CN110812254A - Phospholipase-coated multivesicular liposome and preparation method and application thereof - Google Patents
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
- A61K8/14—Liposomes; Vesicles
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- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
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- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
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- A61Q19/08—Anti-ageing preparations
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Abstract
The invention provides a multi-vesicular liposome coating phospholipase, a preparation method and application thereof, the multi-vesicular liposome coating the phospholipase comprises an inner water phase, an oil phase and an outer water phase, the inner water phase comprises a suitable phospholipase A2, the oil phase comprises an additive and lipid, the outer water phase comprises a thickening agent and water, the inner water phase and the oil phase are mixed and stirred to obtain colostrum, the outer water phase is added into the colostrum and stirred to obtain multiple emulsion, and an organic solvent in the multiple emulsion is removed to obtain the multi-vesicular liposome, the invention utilizes the coating structure of the multi-vesicular liposome to effectively protect the phospholipase A2, is favorable for human body absorption of the phospholipase A2, further can utilize the enzymolysis of the phospholipase A2 entering human skin cells on cell membranes to increase the absorption effect of the cells on other effective components, achieves the aim of enhancing the human skin absorption rate, and overcomes the problem of poor skin absorption rate, has good application prospect.
Description
Technical Field
The invention relates to the technical field of daily chemicals, in particular to a phospholipase-coated multivesicular liposome and a preparation method and application thereof.
Background
The functional cosmetics have important functions as daily chemical articles essential to daily life, and can realize the functions of whitening, moisturizing, resisting wrinkles, preventing ultraviolet rays and the like.
However, the active ingredients for achieving these functions are poor in stability, easily deteriorate under ordinary storage conditions, are inconvenient to store for a long time, and are partially unpleasant in odor when applied directly, which may cause discomfort to the user. In order to improve the stability and the preservation time of the effective substances, the prior art mostly adopts liposome as a carrier, the liposome is formed by encapsulating effective components in a phospholipid bilayer to form a micro vesicle, the liposome not only can effectively protect the effective components and reduce the possibility of deterioration, but also has targeting property, slow release property and cell affinity, the liposome can simultaneously coat various hydrophilic or lipophilic substances, and the effective substances are fed between epidermis and dermis.
However, the liposome has large size, is easy to be demulsified and flocculated, has low stability, and can not effectively deliver some water-soluble effective components into skin cells of human bodies, thereby influencing the absorption effect.
Disclosure of Invention
In view of the above, the present invention provides a phospholipase-coated multivesicular liposome which is smaller in size and more stable, and which is helpful for human skin to absorb water-soluble effective ingredients, and a preparation method and an application thereof.
The technical scheme of the invention is realized as follows: the invention provides a phospholipase-coated multivesicular liposome, which comprises an inner water phase, an oil phase and an outer water phase, wherein the inner water phase comprises water and phospholipase A2 in a mass ratio of (50-2000) to 1, the oil phase comprises an additive and lipid in a mass ratio of (0.1-1) to 1, and the outer water phase comprises a thickening agent and water in a mass ratio of (0.1-0.3) to 1.
On the basis of the technical scheme, preferably, the internal water phase also comprises human oligopeptide-1, cane sugar and chitosan, and the mass ratio of the human oligopeptide-1 to the cane sugar to the chitosan to the phospholipase A2 is (1-25) to (1-5) to 1.
On the basis of the technical scheme, the additive preferably comprises cholesterol, methyl paraben, 2, 6-di-tert-butyl-p-cresol and an organic solvent in the mass ratio of (50-55) to (1-5) to (1-2) to (200-500).
On the basis of the above technical scheme, preferably, the organic solvent is a halogenated hydrocarbon, and particularly preferably includes dichlorohexane, trichloromethane and carbon tetrachloride.
Even more preferably, the lipids include amphiphilic lipids and neutral lipids in a mass ratio of (5-15): 1.
On the basis of the technical scheme, preferably, the amphiphilic lipid is one or more of soybean phospholipid, glycerin monostearate, 16-18 carbon fatty alcohol and isopropyl palmitate.
On the basis of the above technical solution, preferably, the neutral lipid is olive oil.
On the basis of the technical scheme, preferably, the thickening agent is one or more of glucose, sodium chloride, trehalose, cyclodextrin, maltose, pentasodium triphosphate, glycine and lysine.
