Vitamin C lipidosome preparation capable of improving encapsulation efficiency and preparation method of vitamin C lipidosome preparation

Abstract

The invention discloses a preparation method of a vitamin C liposome preparation capable of improving encapsulation efficiency, the vitamin C liposome preparation is prepared by an ethanol injection-high pressure microjet circulation homogenization method, the homogenized liposome is small in particle size and uniform in distribution, and meanwhile, by combining a dialysis purification and buffer agent pH regulation process, the encapsulation efficiency of the vitamin C liposome preparation is improved. The prepared vitamin C liposome preparation can still keep high stability under the condition that cholesterol is not contained, and the problem that in the prior art, a vitamin C liposome preparation is low in encapsulation efficiency is effectively solved.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceutical preparations, and in particular to a vitamin C liposome preparation with improved encapsulation efficiency and a preparation method thereof. Background Art

[0002] Vitamin C, as one of the essential nutrients for the human body, plays an irreplaceable role in maintaining immune system function, promoting collagen synthesis, preventing scurvy, etc. Common vitamin C is mainly in the form of chewable tablets, effervescent tablets, injections, etc. There are two main problems. On the one hand, it has poor stability. It is easily oxidized and decomposed by heat and light during processing and storage, and has poor stability. On the other hand, the absorption rate after ingestion by the human body is low.

[0003] Vitamin C liposome oral solution has unique advantages in solving these two major problems. Liposome technology can protect vitamin C from the external environment by encapsulating vitamin C in liposomes, thereby improving its stability and bioavailability. When the concentration of vitamin C in plasma is maintained at a high level, it has the potential to kill tumor cells and inhibit their metastasis and proliferation. However, direct intake of high doses of vitamin C may have negative effects on intestinal cells, such as excessive gas production or diarrhea. Liposome technology can achieve sustained and controlled release of vitamin C by using liposomes as a drug delivery system, so that vitamin C can be released into the body at a lower and more stable rate over a period of time, thereby avoiding the side effects that may be caused by excessive vitamin C concentration in a short period of time. The application of liposome technology in the domestic medical field has gradually matured, but its application in the field of vitamin supplements still needs to be further expanded. Therefore, the research and development of vitamin C liposome oral solution will help promote the innovation and development of the domestic vitamin C supplement market.

[0004] Foreign countries started research on liposome technology early and the technology is relatively mature. With the continuous change of technology, some internationally renowned pharmaceutical companies have successfully brought various liposome drugs to the market. In 1995, the FDA approved the first liposome product on the market, Doxil, which is a doxorubicin hydrochloride liposome injection developed by Sequus Pharmaceuticals in the United States. Abroad, vitamin C liposome drugs have been clinically used to a certain extent. This preparation has advantages in improving the bioavailability of vitamin C and prolonging the duration of drug action. Studies have shown that oral administration of aqueous solutions and liposome preparations containing 10g of vitamin C, vitamin C liposome C max Higher than vitamin C aqueous solution (303μM vs 180μM), the maximum vitamin C blood concentration (T max ) has a delay time of about 1 hour longer than that of the vitamin aqueous solution (T maxwas 180 min vs 96 min). At the same time, the half-life increased (t 1 / 2 > 6 h vs t 1 / 2 = 4 h) and the AUC increased (81570 μM*min vs 45330 μM*min).

[0005] In recent years, significant progress has indeed been made in the research and production of liposomal drugs in China, especially in the fields of anti-tumor and anti-fungal therapies. However, in the case of vitamin C supplements, the research is not yet complete. The development of vitamin C liposomes in China currently mainly remains at the exploratory research stage of the preparation process, lacking mature preparation processes and pharmacodynamic data, which makes the vitamin C liposome oral solutions on the market mostly rely on imports. Moreover, cholesterol raw materials are added to most formulations. Cholesterol mainly plays a role in maintaining the structure and stability of the liposome membrane, but excessive intake has risks such as causing hyperlipidemia and tumor diseases, and is not suitable for health-conscious people.

