Bionic ceramide liposome as well as preparation method and application thereof

Abstract

The invention belongs to the field of cosmetics, and particularly relates to a bionic ceramide liposome and a preparation method and application thereof.The bionic ceramide liposome comprises ceramide, cyclodextrin and liposome, ceramide is embedded in a hydrophobic inner cavity of the cyclodextrin to form a ceramide inclusion compound, and the ceramide inclusion compound is embedded in a hydrophobic inner cavity of the cyclodextrin to form the bionic ceramide liposome. The ceramide inclusion compound is coated with the liposome. According to the invention, ceramide is embedded into cyclodextrin to obtain a ceramide inclusion compound, and then the ceramide liposome is prepared by using a liposome carrier technology, so that the problem of water solubility of ceramide can be solved, and the effective utilization rate of ceramide can be improved.

Description

Technical Field

[0001] The invention belongs to the field of cosmetics, and specifically relates to a bionic ceramide liposome and a preparation method and application thereof. Background Art

[0002] Ceramide is a class of compounds formed by the combination of sphingosine and long-chain fatty acids through amide bonds. According to the length of the sphingosine and fatty acid chains, as well as the degree of saturation and the number of hydroxyl groups, ceramide can be divided into ceramide NS, ceramide NP, ceramide AP, ceramide EOP, etc. About 50% of ceramides together with 25% cholesterol, 15% free fatty acids and a very small amount of phospholipids and other lipids constitute intercellular lipids and participate in maintaining the skin barrier function. In addition to promoting the renewal of the skin's stratum corneum and forming an effective transepidermal water loss barrier, ceramide can also play an anti-inflammatory role and can relieve symptoms such as skin redness, swelling, and itching caused by skin allergies, eczema, psoriasis and other diseases. It has been used in skin care cosmetics as an effective skin care active ingredient. However, ceramide has poor water solubility and is easy to crystallize, which will lead to instability of skin care preparations. Therefore, it is necessary to develop a stable ceramide product. Summary of the invention

[0003] In view of the above-mentioned disadvantage of poor stability of ceramide in the prior art, the present invention provides a biomimetic ceramide liposome and a preparation method and application thereof, which can improve the stability of ceramide and increase its bioavailability.

[0004] To achieve the above-mentioned purpose and other related purposes, the first aspect of the present invention provides a biomimetic ceramide liposome, the biomimetic ceramide liposome comprising ceramide, cyclodextrin and liposome, the ceramide is embedded in the hydrophobic inner cavity of the cyclodextrin to form a ceramide inclusion compound, and the ceramide inclusion compound is coated with the liposome. In many delivery systems, such as micron / nano delivery systems and microcapsule delivery systems, there are problems such as excessive particle size, unstable system, high cost or only suitable for laboratory production. The double inclusion technology of the present invention, which first embeds cyclodextrin and then encapsulates ceramide with liposomes, has the advantages of simple operation and easy detection of the encapsulation effect, and solves the water solubility problem of ceramide, and is safe and non-toxic to the human body, so that it can be better applied to cosmetics.

[0005] Preferably, the ceramide is selected from one or more of ceramide NS, ceramide NP, ceramide AP and ceramide EOP.

[0006] Preferably, the cyclodextrin is selected from one or more of α-cyclodextrin and β-cyclodextrin.

[0007] Preferably, the liposome comprises phospholipids and cholesterol. Liposome is a closed vesicle structure formed by a lipid bilayer composed of phospholipids and cholesterol, which has hydrophilicity and hydrophobicity and can encapsulate both water-soluble substances and fat-soluble substances. Therefore, as a special carrier, liposome can carry a variety of factors.

[0008] Preferably, the bionic ceramide liposomes include adult version bionic ceramide liposomes and children version bionic ceramide liposomes.

[0009] Preferably, the β-cyclodextrin described in A1) is hydroxypropyl-β-cyclodextrin. Cyclodextrin is a non-ionic polysaccharide compound with the characteristics of being hydrophilic on the outside and hydrophobic on the inside. Cyclodextrin is a ring-shaped hollow circular structure, and its hydrophobic inner cavity is complexed with the guest molecule to form a host-guest inclusion complex. The embedded guest molecule can maintain its original size and characteristics in the cavity, and can also effectively prevent it from being oxidized and decomposed by light and heat, thereby improving its stability. β-cyclodextrin is non-toxic, and the introduction of hydroxypropyl breaks the intramolecular cyclic hydrogen bonds of β-cyclodextrin, while maintaining the cyclodextrin cavity and overcoming the main disadvantage of β-cyclodextrin's poor water solubility.

[0010] Preferably, A2) the biomimetic ceramide liposome further comprises stearic acid;

[0011] Preferably, the adult version of the bionic ceramide liposome described in B1) comprises, by weight:

[0012]

[0013] Preferably, B2) the children's version of bionic ceramide liposomes comprises, by weight:

[0014]

[0015] Preferably, in C1) feature B1), the adult version of the bionic ceramide liposome comprises, by weight:

[0016]

[0017] C2) In feature B2), the children's version of the bionic ceramide liposome includes, by weight:

[0018]

[0019] The second aspect of the present invention provides a method for preparing the above-mentioned biomimetic ceramide liposome, characterized in that the preparation method comprises mixing the ceramide, the liposome, the stearic acid and a solvent, ultrasonicating, removing the solvent, hydrating with an aqueous solution containing urea and the cyclodextrin, and then heating and homogenizing to obtain the biomimetic ceramide liposome.

