess on the results of biological experimental research, and improves the survival rate of cell culture and the accuracy of experimental research. Description of the Drawings

[0040] Figure 1 is the chromatogram of the cholesterol aqueous solution of Example 1 of the present invention;

[0041] Figure 2 is the chromatogram of the cholesterol aqueous solution of Example 2 of the present invention;

[0042] Figure 3 is the chromatogram of the cholesterol aqueous solution of Example 3 of the present invention;

[0043] Figure 4 is the chromatogram of the cholesterol aqueous solution of Example 4 of the present invention;

[0044] Figure 5 is the chromatogram of the cholesterol aqueous solution of Example 7 of the present invention;

[0045] Figure 6 is the chromatogram of the cholesterol aqueous solution prepared by MERK. Detailed Embodiments

[0046] To further elaborate on the technical means and effects adopted by the present invention to achieve the intended invention purpose, a water-soluble cholesterol solution, its lyophilized solid and preparation method according to the present invention are described in detail below in terms of their specific embodiments, features and effects.

[0047] The materials used in this specific embodiment are sourced as follows:

[0048] Cholesterol (plant source): Jiangsu Jare Pharmaceutical Group Co., Ltd., content: 99.2%;

[0049] Methyl-β-cyclodextrin: Adamas-beta, Shandong Binzhou Zhiyuan Biotechnology Co., Ltd.;

[0050] Hydroxypropyl-β-cyclodextrin: Adamas-beta, Shandong Binzhou Zhiyuan Biotechnology Co., Ltd.;

[0051] Sulfobutyl-β-cyclodextrin: Adamas-beta, Shandong Binzhou Zhiyuan Biotechnology Co., Ltd.;

[0052] MERCK product: Water-soluble cholesterol, product number: C4951, 300 mg / mL, with cholesterol proportion: 49 mg / g.

[0053] A preparation method of a water-soluble cholesterol solution provided by this specific embodiment uses a β-cyclodextrin derivative as a solubilizer to encapsulate plant-derived cholesterol; at room temperature, the content of cholesterol in the water-soluble cholesterol solution is 10 - 17 mg / L.

[0054] Its specific steps include:

[0055] S1. Prepare an aqueous solution of a β-cyclodextrin derivative;

[0056] S2. Under heating conditions, dissolve cholesterol in the aqueous solution of the β-cyclodextrin derivative and stir;

[0057] S3. Cool to room temperature, filter to obtain the water-soluble cholesterol solution.

[0058] Among them, the β-cyclodextrin derivative is any one or a mixture of more than one of methyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, or sulfobutyl-β-cyclodextrin.

[0059] In the aqueous solution of the β-cyclodextrin derivative, the concentration of the β-cyclodextrin derivative is 0.5 - 200 g / L.

[0060] The heating temperature is 60 - 100 °C, and the stirring time is 2 - 4 h.

[0061] This specific embodiment also provides a preparation method of a water-soluble cholesterol freeze-dried solid, which prepares the water-soluble cholesterol solution into a freeze-dried product, specifically including the following operating steps:

[0062] P1. Pre-freeze the water-soluble cholesterol solution;

[0063] P2. After pre-freezing, under vacuum conditions, perform sublimation drying;

[0064] P3. After sublimation drying, under vacuum conditions, perform analytical drying.

[0065] Among them, pre-freezing includes reducing the temperature of the product to -35 to -45 °C and maintaining it for 2 hours; subsequently raising the temperature of the product to -5 to -10 °C, followed by low-temperature sublimation drying for 10 to 20 hours. Then, raising the temperature of the sample to 0 °C and performing low-temperature sublimation drying for 4 hours, and evacuating to ≤0.3 mbar; finally, raising the temperature of the product to 25 to 40 °C, performing desorption drying for 2 to 4 hours, and evacuating to ≤0.1 mbar.

[0066] Example 1: A method for preparing a water-soluble cholesterol solution

[0067] The water-soluble cholesterol solution provided in this example uses methyl-β-cyclodextrin as a solubilizer to encapsulate plant-derived cholesterol, and the concentration of cholesterol is 11.3 mg / L.

[0068] The preparation method is as follows:

[0069] S1. Dissolve 1 g of methyl-β-cyclodextrin in 90% of the prescription amount of deionized water;

[0070] S2. Add cholesterol and stir at 80 °C for 4 hours;

[0071] S3. Add deionized water to make up to 1000 ml;

[0072] S4. Filter through a 0.45 μm filter membrane and dispense to obtain the water-soluble cholesterol solution.

[0073] Example 2: A method for preparing a water-soluble cholesterol solution

[0074] The water-soluble cholesterol solution provided in this example uses methyl-β-cyclodextrin as a solubilizer to encapsulate plant-derived cholesterol, and the concentration of cholesterol is 13.8 mg / L.