The invention also provides a preparation method of the phospholipase-coated multivesicular liposome, which comprises the following steps:
step one, mixing water, phospholipase A2, human oligopeptide-1, sucrose and chitosan according to a mass ratio, and stirring at normal temperature for 5-10min to obtain an internal water phase;
mixing cholesterol, methyl paraben, 2, 6-di-tert-butyl-p-cresol, an organic solvent and liposome according to a mass ratio, and stirring at normal temperature for 5-10min to obtain an oil phase;
step three, mixing the thickening agent and water according to the mass ratio, and stirring for 5-10min at normal temperature to obtain an external water phase;
step four, mixing the internal water phase and the oil phase with the same volume, and stirring for 5-15min at the stirring speed of 10000-15000r/min and the temperature of 30-50 ℃ to obtain W/O primary emulsion;
step five, adding the external water phase with the same volume into the W/O primary emulsion, keeping the stirring speed at 500-;
and sixthly, maintaining the rotating speed at 10-50r/min at the temperature of 30-40 ℃, and performing rotary evaporation until no organic solvent is evaporated to obtain the phospholipase-coated multivesicular liposome.
The phospholipase-coated multivesicular liposome can be applied to cosmetics, skin care products, health care products and other related daily chemical products.
The invention also provides a skin care product based on the multi-vesicular liposome coated with the phospholipase, wherein the internal water phase of the multi-vesicular liposome comprises vitamin B1, vitamin B6, vitamin B12, vitamin H, vitamin C and serine, and the mass ratio of the vitamin B1 to the vitamin B6 to the vitamin B12 to the vitamin H to the vitamin C to the serine to the phospholipase A2 is (10-15) to (5-15) to (1-6) to (3-10) to (45-60) to (20-30) to 1.
Among the above technical scheme, the thickening agent of adding in the aquatic of outer aqueous phase can increase the viscosity of outer aqueous phase, and the increase of outer aqueous phase viscosity is favorable to keeping the stability of parcel rete, reduces the surface tension difference between interior aqueous phase and the outer aqueous phase to improve the stability of liposome, utilize the emulsification and the dispersion of thickening agent, can also strengthen the dispersion properties of liposome, avoid the liposome flocculation and reunion each other.
In the technical scheme, the inner water phase is further coated with human oligopeptide-1, sucrose and chitosan, the sucrose and the chitosan can adjust the osmotic pressure of the inner water phase, so that overlarge osmotic pressure difference between an inner water tank and an outer water tank and unstable oil film are prevented, meanwhile, the chitosan has good biocompatibility for a human body and is beneficial to the permeation of phospholipase A2, and meanwhile, after the human oligopeptide-1 enters skin cells of the human body, the proliferation and differentiation of the cells can be promoted, so that the process of replacing aged cells and dead cells with new-born cells is realized, and the anti-aging effect is achieved.
In the technical scheme, 10000-15000r/min of rotation speed is adopted for stirring when the inner aqueous phase and the coating film layer are mixed to form the primary emulsion, so that the liposome of the inner aqueous phase can be more refined, the stirring speed is low when secondary emulsification is carried out, the primary emulsion can keep the original form and cannot be coagulated and aggregated in the process of forming the multiple emulsion, so that more uniform multiple emulsion is formed, the particle size of the multiple-vesicle liposome formed by the multiple emulsion after organic solvent is removed is small, and the multiple emulsion has higher stability.
The phospholipase-coated multivesicular liposome prepared by the invention can be applied to the fields of cosmetics and skin care products.
Compared with the prior art, the phospholipase-coated multivesicular liposome and the preparation method and application thereof have the following beneficial effects:
(1) compared with the conventional multivesicular liposome, the multivesicular liposome coated with the phospholipase has smaller particle size and is more stable, meanwhile, the inner water phase contains the phospholipase A2, and can carry out enzymolysis on epidermal cell membranes, so that epidermal cells have better permeability, thereby being beneficial to the absorption of water-based active ingredients, the phospholipid layer adopted by the oil phase has good biocompatibility and is beneficial to the permeation of the phospholipase A2 into the epidermal cells, and meanwhile, the phospholipid also has certain human cell repair capacity and plays a role in delaying senescence;
(2) the invention also provides a preparation method of the phospholipase-coated multivesicular liposome, compared with the conventional method, the preparation method has the advantages of simplicity and easiness in operation, and the prepared multivesicular liposome is more stable and has good application prospect;
(3) compared with the conventional skin care product, the skin care product has better permeability, cell membranes of epidermal cells are subjected to enzymolysis through the coated phospholipase A2, and the channels of the cell membranes are enlarged, so that more skin care components can enter the epidermal cells more easily, the effect is better, and the human oligopeptide-1 and water-soluble vitamins and serine can effectively prevent the aging of the cells and repair the damaged cells.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Preparation of phospholipase-coated multivesicular liposomes as example materials for testing and examination
Example 1
Weighing 500g of water, 10g of phospholipase A2, 10g of human oligopeptide-1, 10g of sucrose and 10g of chitosan, and stirring at normal temperature for 5min to obtain an internal water phase for later use;
weighing 50g of cholesterol, 1g of methylparaben, 1g of 2, 6-di-tert-butyl-p-cresol, 200g of dichloromethane, 50g of soybean phospholipid and 10g of olive oil, and stirring at normal temperature for 5min to obtain an oil phase for later use;
weighing 10g of glucose and 100g of water, and stirring at normal temperature for 5min to obtain an external water phase for later use;
measuring 200ml of internal water phase and 200ml of oil phase, keeping the temperature at 30 ℃, mixing and stirring at the stirring speed of 10000r/min for 5min to obtain W/O primary emulsion;
weighing 100mlW/O colostrum and 100ml external water phase, adding the external water phase into the W/O colostrum, stirring at 500r/min, and stirring at room temperature for 1min to obtain compound emulsion.