[0006] Reference 1 (Tong Guihong. Research on the Preparation and Properties of Vitamin C Nanoliposomes [D]. Nanchang University, 2011.) discloses a method for preparing vitamin C nanoliposomes by combining the ethanol injection method with the dynamic high-pressure microfluidization technology. Cholesterol was added to the membrane material, and when evaluating the encapsulation efficiency of the nanoliposomes, it was found that the vitamin C nanoliposomes prepared by this method were less than 20%.

[0007] Aiming at the problems of low encapsulation efficiency and formulation dependence on cholesterol existing in the existing ethanol injection-high-pressure microfluidization cyclic homogenization method for preparing vitamin C liposomes, there is an urgent need to find a preparation method to improve the encapsulation efficiency. Summary of the Invention

[0008] In order to solve the above technical problems, the present invention provides a preparation method for a vitamin C liposomal preparation with improved encapsulation efficiency, which can significantly improve the encapsulation efficiency and bioavailability of the prepared vitamin C liposomal preparation.

[0009] A preparation method for a vitamin C liposomal preparation with improved encapsulation efficiency, comprising the following steps:

[0010] (1) Mix soy lecithin with absolute ethanol, heat and stir until completely dissolved to obtain an alcohol phase; dissolve the water-soluble component in water to obtain an aqueous phase, and the water-soluble component is vitamin C;

[0011] (2) Inject the alcohol phase obtained in step (1) into the aqueous phase obtained in step (1), and stir to obtain a crude liposome solution;

[0012] (3) Rotavaporize the crude liposome solution obtained in step (2) to remove the organic solvent, and then perform dialysis purification to obtain a liposome solution;

[0013] (4) Add a buffer to the liposome solution obtained in step (3), adjust the pH to 5.5 - 6.5, and then subject the liposome solution to high-pressure microfluidic homogenization to obtain a vitamin C liposome preparation.

[0014] In the present invention, a vitamin C liposome preparation is prepared by an ethanol injection - high-pressure microfluidic circulation homogenization method. After homogenization, the liposomes have a small particle size and a uniform distribution. At the same time, by combining the processes of dialysis purification and pH adjustment with a buffer, the prepared vitamin C liposome preparation can maintain high stability without cholesterol and effectively solve the problem of low encapsulation efficiency of vitamin C liposome preparations in the prior art.

[0015] Preferably, in step (1), the molar ratio of the water-soluble component to soybean phospholipid is 1:0.018 - 0.07.

[0016] Preferably, in step (1), a fat-soluble component is further added to the alcohol phase, and the fat-soluble component is vitamin D or curcumin.

[0017] In the present invention, a fat-soluble component can also be added to the vitamin C liposome, and vitamin C and vitamin D or curcumin can exert synergistic effects.

[0018] More preferably, the molar ratio of the fat-soluble component, water-soluble component to soybean phospholipid is 0.03 - 0.05:0.05 - 1.2:0.018 - 0.07.

[0019] Preferably, in step (1), the volume ratio of absolute ethanol to water is 0.1 - 0.5:0.8 - 1.5.

[0020] Preferably, in step (2), the temperature of the stirring is 30 - 50 °C.

[0021] Preferably, in step (3), the cut-off molecular weight of the dialysis purification is 2000 - 5000 D, and the dialysis time is 2 - 6 h.

[0022] In the present invention, through dialysis purification treatment, the encapsulated vitamin C in the reaction can be removed.

[0023] Preferably, in step (4), the buffer is a citric acid - sodium citrate solution or a PBS solution.

[0024] In the present invention, when the buffer is selected as a citric acid - sodium citrate solution or a PBS solution, the pH of the system can be adjusted to 5.5 - 6.5, which can increase the stability of the liposomes.

[0025] Preferably, in step (4), the pressure of the high-pressure microfluidic homogenization is 3000 - 10000 psi.

[0026] The present invention also provides a vitamin C liposome preparation prepared by the above method.

[0027] Preferably, the encapsulation efficiency of the vitamin C liposome preparation is > 50%.