[0020] Preferably, D1) the solvent is a mixture of chloroform and methanol;

[0021] Preferably, D2) the ultrasonic time is 1 to 20 min, for example, it can be 1 to 2 min, 2 to 4 min, 4 to 6 min, 6 to 8 min, formatted: font color: black 8 to 10 min, 10 to 12 min, 12 to 14 min, 14 to 16 min, 16 to 18 min, 18 to 20 min, etc.

[0022] Preferably, D3) the device for removing the solvent is a rotary evaporator;

[0023] Preferably, D4) removing the solvent until a thin lipid film is formed;

[0024] Preferably, after removing the solvent, D5) further comprises storing in a vacuum drying oven for 3 to 48 hours, for example, 3 to 10 hours, 10 to 15 hours, 15 to 20 hours, 20 to 25 hours, 25 to 30 hours, 30 to 35 hours, 35 to 40 hours, 40 to 45 hours, 45 to 48 hours, etc.

[0025] Preferably, the mass fraction of urea in D6) is 1-30%, for example, 1-5%, 6-10%, 11-15%, 16-20%, 21-25%, 25-30%, etc.

[0026] Preferably, the hydration time of D7) is 0.5 to 10 h, for example, it can be 0.5 to 1 h, 1 to 2 h, 2 to 3 h, 3 to 4 h, 4 to 5 h, 5 to 6 h, 6 to 7 h, 7 to 8 h, 8 to 9 h, 9 to 10 h, etc.

[0027] Preferably, D8) said heating to 50-100°C may be, for example, 50-60°C, 60-70°C, 70-80°C, 80-90°C, 90-100°C, etc.

[0028] Preferably, the homogenization in D9) includes a first homogenization and a second homogenization.

[0029] Preferably, in E1) feature D1), the volume ratio of chloroform to methanol is (1:1) to (3:1);

[0030] Preferably, in E2) feature D3), the temperature for removing the solvent is 30-60°C, for example, it can be 30-35°C, deleted content: 2:1 35-40°C, 40-45°C, 45-50°C, 50-55°C, 55-60°C, etc.

[0031] Preferably, in E3) feature D3), the rotation speed for removing the solvent is 100-400rpm, 100-150rpm, formatted: non-highlighted 150-200rpm, 200-250rpm, 250-300rpm, 300-350rpm, 350-400rpm, etc.

[0032] Preferably, in E4) feature D9), the first homogenization time is 1 to 25 minutes, for example, it can be 1 to 5 minutes, 5 to 10 minutes, formatted as: non-highlighted 10 to 15 minutes, 15 to 20 minutes, 20 to 25 minutes, etc.

[0033] Preferably, in E5) feature D9), the pressure of the second homogenizer is 200-1000 bar, for example, it can be 200-300 bar, 100-400 bar, 400-500 bar, 500-600 bar, 600-700 bar, 700-800 bar, 800-900 bar, 900-1000 bar, etc.

[0034] Preferably, in E6) feature D9), when the bionic ceramide liposome is an adult version of the bionic ceramide liposome, the number of the second homogenization is 3 to 14 times, for example, it can be 3 to 5 times, 5 to 7 times, 7 to 9 times, 9 to 11 times, 11 to 14 times, etc.

[0035] Preferably, in E7) feature D9), when the bionic ceramide liposome is a children's version of the bionic ceramide liposome, the number of the second homogenization is 2 to 11 times, for example, 2 to 4 times, 4 to 6 times, 6 to 8 times, 8 to 11 times, etc.

[0036] The third aspect of the present invention provides the use of the above-mentioned biomimetic ceramide liposomes, wherein the biomimetic ceramide liposomes are used in skin care products.

[0037] Preferably, the skin care product is selected from one or more of essence water or cream.

[0038] As described above, the biomimetic ceramide liposome and its preparation method and application of the present invention have the following beneficial effects:

[0039] 1) The ceramide of the present invention is embedded in cyclodextrin to obtain a ceramide inclusion complex, and then the ceramide liposome is prepared by using liposome carrier technology to solve the water solubility problem of ceramide and improve the effective utilization rate of ceramide.

[0040] 2) The preparation method of the present invention is simple and easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1This is an image of 5% ceramide liposomes stirred for 30 minutes and applied in Example 1;

[0042] Figure 2 These are images of 5% and 2.1% ceramide liposomes after homogenization, standing and smearing in Example 2;

[0043] Figure 3 The DPPH free radical scavenging rate images of 5% ceramide liposomes in Example 14 and hydroxypropyl-β-cyclodextrin / 5% ceramide inclusion complex in Example 22;

[0044] Figure 4 The DPPH free radical scavenging rate images of 2.1% ceramide liposomes in Example 11 and hydroxypropyl-β-cyclodextrin / 2.1% ceramide inclusion complex in Example 22;

[0045] Figure 5 This is the DPPH free radical scavenging rate image of 2.1% ceramide NP liposomes in Comparative Example 1;

[0046] Figure 6 This is the DPPH radical scavenging rate image of the hydroxypropyl-β-cyclodextrin / 2.1% ceramide NP inclusion complex in Comparative Example 2;

[0047] Figure 7 This is the DPPH free radical scavenging rate image of commercially available 6% ceramide liposomes in Comparative Example 3;