And (3) maintaining the temperature at 30 ℃, adding the multiple emulsion into a rotary evaporator, maintaining the rotating speed at 10r/min, and evaporating at normal temperature until no organic solvent is evaporated to obtain the phospholipase-coated multivesicular liposome 1.
Example 2
Weighing 2000g of water, 1g of phospholipase A2, 25g of human oligopeptide-1, 5g of sucrose and 5g of chitosan, and stirring at normal temperature for 10min to obtain an inner water phase for later use;
weighing 55g of cholesterol, 5g of methyl p-hydroxybenzoate, 2g of 2, 6-di-tert-butyl-p-cresol, 500g of trichloromethane, 15g of glyceryl monostearate and 1g of olive oil, and stirring at normal temperature for 10min to obtain an oil phase for later use;
weighing 10g of sodium chloride, 10g of trehalose and 100g of water, and stirring at normal temperature for 10min to obtain an external water phase for later use;
measuring 200ml of internal water phase and 200ml of oil phase, keeping the temperature at 50 ℃, mixing and stirring at the stirring speed of 15000r/min for 15min to obtain W/O primary emulsion;
weighing 100mlW/O colostrum and 100ml external water phase, adding the external water phase into the W/O colostrum, stirring at 1000r/min, and stirring at room temperature for 5min to obtain compound emulsion.
And (3) keeping the temperature at 40 ℃, adding the multiple emulsion into a rotary evaporator, keeping the rotating speed at 50r/min, and evaporating at normal temperature until no organic solvent is evaporated to obtain the phospholipase-coated multivesicular liposome 2.
Example 3
Weighing 1000g of water, 1g of phospholipase A2, 10g of human oligopeptide-1, 2g of sucrose and 2g of chitosan, and stirring at normal temperature for 7min to obtain an inner water phase for later use;
weighing 52g of cholesterol, 2g of methylparaben, 1g of 2, 6-di-tert-butyl-p-cresol, 300g of carbon tetrachloride, 20g of 16-18 carbon fatty alcohol and 2.5g of olive oil, and stirring at normal temperature for 8min to obtain an oil phase for later use;
weighing 15g of cyclodextrin, 15g of maltose and 100g of water, and stirring at normal temperature for 5min to obtain an external water phase for later use;
weighing 200ml of internal water phase and 200ml of oil phase, keeping the temperature at 40 ℃, mixing and stirring at the stirring speed of 12000r/min for 5min to obtain W/O primary emulsion;
weighing 100mlW/O colostrum and 100ml external water phase, adding the external water phase into the W/O colostrum, stirring at 800r/min, and stirring at room temperature for 3min to obtain compound emulsion.
And (3) maintaining the temperature at 35 ℃, adding the multiple emulsion into a rotary evaporator, maintaining the rotating speed at 40r/min, and evaporating at normal temperature until no organic solvent is evaporated to obtain the phospholipase-coated multivesicular liposome 3.
Example 4
Weighing 500g of water, 1g of phospholipase A2, 15g of human oligopeptide-1, 3g of sucrose and 3g of chitosan, and stirring at normal temperature for 5min to obtain an inner water phase for later use;
weighing 54g of cholesterol, 4g of methyl paraben, 292, 6-di-tert-butyl-p-cresol, 400g of dichloromethane, 15g of isopropyl palmitate and 1.25g of olive oil, and stirring at normal temperature for 5min to obtain an oil phase for later use;
weighing 10g of pentasodium triphosphate, 5g of glycine, 5g of lysine and 100g of water, and stirring at normal temperature for 6min to obtain an external water phase for later use;
weighing 200ml of internal water phase and 200ml of oil phase, keeping the temperature at 30 ℃, mixing and stirring at the stirring speed of 14000r/min for 5min to obtain W/O primary emulsion;
weighing 100mlW/O colostrum and 100ml external water phase, adding the external water phase into the W/O colostrum, stirring at 200r/min, and stirring at room temperature for 1min to obtain compound emulsion.
