Liposome vesicle for inhibiting ergothioneine degradation and preparation method thereof

Abstract

The invention discloses a liposome vesicle for inhibiting ergothioneine degradation and a preparation method thereof, and belongs to the technical field of cosmetics. According to the ergothioneine liposome and the preparation method thereof disclosed by the invention, by constructing an embedding structure, the ergothioneine is embedded in a phospholipid bimolecular layer of the liposome vesicle, and the outer layer of the phospholipid bimolecular layer is also wrapped with a biocompatible high-molecular polymer shell, so that the ergothioneine is doubly wrapped by the phospholipid bimolecular layer and the high-molecular polymer shell of the ergothioneine liposome; and the degradation of ergothioneine is reduced. And the phospholipid bilayer base material for preparing the liposome vesicles can generate ionic interaction with trimethylamine degraded by the ergothioneine, so that the odor problem of the ergothioneine is effectively reduced.

Description

Technical Field

[0001] The invention relates to a liposome vesicle for inhibiting ergothioneine degradation and a preparation method thereof, and belongs to the technical field of cosmetics. Background Art

[0002] Ergothioneine is a natural antioxidant with excellent free radical scavenging ability. It protects cells from free radical damage through antioxidant effect and has anti-aging and anti-wrinkle effects. It inhibits the activity of tyrosinase and inhibits the production of melanin, which has the effect of whitening and lightening spots. In addition, ergothioneine has many functions such as anti-inflammatory, detoxification, and anti-radiation. With its unique biological and pharmacological properties and safety advantages, it has broad application prospects in the fields of cosmetics, functional foods, and biomedicine.

[0003] However, the trimethylamine structure in ergothioneine is easily degraded by light and high temperature. Its degradation product trimethylamine and other amine gases, even at extremely low concentrations, can produce strong smells, affecting the application of the product, resulting in a low addition of ergothioneine. Therefore, how to further improve stability, neutralize or adsorb the amine gases of trace decomposition is an industry difficulty to be solved urgently. In order to reduce the generation of amine taste, at present, mainly by compounding ergothioneine and other raw materials. For example, patent CN115363980A discloses a composition containing ergothioneine and persimmon fruit extract and its application, patent CN116509743A discloses a composition, raw material and external skin preparation for whitening skin, CN117752539A discloses a composition and its application for inhibiting ergothioneine degradation and smell, respectively by compounding persimmon fruit extract, ferulic acid, licorice extract to achieve the effect of inhibiting ergothioneine smell, but by compounding raw materials to suppress the smell of ergothioneine, there is a short time to suppress the smell, and the solution system turns yellow. Summary of the invention

[0004] [Technical issues]

[0005] The invention aims to solve the problems that thioneine has poor light stability, poor thermal stability, generates amine odor, and has a short odor suppression time by compounding thioneine with other raw materials.

[0006] [Technical solution]

[0007] In order to solve the above problems, the present invention provides a liposome vesicle for inhibiting the degradation of thioneine and a preparation method thereof, the liposome vesicle can not only solve the pain point problems of thioneine light instability and thermal instability, but also can keep thioneine from emitting amine taste for a long time, and the preparation method of the liposome vesicle for inhibiting the degradation of thioneine is easy to batch produce.

[0008] The invention provides a liposome vesicle for inhibiting the degradation of ergothioneine. The ergothioneine is embedded in the interior of the phospholipid bilayer of the liposome vesicle by constructing an embedding structure, and the outer layer of the phospholipid bilayer is also wrapped with a biocompatible high molecular polymer shell.

[0009] In one embodiment of the present invention, the substrate used for the phospholipid bilayer is one or more of stearyl phosphate, soybean lecithin, egg yolk lecithin, cetearyl phosphate, tristearyl phosphate, and phospholipids.

[0010] In one embodiment of the present invention, the substrate used for the phospholipid bilayer is stearyl alcohol phosphate and egg yolk lecithin, and the mass ratio of stearyl alcohol phosphate to egg yolk lecithin is 4-10:11.

[0011] In one embodiment of the present invention, the mass ratio of stearyl alcohol phosphate to egg yolk lecithin is 8:11.

[0012] In one embodiment of the present invention, the content of phosphatidylcholine in egg yolk lecithin is above 70%.

[0013] In one embodiment of the present invention, the mass ratio of the substrate used in the phospholipid bilayer to ergothioneine is 15-21:64-112.

[0014] In one embodiment of the present invention, the mass ratio of the substrate used in the phospholipid bilayer to ergothioneine is 19:80.

[0015] In one embodiment of the present invention, the biocompatible high molecular polymer is one or more of maltose, cellulose and its derivatives, starch and its derivatives, polyvinyl alcohol, polyamino acids, dextran, sodium alginate, polyvinyl pyrrolidone, sodium hyaluronate, chitosan, and polyethylene glycol.

[0016] In one embodiment of the present invention, the biocompatible high molecular polymer is polyvinyl alcohol and polyvinyl pyrrolidone, and the mass ratio of polyvinyl alcohol to polyvinyl pyrrolidone is 1-4:1-3.

[0017] In one embodiment of the present invention, the mass ratio of polyvinyl alcohol to polyvinyl pyrrolidone is 3:2.