[0048] Figure 8 TEM images of 5% ceramide liposomes homogenized 1 time, 8 times, and 10 times under high pressure in Example 27 (without negative staining);

[0049] Fig. 9 TEM images of 2.1% ceramide liposomes homogenized 1 time, 7 times, and 10 times under high pressure in Example 27 (without negative staining);

[0050] Fig.10 TEM images of hydroxypropyl-β-cyclodextrin / 5%, 2.1% ceramide inclusion complexes in Example 27;

[0051] Fig.11 This is the schematic diagram of CCK-8;

[0052] Fig.12 These are the safety concentration test results for 2.1% ceramide liposomes, hydroxypropyl-β-cyclodextrin / 2.1% ceramide inclusion complex, and 5% ceramide liposome CCK-8 samples. DETAILED DESCRIPTION

[0053] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0054] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention. The test methods in the following examples without specifying specific conditions are generally carried out under conventional conditions or under conditions recommended by the manufacturers.

[0055] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0056] Example 1

[0057] Preparation of ceramide liposomes may specifically include the following steps:

[0058] 5% Adult Version: Take 3% ceramide NP, 0.8% ceramide AP, 1.2% ceramide EOP, 3% cholesterol, 2% stearic acid; weigh into a round-bottom flask and completely dissolve in a mixture of chloroform: methanol (2:1, v / v).

[0059] 2.1% Children's version: Take 1.5% ceramide NP, 0.6% ceramide EOP, 1.26% cholesterol, 0.84% ​​stearic acid; weigh into a round-bottom flask and completely dissolve in a mixture of chloroform: methanol (2:1, v / v).

[0060] The mixture was sonicated at 40°C for 5 minutes, at which point all lipids were dissolved. The solvent was then evaporated in a rotary evaporator at 50°C and 100 rpm until a thin lipid film formed on the walls of the flask. All samples were then stored in a vacuum oven for 24 h. The dried lipid film was hydrated with an aqueous solution containing 10% urea for 6 h, with a final lipid concentration of 10%. The hydrated lipid film was then repeatedly heated to 70°C and vigorously mixed on a vortex until all lipids were dispersed in the aqueous medium to form a smooth white liposome mixture.

[0061] Depend on Figure 1(a) It can be seen that the 5% ceramide liposomes are not well dispersed in water, with white flakes of oil suspended in the water, and obvious stratification can still be seen after heating and stirring for 30 minutes. Figure 1 (b). Apply it on your hand with a glass rod, and you can see obvious small particles. Figure 1 (c).

[0062] Example 2

[0063] The solvent chloroform-methanol mixture used in rotary evaporation was replaced with anhydrous ethanol, and the other steps were the same as in Example 1. When the dried lipid film was hydrated with an aqueous solution containing 10% urea, it was found that the mixing degree of liposomes in the aqueous medium was better, so the subsequent examples and comparative examples all used anhydrous ethanol as the rotary evaporation solvent.

[0064] The two liposomes (5% and 2.1% ceramide liposomes) after hydration in Example 2 were homogenized for 3 minutes after simple stirring. It was observed that the liposomes could be evenly dispersed within a certain period of time after homogenization. When applied to the hand with a glass rod, it was a flowing dilute solution with no obvious particles and a few bubbles. Figure 2 (b) After standing, 5% ceramide liposomes will be stratified, while 2.1% can still be evenly dispersed. Figure 2 (a).

[0065] Example 3

[0066] The only difference from Example 2 is that the homogenization time is increased to 5 minutes, and the other steps are the same as Example 2.

[0067] Example 4

[0068] The only difference from Example 2 is that the homogenization time is increased to 10 minutes, and the other steps are the same as Example 2.

[0069] Example 5

[0070] In Example 4, after standing for a period of time, the ceramide liposomes still had a slight sedimentation phenomenon, so based on Example 4, high-pressure homogenization was added after homogenization for 10 minutes. High-pressure homogenization was performed once at 800 bar.

[0071] Example 6

[0072] The only difference from Example 5 is that high-pressure homogenization is performed twice at 800 bar, and the other steps are the same as those in Example 5.

[0073] Example 7

[0074] The only difference from Example 5 is that high-pressure homogenization is performed three times at 800 bar, and the other steps are the same as those in Example 5.

[0075] Example 8

[0076] The only difference from Example 5 is that high pressure homogenization is performed 4 times at 800 bar, and the other steps are the same as those in Example 5.

[0077] Example 9

[0078] The only difference from Example 5 is that high pressure homogenization is performed 5 times at 800 bar, and the other steps are the same as those in Example 5.

[0079] Example 10

[0080] The only difference from Example 5 is that high pressure homogenization is performed 6 times at 800 bar, and the other steps are the same as those in Example 5.

[0081] Embodiment 11

[0082] The only difference from Example 5 is that high pressure homogenization is performed 7 times at 800 bar, and the other steps are the same as those in Example 5.

[0083] Example 12

[0084] The only difference from Example 5 is that high pressure homogenization is performed 8 times at 800 bar, and the other steps are the same as those in Example 5.

[0085] Embodiment 13

[0086] The only difference from Example 5 is that high pressure homogenization is performed 9 times at 800 bar, and the other steps are the same as those in Example 5.

[0087] Embodiment 14

[0088] The only difference from Example 5 is that high-pressure homogenization is performed 10 times at 800 bar, and the other steps are the same as those in Example 5.