And (3) keeping the temperature at 45 ℃, adding the multiple emulsion into a rotary evaporator, keeping the rotating speed at 30r/min, and evaporating at normal temperature until no organic solvent is evaporated to obtain the phospholipase-coated multivesicular liposome 4.
The phospholipase-coated multivesicular liposomes 1 to 4 were subjected to particle size measurement, and the structure of the sample was observed by an optical microscope, and the average particle size of the phospholipase-coated multivesicular liposomes was evaluated by measuring 100 particles with a low particle size under a light microscope.
Respectively taking a certain amount of phospholipase-coated multivesicular liposome 1-4, placing in a centrifugal tube with scales, centrifuging for 15min, observing layering conditions, reading the total volume of the emulsion in the tube and the volume of the outer water phase sucked out, and calculating the retention rate of the emulsion layer.
Respectively taking a certain amount of the phospholipase-coated multivesicular liposome 1-4, standing at 4 ℃ for 2 months, and observing the retention rate of the emulsion layer.
Diluting 25ml of phospholipase-coated multivesicular liposome with deionized water by 5 times, centrifuging for 20min, sucking 5ml of upper layer emulsion, adding ethanol-cyclohexane (1: 1), shaking until a coating film layer is completely separated out, centrifuging for 20min, sucking 1ml of water layer, detecting the total content of phospholipase A2, sucrose, chitosan and human oligopeptide-1 in the water layer by liquid chromatography, and calculating the embedding rate.
The results of the tests are shown in the following table:
the data show that the phospholipase-coated multivesicular liposome has small particle size, can effectively avoid the austenite curing effect, has higher stability, has higher emulsion layer retention rate under the centrifugal condition or the standing condition, is not easy to break emulsion, and can keep the activity of the vitamin and the human oligopeptide-1 coated by the inner water phase for a long time.
Example 5
Weighing 1000g of water, 1g of phospholipase A2, 10g of human oligopeptide-1, 2g of sucrose, 2g of chitosan, 10g of vitamin B1, 5g of vitamin B6, 1g of vitamin B12, 3g of vitamin H, 45g of vitamin C and 20g of serine, and stirring at normal temperature for 6min to obtain an inner water phase for later use;
weighing 52g of cholesterol, 2g of methylparaben, 1g of 2, 6-di-tert-butyl-p-cresol, 300g of dichloromethane, 20g of 16-18 carbon fatty alcohol and 2.5g of olive oil, and stirring at normal temperature for 8min to obtain an oil phase for later use;
weighing 15g of cyclodextrin, 15g of maltose and 100g of water, and stirring at normal temperature for 5min to obtain an external water phase for later use;
weighing 200ml of internal water phase and 200ml of oil phase, keeping the temperature at 40 ℃, mixing and stirring at the stirring speed of 12000r/min for 5min to obtain W/O primary emulsion;
weighing 100mlW/O colostrum and 100ml external water phase, adding the external water phase into the W/O colostrum, stirring at 800r/min, and stirring at room temperature for 3min to obtain compound emulsion.
And (3) keeping the temperature at 35 ℃, adding the multiple emulsion into a rotary evaporator, keeping the rotating speed at 40r/min, and evaporating at normal temperature until no organic solvent is evaporated to obtain the phospholipase-coated multivesicular liposome 5.
Example 6
Weighing 1000g of water, 1g of phospholipase A2, 10g of human oligopeptide-1, 2g of sucrose, 2g of chitosan, 15g of vitamin B1, 15g of vitamin B6, 6g of vitamin B12, 10g of vitamin H, 60g of vitamin C and 30g of serine, and stirring at normal temperature for 6min to obtain an inner water phase for later use;
weighing 52g of cholesterol, 2g of methylparaben, 1g of 2, 6-di-tert-butyl-p-cresol, 300g of dichloromethane, 20g of 16-18 carbon fatty alcohol and 2.5g of olive oil, and stirring at normal temperature for 8min to obtain an oil phase for later use;
weighing 15g of cyclodextrin, 15g of maltose and 100g of water, and stirring at normal temperature for 5min to obtain an external water phase for later use;
weighing 200ml of internal water phase and 200ml of oil phase, keeping the temperature at 40 ℃, mixing and stirring at the stirring speed of 12000r/min for 5min to obtain W/O primary emulsion;
weighing 100mlW/O colostrum and 100ml external water phase, adding the external water phase into the W/O colostrum, stirring at 800r/min, and stirring at room temperature for 3min to obtain compound emulsion.