[0075] The preparation method is as follows:

[0076] S1. Dissolve 5 g of methyl-β-cyclodextrin in 90% of the prescription amount of deionized water;

[0077] S2. Add cholesterol and stir at 80 °C for 4 hours;

[0078] S3. Add deionized water to make up to 1000 ml;

[0079] S4. Filter through a 0.45 μm filter membrane and dispense to obtain the water-soluble cholesterol solution.

[0080] Example 3: A method for preparing a water-soluble cholesterol solution

[0081] The water-soluble cholesterol solution provided in this example uses methyl-β-cyclodextrin as a solubilizer to encapsulate plant-derived cholesterol, and the concentration of cholesterol is 14.4 mg / L.

[0082] The preparation method is as follows:

[0083] S1. Dissolve 50 g of methyl-β-cyclodextrin in 90% of the prescription amount of deionized water;

[0084] S2. Add cholesterol and stir at 80 °C for 4 h;

[0085] S3. Add deionized water to make the volume up to 1000 ml;

[0086] S4. Filter with a 0.45-μm filter membrane and dispense to obtain a water-soluble cholesterol solution.

[0087] Example 4: A preparation method of a freeze-dried solid of water-soluble cholesterol

[0088] First, prepare a water-soluble cholesterol solution. The water-soluble cholesterol solution provided in this example uses methyl-β-cyclodextrin as a solubilizer to encapsulate plant-derived cholesterol. Among them, the concentration of cholesterol is 15.6 mg / L, and its preparation method is as follows:

[0089] S1. Dissolve 100 g of methyl-β-cyclodextrin in 90% of the prescription amount of deionized water;

[0090] S2. Add cholesterol and stir at 80 °C for 4 h;

[0091] S3. Add deionized water to make the volume up to 1000 ml;

[0092] S4. Filter with a 0.45-μm filter membrane and dispense to obtain a water-soluble cholesterol solution.

[0093] Then, prepare the water-soluble cholesterol solution into a freeze-dried solid of water-soluble cholesterol, and the method is as follows:

[0094] P1. Pre-freeze the above-obtained water-soluble cholesterol solution: within 2 h, lower the system temperature from room temperature to -40 °C and maintain it for 2 h;

[0095] P2. Sublimation drying: raise the system temperature to -10 °C in 3 h and maintain it for 10 h, and evacuate to 0.3 mbar; then, within 1 h, raise the system temperature to 0 °C and maintain it for 4 h, maintaining a vacuum degree of 0.3 mbar;

[0096] P3. Desorption drying: raise the system temperature to 25 °C in 1 h and maintain it for 4 h, and evacuate to 0.1 mbar to obtain a freeze-dried solid of water-soluble cholesterol.

[0097] Example 5: A preparation method of a water-soluble cholesterol solution

[0098] The water-soluble cholesterol solution provided in this example uses methyl-β-cyclodextrin as a solubilizer to encapsulate plant-derived cholesterol, and the concentration of cholesterol is 11.3 mg / L.

[0099] The preparation method is as follows:

[0100] S1. Dissolve 5 g of methyl-β-cyclodextrin in 90% of the prescription amount of deionized water;

[0101] S2. Add cholesterol and stir at 100 °C for 4 h;

[0102] S3. Add deionized water to make up the volume to 1000 ml;

[0103] S4. Filter through a 0.45-μm filter membrane and dispense to obtain the water-soluble cholesterol solution.

[0104] Example 6: A preparation method of a water-soluble cholesterol solution

[0105] The water-soluble cholesterol solution provided in this example uses methyl-β-cyclodextrin as a solubilizer to encapsulate plant-derived cholesterol, and the concentration of cholesterol is 12.5 mg / L.

[0106] The preparation method is as follows:

[0107] S1. Dissolve 5 g of methyl-β-cyclodextrin in 90% of the prescription amount of deionized water;

[0108] S2. Add cholesterol and stir at 100 °C for 4 h;

[0109] S3. Add deionized water to make up the volume to 1000 ml;

[0110] S4. Filter through a 0.45-μm filter membrane and dispense to obtain the water-soluble cholesterol solution.

[0111] Example 7: A preparation method of a water-soluble cholesterol solution

[0112] The water-soluble cholesterol solution provided in this example uses hydroxypropyl-β-cyclodextrin as a solubilizer to encapsulate plant-derived cholesterol, and the concentration of cholesterol is 10.6 mg / L.