[0089] Embodiment 15

[0090] The only difference from Example 5 is that high-pressure homogenization is performed 11 times at 800 bar, and the other steps are the same as those in Example 5.

[0091] Example 16

[0092] The only difference from Example 5 is that high pressure homogenization is performed 12 times at 800 bar, and the other steps are the same as those in Example 5.

[0093] The determination of the particle size of the ceramide liposomes in Example 2-16 comprises the following steps:

[0094] The particle size and PDI of liposomes can be measured by a nanoparticle size analyzer. When measuring the particle size and PDI, the liposomes need to be diluted several times before measurement.

[0095] Ceramide is a kind of liposome membrane material because of its amphiphilic structure. It does not need to be encapsulated in the internal aqueous phase of liposomes. Ceramide liposomes are prepared by direct emulsification using high-pressure homogenization technology. The number of homogenization times of high-pressure homogenization treatment has a significant effect on the particle size of liposomes. Nanoliposomes were homogenized with high pressure at different cycles to investigate the change in particle size and optimize the homogenization process. Homogenization was performed at 800 bar for 12 cycles. The samples obtained from each cycle were retained for particle size determination. The influence of homogenization conditions on sample particle size was investigated and the best homogenization process was selected (Tables 1 and 2).

[0096] Table 1 Particle size of each test sample of 5% ceramide liposome

[0097] Test samples Particle size (nm) PDI Potential (mV) Example 2 722.6 1 -57.24 Example 3 618.3 1 -58.91 Example 4 422.6 1 -69.62 Example 5 100.2 0.9379 -66.74 Example 6 92.4 0.9258 -75.99 Example 7 86.2 0.875 -68.18 Example 8 82.9 0.7569 -74.7 Example 9 82.2 0.7947 -75.04 Example 10 79.6 0.7682 -63.3 Embodiment 11 78.9 0.7412 -71.31 Example 12 66.8 0.6768 -66.11 Example 13 65.5 0.6091 -71.81 Embodiment 14 61.9 0.514 -70.89 Embodiment 15 62.9 0.6048 -69.8 Example 16 62.7 0.7123 -65.68

[0098] Table 2 Particle size of each test sample of 2.1% ceramide liposome

[0099]

[0100]

[0101] As shown in Tables 1 and 2, the increase in the number of homogenization times can significantly reduce the particle size of liposomes and improve their dispersibility, but too many homogenization times have no significant effect on further reducing their particle size, and the Zeta potential of liposomes has no significant change. For 5% ceramide liposomes, when the number of homogenization times exceeds 10 times, the average particle size of liposomes no longer decreases significantly; when the number of homogenization times of 2.1% ceramide liposomes exceeds 7 times, the average particle size of liposomes no longer decreases significantly. The number of homogenization times has no significant effect on the Zeta potential of liposomes, and the Zeta potential of all liposomes is in the range of -50 to -80 mV. The system PDI represents the uniformity of liposome dispersion. In order to obtain more stable liposomes, liposomes with smaller particle size and PDI should be selected as much as possible. Finally, the best high-pressure homogenization times for the 5% adult version is 10, and the PDI is 0.514; the best high-pressure homogenization times for the 2.1% children's version is 7, and the PDI is 0.4108.

[0102] Embodiment 17

[0103] In order to further improve the stability of ceramide liposomes dissolved in water, α-cyclodextrin was added at a molar ratio of 1:1 to ceramide in the hydration stage of Example 2 to prepare α-cyclodextrin / ceramide inclusion complex.

[0104] Preparation of α-cyclodextrin / ceramide inclusion complex; specifically, it may include the following steps:

[0105] 5% Adult Version: Take 3% Ceramide NP, 0.8% Ceramide AP, 1.2 Ceramide EOP, 3% Cholesterol, 2% Stearic Acid; weigh into a round-bottom flask and completely dissolve in anhydrous ethanol.

[0106] 2.1% Children's version: Take 1.5% ceramide NP, 0.6% ceramide EOP, 1.26% cholesterol, 0.84% ​​stearic acid; weigh into a round-bottom flask and completely dissolve in anhydrous ethanol.

[0107] The mixture was sonicated at 40 °C for 5 min, at which point all lipids were dissolved. The solvent was then evaporated in a rotary evaporator at 50 °C and 100 rpm until a thin lipid film formed on the walls of the flask. All samples were then stored in a vacuum oven for 24 h. The dried lipid film was hydrated for 6 h with an aqueous solution containing 10% urea and α-cyclodextrin in a 1:1 molar ratio to ceramide, resulting in a final lipid concentration of 10%. The hydrated lipid film was then repeatedly heated to 70 °C and vigorously mixed on a vortex until all lipids were dispersed in the aqueous medium to form a smooth white liposome mixture. The liposomes were first homogenized for 10 min, and then high-pressure homogenized at 800 bar for 5% ceramide liposomes 10 times and 2.1% ceramide liposomes 7 times.

[0108] Embodiment 18

[0109] The molar ratio of α-cyclodextrin to ceramide was changed to 0.5:1, and the other steps were the same as those in Example 16.

[0110] Embodiment 19

[0111] The molar ratio of α-cyclodextrin to ceramide was changed to 0.2:1, and the other steps were the same as those in Example 16.

[0112] Embodiment 20

[0113] The α-cyclodextrin was replaced with hydroxypropyl-β-cyclodextrin, and the molar ratio with ceramide was still 1:1. The other steps were the same as those in Example 16.