And (3) keeping the temperature at 35 ℃, adding the multiple emulsion into a rotary evaporator, keeping the rotating speed at 40r/min, and evaporating at normal temperature until no organic solvent is evaporated to obtain the phospholipase-coated multivesicular liposome 6.
Comparative example: a commercially available skin care product was used.
30 female volunteers aged 22-28 years were recruited, examples 5-6 and comparative example were applied to the backs of the hands of 10 different volunteers, one each in the morning and evening, for 10 consecutive days, and moisture testing and appearance recording were performed on the dorsal skin of the hands using a fiscal tester, with the following results:
the data result shows that the skin care product prepared by the phospholipase-coated multi-microcapsule liposome has a good using effect, and the effect is particularly obvious compared with the conventional skin care product.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. The phospholipase-coated multivesicular liposome is characterized by comprising an inner water phase, an oil phase and an outer water phase, wherein the inner water phase comprises water and phospholipase A2 in a mass ratio of (50-2000) to 1, the oil phase comprises an additive and lipid in a mass ratio of (0.1-1) to 1, and the outer water phase comprises a thickening agent and water in a mass ratio of (0.1-0.3) to 1.
2. The phospholipase-coated multivesicular liposome of claim 1, wherein the internal aqueous phase further comprises human oligopeptide-1, sucrose and chitosan, and wherein the mass ratio of human oligopeptide-1: sucrose: chitosan: phospholipase A2 is (1-25): 1-5: 1.
3. The phospholipase-coated multivesicular liposome of claim 1, wherein the additive comprises cholesterol, methyl paraben, 2, 6-di-t-butyl-p-cresol, and an organic solvent in a mass ratio of (50-55) to (1-5) to (1-2) to (200-500).
4. The phospholipase-coated multivesicular liposome of claim 3, wherein the organic solvent is a halogenated hydrocarbon.
5. The phospholipase-coated multivesicular liposome of claim 1, wherein the lipid comprises amphipathic and neutral lipids in a mass ratio of (5-15): 1.
6. The phospholipase-coated multivesicular liposome of claim 5, wherein the amphiphilic lipid is one or more of soybean phospholipid, glycerol monostearate, 16-18 carbon fatty alcohol, and isopropyl palmitate.
7. The phospholipase-coated multivesicular liposome of claim 5, wherein the neutral lipid is olive oil.
8. The phospholipase-coated multivesicular liposome of claim 1, wherein the thickening agent is one or more of glucose, sodium chloride, trehalose, cyclodextrin, maltose, pentasodium triphosphate, glycine, and lysine.
9. The preparation method of the phospholipase-coated multivesicular liposome is characterized by comprising the following steps:
step one, mixing water, phospholipase A2, human oligopeptide-1, sucrose and chitosan according to a mass ratio, and stirring at normal temperature for 5-10min to obtain an internal water phase;
mixing cholesterol, methyl paraben, 2, 6-di-tert-butyl-p-cresol, an organic solvent and lipid according to a mass ratio, and stirring at normal temperature for 5-10min to obtain an oil phase;
step three, mixing the thickening agent and water according to the mass ratio, and stirring for 5-10min at normal temperature to obtain an external water phase;
step four, mixing the internal water phase and the oil phase with the same volume, and stirring for 5-15min at the stirring speed of 10000-15000r/min and the temperature of 30-50 ℃ to obtain W/O primary emulsion;
step five, adding the external water phase with the same volume into the W/O primary emulsion, keeping the stirring speed at 500-;
and sixthly, maintaining the rotating speed at 10-50r/min at the temperature of 30-40 ℃, and performing rotary evaporation until no organic solvent is evaporated to obtain the phospholipase-coated multivesicular liposome.
10. The use of the phospholipase-coated multivesicular liposome of claim 1 in a skin care product, wherein the internal aqueous phase further comprises vitamin B1, vitamin B6, vitamin B12, vitamin H, vitamin C, and serine, and the mass ratio of vitamin B1, vitamin B6, vitamin B12, vitamin H, vitamin C, serine, and phospholipase A2 is (10-15), (5-15), (1-6), (3-10), (45-60), (20-30), and (1).
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