[0028] In the present invention, by combining the processes of dialysis purification and pH adjustment with a buffer, while removing free vitamin C, the pH is adjusted to 5.5 - 6.5. Without relying on cholesterol, the stability of the liposome can be improved, the encapsulation efficiency of the vitamin C liposome preparation can be significantly increased, and the increase in the encapsulation efficiency can further improve the bioavailability of the vitamin C liposome preparation.

[0029] Preferably, the particle size of the vitamin C liposome preparation is 30 - 200 nm.

[0030] The present invention also provides the use of the above vitamin C liposome preparation in the preparation of a vitamin C supplement. A vitamin C supplement includes the above vitamin C liposome preparation.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0032] In the present invention, a vitamin C liposome preparation is prepared by the ethanol injection - high - pressure microfluidic circulation homogenization method. After homogenization, the liposome has a small particle size and a uniform distribution. At the same time, by combining the processes of dialysis purification and pH adjustment with a buffer, the prepared vitamin C liposome preparation can still maintain high stability without cholesterol, effectively solve the problem of low encapsulation efficiency of vitamin C liposome preparations in the prior art, and improve the bioavailability of vitamin C liposome preparations. Description of the Drawings

[0033] Figure 1 It is a particle size distribution diagram of the vitamin C liposome preparation prepared in Example 1.

[0034] Figure 2 It is a plasma - time curve of the vitamin C liposome preparation prepared in Example 1. Detailed Embodiments

[0035] The following combines examples to further illustrate the present invention in detail, but the embodiments of the present invention are not limited to the following examples.

[0036] The raw materials used in the present invention are all commercially available.

[0037] Example 1

[0038] (1) After mixing soybean phospholipids (150 mg, 0.2 mmol) with absolute ethanol (1.5 mL), heat to 50 °C and stir thoroughly until completely dissolved to obtain an alcohol phase; dissolve vitamin C in water (10 mL) to obtain an aqueous phase, where the molar ratio of soybean phospholipids to vitamin C is 0.035:1;

[0039] (2) Inject the alcohol phase obtained in step (1) into the aqueous phase obtained in step (1), and stir for 1 h at 50 °C and 600 r / min to obtain a crude liposome solution;

[0040] (3) After removing the organic solvent from the crude liposome solution obtained in step (2) by rotary evaporation at 30 °C, use a dialysis bag with a molecular weight cut-off of 3500 D for dialysis purification for 4 h to obtain a liposome solution;

[0041] (4) Mix the liposome solution obtained in step (3) with a citric acid - sodium citrate solution (pH = 6.4), adjust the pH of the liposome solution to 6.0, and then subject the liposome solution to high-pressure microfluidic homogenization (8000 psi) 3 times to obtain a vitamin C liposome preparation.

[0042] Example 2

[0043] (1) After mixing soybean phospholipids (150 mg, 0.2 mmol) with absolute ethanol (1.5 mL), heat to 50 °C and stir thoroughly until completely dissolved to obtain an alcohol phase; dissolve vitamin C in water (10 mL) to obtain an aqueous phase, where the molar ratio of soybean phospholipids to vitamin C is 0.035:1;

[0044] (2) Inject the alcohol phase obtained in step (1) into the aqueous phase obtained in step (1), and stir for 1 h at 50 °C and 600 r / min to obtain a crude liposome solution;

[0045] (3) After removing the organic solvent from the crude liposome solution obtained in step (2) by rotary evaporation at 30 °C, use a dialysis bag with a molecular weight cut-off of 3500 D for dialysis purification for 4 h to obtain a liposome solution;

[0046] (4) Mix the liposome solution obtained in step (3) with a PBS solution (pH = 7.4), adjust the pH of the liposome solution to 6.0, and then subject the liposome solution to high-pressure microfluidic homogenization (4000 psi) 3 times to obtain a vitamin C liposome preparation.