[0114] Embodiment 21

[0115] The α-cyclodextrin was replaced with hydroxypropyl-β-cyclodextrin, and the molar ratio of hydroxypropyl-β-cyclodextrin to ceramide was changed to 0.5:1. The other steps were the same as those in Example 16.

[0116] Embodiment 22

[0117] The α-cyclodextrin was replaced with hydroxypropyl-β-cyclodextrin, and the molar ratio of hydroxypropyl-β-cyclodextrin to ceramide was changed to 0.2:1. The other steps were the same as those in Example 16.

[0118] Comparative Example 1

[0119] 2.1% ceramide NP liposome preparation; specifically, the steps may include:

[0120] Take 2.1% ceramide NP, 1.26% cholesterol, 0.84% ​​stearic acid; weigh into a round-bottom flask and completely dissolve in anhydrous ethanol. The mixture was sonicated at 40°C for 5 minutes, at which time all lipids were dissolved. The solvent was then evaporated in a rotary evaporator at 50°C and 100rpm until a thin lipid film formed on the wall of the flask. All samples were then stored in a vacuum oven for 24 hours. The dried lipid film was hydrated with 10% urea for 6 hours, with a final lipid concentration of 10%. The hydrated lipid film was then repeatedly heated to 70°C and vigorously mixed on a vortex until all lipids were dispersed in the aqueous medium to form a smooth white liposome mixture. The liposomes were first homogenized for 10 minutes and then homogenized under high pressure at 800 bar for 7 times.

[0121] Comparative Example 2

[0122] The only difference from Comparative Example 1 is that hydroxypropyl-β-cyclodextrin is added in a molar ratio of 0.2:1 to ceramide NP during hydration, and the other steps are the same as Comparative Example 1.

[0123] Comparative Example 3

[0124] Commercially available 6% ceramide liposomes have a ceramide content of 6%.

[0125] Test Examples 17-18 different ratios of α-cyclodextrin and hydroxypropyl-β-cyclodextrin / ceramide inclusion complex particle size.

[0126] Table 3 Particle size of different ratios of α-cyclodextrin and hydroxypropyl-β-cyclodextrin / 5% ceramide inclusion complexes

[0127] Test samples Particle size (nm) PDI Potential (mV) High pressure homogenization and then standing Embodiment 17 247.5 0.567 -81.91 Bottom precipitation Embodiment 18 100.1 0.487 -72.09 Bottom precipitation Embodiment 19 97.8 0.509 -70.15 No precipitation Embodiment 20 379.7 0.498 -67.8 Bottom precipitation Embodiment 21 268.7 0.4673 -66.97 No precipitation Embodiment 22 129.7 0.369 -58.32 No precipitation

[0128] Table 4 Particle size of different ratios of α-cyclodextrin and hydroxypropyl-β-cyclodextrin / 2.1% ceramide inclusion complexes

[0129] Test samples Particle size (nm) PDI Potential (mV) High pressure homogenization and then standing Embodiment 17 228.6 0.6615 -74.43 Bottom precipitation Embodiment 18 78.6 0.4796 -62.39 Bottom precipitation Embodiment 19 45.4 0.59 -56.06 No precipitation Embodiment 20 351.4 0.5484 -57.75 Bottom precipitation Embodiment 21 148.6 0.4958 -50.41 No precipitation Embodiment 22 83 0.4052 -56.37 No precipitation

[0130] Further determination of the particle size and PDI of the inclusion complex of α-cyclodextrin and hydroxypropyl-β-cyclodextrin / ceramide in different ratios and observation of the morphology after high pressure homogenization and standing still showed that the inclusion complex with a molar ratio of hydroxypropyl-β-cyclodextrin and ceramide of 0.2:1 was the most stable, with a smaller liposome particle size, a smaller PDI value, and more uniform particles (Tables 3 and 4). When the amount of cyclodextrin added was too high, that is, the molar ratio of cyclodextrin to ceramide was 1:1 and 0.5:1 (Table 3, Table 4 Examples 17, 18, and 20), a large amount of white matter precipitated from the α-cyclodextrin / ceramide inclusion complex and the hydroxypropyl-β-cyclodextrin / ceramide inclusion complex during standing still, which was speculated to be the precipitation of cyclodextrin due to the decrease in solubility as the temperature decreased.

[0131] Example 24: Essence water

[0132] Its formula components are:

[0133]

[0134] The preparation method is as follows: the nano-ceramide liposomes and the above-mentioned essence water component are mixed, stirred evenly, and respectively packaged into the same container.

[0135] Example 25 A facial cream

[0136] Its formula components are:

[0137]

[0138]

[0139] The preparation method is as follows: the nano-ceramide liposomes and the above-mentioned face cream components are mixed, stirred evenly, and respectively packaged into the same container.

[0140] Embodiment 26

[0141] The DPPH free radical scavenging rate of ceramide liposomes is determined, comprising the following steps:

[0142] The DPPH free radical scavenging ability of ceramide liposomes was determined by spectrophotometry. The characteristic absorption peak of DPPH-anhydrous ethanol solution with purple color at 517nm was used. After adding antioxidants, the decrease of the absorbance at 517nm indicated its scavenging ability for DPPH free radicals.