[0113] The preparation method is as follows:

[0114] S1. Dissolve 100 g of hydroxypropyl-β-cyclodextrin in 90% of the prescription amount of deionized water;

[0115] S2. Add cholesterol and stir at 80 °C for 4 h;

[0116] S3. Add deionized water to make up the volume to 1000 ml;

[0117] S4. Filter through a 0.45 μm filter membrane and dispense to obtain a water-soluble cholesterol solution.

[0118] Example 8: A method for preparing a water-soluble cholesterol solution

[0119] The water-soluble cholesterol solution provided in this example uses sulfobutyl-β-cyclodextrin as a solubilizer to encapsulate plant-derived cholesterol, and the concentration of cholesterol is 11.0 mg / L.

[0120] The preparation method is as follows:

[0121] S1. Dissolve 100 g of sulfobutyl-β-cyclodextrin in 90% of the prescription amount of deionized water;

[0122] S2. Add cholesterol and stir at 80 °C for 4 h;

[0123] S3. Add deionized water to make up to 1000 ml;

[0124] S4. Filter through a 0.45 μm filter membrane and dispense to obtain a water-soluble cholesterol solution.

[0125] Performance test

[0126] Perform chromatographic analysis on the cholesterol aqueous solutions obtained in Examples 1-4 and Example 7 and the cholesterol aqueous solution produced by MERK, and use a bench-top turbidimeter (model: TL2350, manufacturer: HACH) to test the precipitation rate of water-soluble cholesterol. The test results are shown in the following table; among them, the specific operation method used in this performance test is to dilute or dissolve the water-soluble cholesterol solutions obtained in Examples 1-4 and Example 7 and the cholesterol aqueous solution produced by MERK to 10 mg / L. The specific experimental data are shown in Figures 1 to 6 and Table 1.

[0127] Table 1. Turbidity test results

[0128]

[0129]

[0130] According to Figures 1 to 6 and the test results in Table 1, it can be seen that compared with the commercially available products, the water-soluble cholesterol provided by the present invention not only has higher water solubility, but also has no interference from organic reagents, effectively solves the problem of cholesterol precipitation during cell culture of commercially available products, and is more suitable for cell culture.

[0131] The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Although the present invention has been shown above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to equivalent embodiments by using the disclosed technical content within the scope of the technical solution of the present invention. However, as long as it does not depart from the content of the technical solution of the present invention, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A water-soluble cholesterol solution, characterized in that, it contains cholesterol from plant sources, β-cyclodextrin derivatives and water, wherein the molar ratio of the cholesterol from plant sources to the β-cyclodextrin derivatives is (1:7) to (1:30).

2. The water-soluble cholesterol solution according to claim 1, characterized in that, the concentration of the cyclodextrin derivatives is 0.5 to 200 g / L.

3. The water-soluble cholesterol solution according to claim 1, characterized in that, the β-cyclodextrin derivative is any one or a mixture of more than one of methyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin or sulfobutyl-β-cyclodextrin.

4. A method for preparing the water-soluble cholesterol solution according to any one of claims 1 to 3, characterized in that, specifically includes: preparing an aqueous solution of β-cyclodextrin derivatives; dissolving cholesterol in the aqueous solution of β-cyclodextrin derivatives under heating conditions and stirring; cooling to room temperature, filtering to obtain the water-soluble cholesterol solution.

5. The method for preparing the water-soluble cholesterol solution according to claim 4, characterized in that, the heating temperature is 60 to 100 °C and the stirring time is 2 to 4 h.

6. The method for preparing the water-soluble cholesterol solution according to claim 4, characterized in that, the filtering is specifically carried out by filtering with a 0.45 μm filter membrane.

7. A method for preparing a freeze-dried solid based on the water-soluble cholesterol solution according to any one of claims 4 to 6, characterized in that, specifically includes the following operation steps: pre-freezing the water-soluble cholesterol solution; after pre-freezing, under vacuum conditions, carrying out sublimation drying; after sublimation drying, under vacuum conditions, carrying out desorption drying.

8. The method for preparing a freeze-dried solid of water-soluble cholesterol according to claim 7, characterized in that, the pre-freezing includes reducing the temperature to -35 to -45 °C and maintaining for 2 h.

9. The method for preparing a freeze-dried solid of water-soluble cholesterol according to claim 7, characterized in that, the sublimation drying specifically includes: after raising the temperature to -5 to -10 °C, carrying out low-temperature sublimation drying for 10 to 20 h, then raising the temperature to 0 °C and carrying out low-temperature sublimation drying for 4 h, and evacuating to ≤ 0.3 mbar.

10. The method for preparing a freeze-dried solid of water-soluble cholesterol according to claim 7, characterized in that, the desorption drying specifically includes: raising the temperature to 25 to 40 °C, carrying out desorption drying for 2 to 4 h, and evacuating to ≤ 0.1 mba.

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