[0047] Example 3

[0048] (1) After mixing soybean phospholipids (150 mg, 0.2 mmol) and vitamin D (75 mg, 0.2 mmol) with absolute ethanol (1.5 mL), heat to 50 °C and stir thoroughly until completely dissolved to obtain an alcohol phase; dissolve vitamin C in water (10 mL) to obtain an aqueous phase, where the molar ratio of vitamin C, vitamin D, and soybean phospholipids is 5.7:0.2:0.2;

[0049] (2) Inject the alcohol phase obtained in step (1) into the aqueous phase obtained in step (1), and stir at 50 °C and 600 r / min for 1 h to obtain a crude liposome solution;

[0050] (3) After removing the organic solvent from the crude liposome solution obtained in step (2) by rotary evaporation at 30 °C, perform dialysis purification for 4 h using a dialysis bag with a molecular weight cut-off of 3500 D to obtain a liposome solution;

[0051] (4) Mix the liposome solution obtained in step (3) with a citric acid-sodium citrate solution (pH = 6.4), adjust the pH of the liposome solution to 6.0, and then subject the liposome solution to high-pressure microfluidic homogenization (4000 psi) 3 times to obtain a vitamin C liposome preparation.

[0052] Example 4

[0053] (1) After mixing soybean phospholipids (150 mg, 0.2 mmol) and curcumin (80 mg, 0.2 mmol) with absolute ethanol (1.5 mL), heat to 50 °C and stir thoroughly until completely dissolved to obtain an alcohol phase; dissolve vitamin C in water (10 mL) to obtain an aqueous phase, where the molar ratio of vitamin C, curcumin, and soybean phospholipids is 5.7:0.2:0.2;

[0054] (2) Inject the alcohol phase obtained in step (1) into the aqueous phase obtained in step (1), and stir at 50 °C and 600 r / min for 1 h to obtain a crude liposome solution;

[0055] (3) After removing the organic solvent from the crude liposome solution obtained in step (2) by rotary evaporation at 30 °C, perform dialysis purification for 4 h using a dialysis bag with a molecular weight cut-off of 3500 D to obtain a liposome solution;

[0056] (4) Mix the liposome solution obtained in step (3) with a PBS solution (pH = 7.4), adjust the pH of the liposome solution to 6.0, and then subject the liposome solution to high-pressure microfluidic homogenization (4000 psi) 3 times to obtain a vitamin C liposome preparation.

[0057] Sample Analysis

[0058] I. Encapsulation Efficiency Test

[0059] The encapsulation efficiency of the vitamin C liposome preparation obtained in Example 1 was tested. The specific steps are as follows: precisely measure 600 μL of the VC liposome and place it in an ultrafiltration centrifuge tube. Centrifuge at 3500 r / min for 20 min. Transfer 50 μL of the solution in the lower layer of the ultrafiltration centrifuge tube, dilute it appropriately with 0.1% formic acid water, filter through a 0.22 μm membrane, and measure the concentration of free vitamin C by HPLC method, denoted as W_free; calculate according to the formula for calculating the liposome encapsulation efficiency in the Chinese Pharmacopoeia.

[0060]

[0061] W 总 is the total amount of drug input, and W 游 is the amount of free drug not encapsulated in the liposome.

[0062] The results are shown in Table 1.

[0063] Table 1: Encapsulation efficiency results of three parallel experiments in Example 1

[0064]

[0065] As shown in Table 1, the vitamin C liposome preparation obtained in Example 1 had an encapsulation efficiency of 55.65% (RSD = 1.0%), which was significantly higher than that of the ethanol injection - high - pressure microfluidic circulation homogenization method in the prior art (less than 20%), and had good reproducibility.

[0066] II. Particle size test

[0067] The particle size of the vitamin C liposome preparation obtained in Example 1 was analyzed. Take the vitamin C liposome in a cuvette and place it in a Malvern Zetasizer Pro laser particle size analyzer to measure the average particle size of the vitamin C liposome. The results are as Figure 1 shown.

[0068] Figure 1 is the particle size distribution diagram of the vitamin C liposome preparation obtained in Example 1. As shown in the figure, the particle size of the vitamin C liposome preparation obtained in Example 1 was in the range of 30 - 200 nm, showing a normal distribution, and the mean value was 71 nm.