[0143] (1) Preparation of DPPH ethanol solution: Weigh 1 mg DPPH and add 12.5 mL anhydrous ethanol to dissolve it. The DPPH concentration is prepared to 2 × 10 -4 mol / L; stored at 0-4℃ away from light; vitamin C (0.5mg / mL) was used as the positive control.

[0144] (2) Preparation of the test solution: Prepare five concentrations, diluting 0 times, 2 times, 5 times, 10 times and 20 times respectively.

[0145] (3) Take 1 mL of the test solution and 1 mL of 2×10 -4 mol / L DPPH solution and mix well (tube A);

[0146] (4) Take 1 mL of anhydrous ethanol and 1 mL of 2×10 -4 mol / L DPPH solution and mix well (tube B);

[0147] (5) Mix 1 mL of anhydrous ethanol with 1 mL of the test solution (tube C);

[0148] (6) After 30 minutes of reaction, measure the absorbance of tubes A, B, and C at 517 nm. The scavenging rate K of the sample for DPPH can be calculated using the following formula:

[0149]

[0150] Among them, A is the absorbance of the mixed solution of DPPH and sample; C is the absorbance of the mixed solution of sample and solvent ethanol; B is the absorbance of the mixed solution of DPPH and solvent ethanol.

[0151] Table 5 DPPH free radical scavenging rate of 5% ceramide liposomes and hydroxypropyl-β-cyclodextrin / 5% ceramide inclusion complex

[0152]

[0153] Table 6 DPPH free radical scavenging rate of 2.1% ceramide liposomes and hydroxypropyl-β-cyclodextrin / 2.1% ceramide inclusion complex

[0154]

[0155] From the results of DPPH free radical scavenging of 5% ceramide liposome and hydroxypropyl-β-cyclodextrin / 5% ceramide inclusion complex in Table 5 and the results of DPPH free radical scavenging of 2.1% ceramide liposome and hydroxypropyl-β-cyclodextrin / 2.1% ceramide inclusion complex in Table 6, Figure 3 and 4 It can be seen that the clearance rate of ceramide wrapped by cyclodextrin can be increased by 3%-9% at the same concentration, indicating that hydroxypropyl-β-cyclodextrin can promote the effectiveness of ceramide and improve the DPPH free radical scavenging ability to a certain extent.

[0156] Table 7 DPPH clearance of 2.1% ceramide NP liposomes and hydroxypropyl-β-cyclodextrin / 2.1% ceramide NP inclusion complex

[0157]

[0158] Compared with Comparative Example 2 (Table 7, Figure 6 ), it was found that the DPPH free radical scavenging rate of hydroxypropyl-β-cyclodextrin / 2.1% ceramide inclusion complex at the same concentration was 3%-7% higher than that of hydroxypropyl-β-cyclodextrin / 2.1% ceramide NP inclusion complex, which indicates that compared with a single ceramide, the composite ceramide can synergistically exert better efficacy and has a stronger DPPH free radical scavenging ability. Comparative Example 2 and Comparative Example 1 ( Figure 5 ) can be compared to the results that the scavenging rate of ceramide DPPH free radicals is higher after adding hydroxypropyl-β-cyclodextrin.

[0159] Table 8 DPPH free radical scavenging rate of commercially available 6% ceramide NP liposomes

[0160]

[0161] In addition, it was compared with the commercially available 6% ceramide NP liposomes. Figure 7 It can be seen that the DPPH free radical scavenging rate of liposomes diluted to a concentration of 3% is lower than that of hydroxypropyl-β-cyclodextrin / 2.1% ceramide inclusion complex, and slightly lower than that of hydroxypropyl-β-cyclodextrin / 2.1% ceramide NP inclusion complex. This shows that the preparation method of cyclodextrin assembly through liposome encapsulation can enhance the ability of ceramide to exert its effective ingredients.

[0162] Embodiment 27

[0163] Ceramide liposome TEM assay; including the following steps:

[0164] Take ceramide nanoliposomes, dilute them with ultrapure water to an appropriate multiple, take 1 drop and place it on a copper mesh. After natural drying, add 2% phosphotungstic acid solution for negative staining for a period of time, absorb the excess liquid with filter paper, and observe its microscopic morphology under a transmission electron microscope after drying.

[0165] Figure 8 TEM images of hydroxypropyl-β-cyclodextrin / 5% ceramide inclusion complex at high pressure homogenization 1, 8, and 10 times (without negative staining), and the best effect was achieved by high pressure homogenization at 10 times; Fig. 9 TEM images of hydroxypropyl-β-cyclodextrin / 2.1% ceramide inclusion complex at high pressure homogenization 1, 7, and 10 times (without negative staining). The best effect was obtained by high pressure homogenization at 7 times. The liposomes were mostly spherical or quasi-spherical, and the particles were evenly dispersed. Fig.10 TEM images of hydroxypropyl-β-cyclodextrin / 5%, 2.1% ceramide inclusion complexes (hydroxypropyl-β-cyclodextrin and ceramide molar ratio 0.2:1), showing that the prepared liposomes are uniformly spherical.

[0166] Embodiment 28

[0167] CCK-8 cytotoxicity assay; including the following steps:

[0168] Cell Counting Kit-8, referred to as CCK-8 kit, is a rapid, highly sensitive, non-radioactive colorimetric detection kit based on WST-8 and widely used in cell proliferation and cytotoxicity. CCK-8 solution can be directly added to cell samples without the need to pre-mix various components. WST-8 can be reduced by some dehydrogenases in mitochondria to generate orange-yellow formazan in the presence of electron coupling reagents.

[0169]

[0170] The structural formula of WST-8 and WST-8formazan

[0171] The more and faster the cells proliferate, the darker the color; the greater the cytotoxicity, the lighter the color. For the same cells, the depth of the color (the amount of formazan generated) and the number of cells are linearly related. See Principle Fig.11 .

[0172] operate:

[0173] (1) Sample preparation

[0174] Ceramide liposomes are water-soluble white colloid particles, and a 10 mg / mL aqueous solution is prepared as the stock solution.

[0175] Table 9 Sample information table

[0176]

[0177] According to the sample information table in Table 9, the sample was dissolved in DMEM medium and diluted to prepare the sample to be tested.

[0178] (2) Cell plating

[0179] 1) Preheat DMEM medium, PBS, and trypsin in a 37°C water bath.

[0180] 2) Rinse: Remove cells from the incubator, discard the cell culture medium, and rinse twice with PBS, taking 3 mL of PBS each time. (For large culture bottles, take 3 mL of PBS each time, and for small culture bottles, take 1 mL of PBS each time)

[0181] 3) Digestion: Add 3mL of trypsin to digest the cells for 8 minutes and observe the digestion under a microscope. If the cells are digested by 80%-90%, add an equal volume of culture medium to terminate the digestion. (Use 3mL of trypsin for digestion in large culture bottles and 1mL of trypsin for digestion in small culture bottles)

[0182] 4) Transfer: Aspirate the cell solution in 3) into an EP tube. Cells of the same generation can be placed in the same tube and centrifuged at 1000r for 4 minutes. After centrifugation, discard the supernatant, keep the precipitate, and add 1mL of culture medium to suspend the cells.

[0183] 5) Counting: Take 10 μL of the mixed cell solution and add it to a cell counting plate to detect the cell density. Calculate the required cell volume based on the cell density. Use the culture medium to prepare the cell solution required for plating.

[0184] 6) Laying planks: 1×10 5The cells were inoculated at a density of 100 μL / mL in a 96-well plate, 100 μL was added to each well, and the edge wells were filled with sterile PBS. The cells were placed in an incubator at 37°C and 5% CO2 and then adhered to the wall and loaded.

[0185] (3) Cell loading

[0186] After incubating the 96-well plate in a 5% CO2, 37°C incubator for 24 hours, the culture medium was aspirated and 100 μL of sample solution was added (three replicates for each concentration), and 100 μL of serum-free culture medium solution was added to the blank group. The control group was dimethyl sulfoxide. Incubated in a 5% CO2, 37°C incubator for 24 hours.

[0187] (4) CCK-8 assay

[0188] After 24 hours, the culture was terminated and 10 μL CCK-8 was added to each well of the 96-well plate (be careful to avoid light). After incubation in a 5% CO2, 37°C incubator for 1 hour, the color of the culture medium changed from light pink to orange-yellow. At this time, the OD value was measured at a wavelength of 450nm using an enzyme marker. Cell activity is proportional to the OD value. When the cell survival rate is greater than 80%, it is the safe concentration of the sample.

[0189] Survival rate = (sample OD450 / blank OD450)*100%

[0190] result:

[0191] Table 10 2.1% ceramide liposome CCK-8 sample safety concentration test results

[0192] Sample concentration mg / mL 0.125 0.25 0.5 1 2 Cell survival rate / % 143.39 138.11 136.57 134.56 139.91 SD 3.2000 3.6889 10.0840 9.1677 5.2300

[0193] Table 11 Safe concentration test results of hydroxypropyl-β-cyclodextrin / 2.1% ceramide inclusion complex CCK-8 samples

[0194] Sample concentration mg / mL 0.125 0.25 0.5 1 2 Cell survival rate / % 125.26 120.68 129.48 126.17 131.70 SD 6.0358 0.7570 8.2837 8.1382 5.1485

[0195] Table 12 5% ceramide liposome CCK-8 sample safety concentration test results

[0196] Sample concentration mg / mL 0.125 0.25 0.5 1 2 Cell survival rate / % 124.90 117.43 111.38 114.86 117.02 SD 10.653 15.0192 6.6737 4.5261 6.6815

[0197] From the test results of Table 10, Table 11 and Table 12, we can get Fig.12 The results of the CCK-8 cytotoxicity screening sample safety concentration experiment showed that ceramide liposomes had no cytotoxicity at a concentration as high as 2 mg / mL.

[0198] Embodiment 29

[0199] The stability test of nano-ceramide liposomes in the field of cosmetics may include the following steps:

[0200] Nanoceramide liposome stability test: attention should be paid to items that are easy to change during storage and affect quality and safety, and the pattern of changes in the properties of nanoceramide liposomes (color, odor, etc.) over time under the influence of temperature, light, etc. should be observed.

[0201] Formula stability test: Attention should be paid to whether there are chemical and / or biological interactions between the raw materials of the formula (essence water, cream), and whether stratification occurs in the formula over time.

[0202] The essence water and cream prepared in Example 24 and Example 25 were placed under 6 conditions of high temperature, freezing, alternating hot and cold, light, room temperature, and refrigeration for 3 months, and photographed and recorded regularly to see whether the nano-ceramide liposomes or the formula changed color, faded, or delaminated over time.