[0069] III. Oral absorption test

[0070] To evaluate the absorption effect of liposomal vitamin C in rats, a pre-experiment of animal experiment with liposomal vitamin C was carried out. The steps and results of the pre-experiment are as follows: SD rats at 5 weeks of age, half male and half female, were selected and adapted for 1 week. They were fasted 24 h before the experiment, and on the morning of the experimental day, the test preparation (liposomal vitamin C prepared in Example 1, denoted as "T" in the figure) and the reference preparation (purchased liposomal vitamin C, denoted as "R" in the figure) were taken on an empty stomach. Eight healthy mice were used as their own controls and divided into two groups. After taking the test preparation and the reference preparation at the same concentration and dose, all plasma samples were determined by HPLC-MS method. The plasma concentration-time curves of the two preparations were plotted. T max and C max Using the measured values, the AUC 0-t value was calculated using DAS 2.0 software. The obtained parameters are listed in the table. According to the AUC 0-t values of vitamin C in the test preparation and the reference preparation, the relative bioavailability F was calculated according to the following formula,

[0071]

[0072] Table 2: Pharmacokinetic parameters of liposomal vitamin C preparations of Example 1 and commercially available products in rats

[0073]

[0074] Figure 2 is the plasma concentration-time curve of the liposomal vitamin C preparation obtained in Example 1. As shown in Figure 2 and Table 2, the plasma concentration of the liposomal vitamin C preparation of Example 1 was significantly higher than that of the commercially available liposomal vitamin C preparation from 1 to 6 hours after oral administration.

[0075] The above is only the preferred embodiment of the present invention and is not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of a vitamin C liposome preparation with improved encapsulation efficiency, characterized in that, It includes the following steps: (1) After mixing soybean phospholipids with absolute ethanol, heat and stir until completely dissolved to obtain an alcohol phase; dissolve the water-soluble component in water to obtain an aqueous phase, and the water-soluble component is vitamin C; (2) Inject the alcohol phase obtained in step (1) into the aqueous phase obtained in step (1), and stir to obtain a crude liposome solution; (3) After rotary evaporation of the crude liposome solution obtained in step (2) to remove the organic solvent, perform dialysis purification to obtain a liposome solution; (4) Add a buffer to the liposome solution obtained in step (3), adjust the pH to 5.5 - 6.5, and then subject the liposome solution to high-pressure microfluidization homogenization to obtain a vitamin C liposome preparation.

2. The preparation method of the vitamin C liposome preparation with improved encapsulation efficiency according to claim 1, characterized in that, In step (1), the molar ratio of the water-soluble component to soybean phospholipids is 1:0.018 - 0.

07.

3. The preparation method of the vitamin C liposome preparation with improved encapsulation efficiency according to claim 1, characterized in that In step (1), a fat-soluble component is further added to the alcohol phase, and the fat-soluble component is vitamin D or curcumin.

4. The preparation method of the vitamin C liposome preparation with improved encapsulation rate according to claim 3, characterized in that, The molar ratio of the fat-soluble component, water-soluble component to soybean phospholipids is 0.03 - 0.05:0.05 - 1.2:0.018 - 0.

07.

5. The preparation method of the vitamin C liposome preparation with improved encapsulation efficiency according to claim 1, characterized in that, In step (3), the cut-off molecular weight for dialysis purification is 2000 - 5000 D, and the dialysis time is 2 - 6 h.

6. The preparation method of the vitamin C liposome preparation with improved encapsulation rate according to claim 1, characterized in that, In step (4), the buffer is a citric acid-sodium citrate solution or a PBS solution.

7. The preparation method of the vitamin C liposome preparation with improved encapsulation efficiency according to claim 1, characterized in that, In step (4), the pressure of the high-pressure microfluidization homogenization is 3000 - 10000 psi.

8. A vitamin C liposome preparation prepared by the preparation method according to any one of claims 1 - 7.

9. The liposomal preparation of vitamin C according to claim 8, characterized in that, The encapsulation efficiency of the vitamin C liposome preparation is > 50%; the particle size of the vitamin C liposome preparation is 30 - 200 nm.

10. A vitamin C supplement, characterized in that, It includes the vitamin C liposome preparation according to claim 8 or 9.

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