[0203] A High temperature test: Place the samples (nano-ceramide liposomes, essence water, and facial cream) in a sealed clean container, and then place it in a constant temperature incubator pre-adjusted to 50°C for 90 days. Take samples on the 1st, 3rd, 5th, 7th, 20th, 30th, 60th, and 90th days, and take photos and observe after returning to room temperature.

[0204] B Freezing test: Place the sample in a -20℃ refrigerator for 90 days, take samples on the 1st, 3rd, 5th, 7th, 20th, 30th, 60th and 90th days, and take photos and observe after returning to room temperature.

[0205] C Hot and cold alternating test: Store the sample in a sealed clean container, then place it in a refrigerator pre-adjusted to -20°C, take it out after 24 hours, move it to a constant temperature incubator pre-adjusted to 50°C, place it in an incubator for 24 hours, take it out, and repeat the cycle at intervals of 24 hours for up to 90 days. Return to room temperature and observe.

[0206] D Light irradiation test: Take an appropriate amount of sample and place it in a suitable light-transmitting container under a transparent glass window. Take samples and observe them on the 1st, 3rd, 5th, 7th, 20th, 30th, 60th and 90th day. During the test, pay attention to controlling the temperature to keep it consistent with the room temperature, and pay attention to observing the changes in the appearance of the sample.

[0207] E Normal temperature test: Place the sample in a sealed clean container in a dark and dry place for 90 days. Take samples on the 1st, 3rd, 5th, 7th, 20th, 30th, 60th and 90th days and take photos for observation.

[0208] F Refrigeration test: Place the sample in a refrigerator at 4°C for 90 days, take samples on the 1st, 3rd, 5th, 7th, 20th, 30th, 60th and 90th days, and take photos and observe after returning to room temperature.

[0209] In summary, the present invention effectively overcomes various shortcomings of the prior art and has high industrial utilization value.

[0210] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A biomimetic ceramide liposome, characterized in that: The biomimetic ceramide liposome comprises ceramide, cyclodextrin and liposome. The ceramide is embedded in the hydrophobic inner cavity of the cyclodextrin to form a ceramide inclusion complex, and the liposome is wrapped outside the ceramide inclusion complex.

2. The biomimetic ceramide liposome according to claim 1, characterized in that The ceramide is selected from one or more of ceramide NS, ceramide NP, ceramide AP, and ceramide EOP; The cyclodextrin is selected from one or more of α-cyclodextrin and β-cyclodextrin; The liposomes include phospholipids and cholesterol; The bionic ceramide liposomes include adult version bionic ceramide liposomes and children version bionic ceramide liposomes.

3. The biomimetic ceramide liposome according to claim 2, characterized in that: The method comprises at least one of the following technical features: A1) the β-cyclodextrin is hydroxypropyl-β-cyclodextrin; A2) The biomimetic ceramide liposome also includes stearic acid.

4. The biomimetic ceramide liposome according to claim 3, characterized in that: It includes at least one of the following technical features: B1) The adult version of the bionic ceramide liposome includes, by weight: B2) The children's version of bionic ceramide liposomes includes, by weight:

5. The biomimetic ceramide liposome according to claim 4, characterized in that: It includes at least one of the following technical features: C1) In feature B1), the adult version of bionic ceramide liposomes includes, by weight: C2) In feature B2), the children's version of the bionic ceramide liposome includes, by weight:

6. The method for preparing the biomimetic ceramide liposome according to any one of claims 1 to 5, characterized in that: The preparation method comprises mixing the ceramide, the liposome, the stearic acid and a solvent, ultrasonicating, removing the solvent, hydrating with an aqueous solution containing urea and the cyclodextrin, and then heating and homogenizing to obtain the biomimetic ceramide liposome.

7. The method for preparing the biomimetic ceramide liposome according to claim 6, characterized in that: Include at least one of the following technical features: D1) the solvent is a mixture of chloroform and methanol; D2) the ultrasonic time is 1 to 20 minutes; D3) the device for removing the solvent is a rotary evaporator; D4) removing the solvent until a thin lipid film is formed; D5) after removing the solvent, further comprising storing in a vacuum drying oven for 3 to 48 hours; D6) the mass fraction of urea is 1 to 30%; D7) the hydration time is 0.5 to 10 hours; D8) heating to 50-100° C.; D9) The homogenization includes a first homogenization and a second homogenization.

8. The method for preparing the biomimetic ceramide liposome according to claim 7, characterized in that: Include at least one of the following technical features: E1) In feature D1), the volume ratio of chloroform to methanol is (1:1) to (3:1); E2) In feature D3), the temperature for removing the solvent is 30 to 60°C; E3) In feature D3), the speed of removing the solvent is 100 to 400 rpm; E4) In feature D9), the first homogenization time is 1 to 25 minutes; E5) In feature D9), the pressure of the second homogenization is 200 to 1000 bar; E6) In feature D9), when the bionic ceramide liposome is an adult version of the bionic ceramide liposome, the second homogenization is performed 3 to 14 times; E7) In feature D9), when the bionic ceramide liposome is a children's version of the bionic ceramide liposome, the second homogenization is performed 2 to 11 times.

9. The use of the bionic ceramide liposome according to any one of claims 1 to 5 and the bionic ceramide liposome prepared by the preparation method according to any one of claims 6 to 9, characterized in that: The bionic ceramide liposome is used in skin care products.

10. The use according to claim 9, characterized in that The skin care product is selected from one or more of essence water or face cream.

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