[0018] In one embodiment of the present invention, the alcoholysis degree of the biocompatible high molecular polymer polyvinyl alcohol is 87.0-89.0%, mol / mol.

[0019] In one embodiment of the present invention, the molecular weight of the biocompatible high molecular weight polymer polyvinyl pyrrolidone is 45,000-55,000.

[0020] The present invention provides a method for preparing a liposome vesicle for inhibiting degradation of ergothioneine, comprising the following steps:

[0021] (1) adding anhydrous ethanol to the phospholipid bilayer substrate and cholesterol and stirring until completely dissolved to prepare an oil phase A;

[0022] (2) removing anhydrous ethanol from the oil phase A to prepare a liposome film;

[0023] (3) adding thioneine aqueous solution and glycerol to the above-mentioned film and rotating until the film is completely dissolved and ice bath ultrasound is prepared into liposome B;

[0024] (4) filtering the liposome B to prepare liposome C;

[0025] (5) adding a biocompatible polymer into PBS to prepare solvent D;

[0026] (6) slowly dripping an equal volume of solvent D into liposome C, stirring, leaving standstill, and preparing thioneine liposome vesicle.

[0027] In one embodiment of the present invention, in step (1), the mass ratio of the phospholipid bilayer substrate to cholesterol is 15-21:1-8.

[0028] In one embodiment of the present invention, in step (1), the mass ratio of the phospholipid bilayer substrate to cholesterol is 19:2.

[0029] In one embodiment of the present invention, in step (3), the mass concentration of the ergothioneine aqueous solution is 8-10%.

[0030] In one embodiment of the present invention, in step (3), the mass ratio of the ergothioneine aqueous solution to glycerol is 2:1-4.

[0031] In one embodiment of the present invention, in step (3), the mass ratio of the ergothioneine aqueous solution to glycerol is 1:1.

[0032] In one embodiment of the present invention, in step (6), the stirring speed is 400-1600 rpm, more preferably 800 rpm.

[0033] In one embodiment of the present invention, in step (6), the stirring temperature is 4-25°C, more preferably 4°C.

[0034] The present invention also provides application of the liposome vesicle for inhibiting degradation of ergothioneine in the field of cosmetics.

[0035] In one embodiment of the present invention, the cosmetic may be a cream, a lotion, a water-based solution, a gel, or an oily solution.

[0036] [Beneficial Effects]

[0037] (1) the present invention carries out double wrapping by thioneine of liposome phospholipid bilayer and high molecular polymer shell, thioneine can be protected, light instability, thermal instability of thioneine are prevented, and the trimethylamine produced by the degradation of thioneine is reduced. And the phospholipid bilayer substrate (stearyl alcohol phosphate and egg yolk lecithin) for preparing liposome vesicle can produce ionization with the trimethylamine of thioneine degradation, so that a small amount of trimethylamine produced can be stably wrapped in liposome vesicle and combined with phospholipid bilayer substrate, and will not leak outside liposome vesicle, so as to minimize the smell problem of thioneine.

[0038] (2) In the present invention, the outer layer of the liposome is wrapped with a polymer shell to prepare a liposome vesicle, which can maintain the morphology of the liposome vesicle and prevent the rupture of the liposome membrane in an environment such as high temperature and light.

[0039] (3) The thioneine liposome vesicle prepared in the present invention also has the advantages of enhancing the solubility of thioneine in polyols and increasing the transdermal efficiency of thioneine.

[0040] (4) The particle size distribution of the ergothioneine liposome vesicle prepared by the present invention is uniform, and the maximum embedding rate can reach 90.35%.

[0041] (5) The liposome vesicle of the present invention can be used as a component of cosmetics, and has the effect of preventing thioneine from being degraded and emitting an amine smell, thereby solving the problem that a high amount of thioneine added in current cosmetics will produce a very obvious amine smell.

[0042] (6) The present invention carries out the stability evaluation of thioneine liposome vesicle and the liposome vesicle preparation formula, proves that the liposome vesicle prepared in the present invention and its formula have no obvious amine smell under illumination conditions within 3 months, solves the problem that the prior art suppresses the odor by compounding thioneine and other raw materials with short time effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 A schematic diagram of a liposome vesicle for inhibiting the degradation of ergothioneine according to Example 1 of the present invention;

[0044] Figure 2 It is a physical diagram of a liposome vesicle that inhibits the degradation of ergothioneine in Example 1 of the present invention;

[0045] Figure 3 The transdermal ability of the essence water prepared by the liposome vesicles prepared in Example 1 and the essence water prepared by ergothioneine powder in the present invention;

[0046] Figure 4The malondialdehyde (MDA) level of mouse skin using the essence water prepared by the liposome vesicles prepared in Example 1 and the essence water prepared by ergothioneine powder;

[0047] Figure 5 The hydroxyproline HYP level of mouse skin using the essence water prepared by the liposome vesicles prepared in Example 1 and the essence water prepared by ergothioneine powder;

[0048] Figure 6 The Massons staining pictures of mouse skin using the essence water prepared by the liposome vesicles prepared in Example 1 and the essence water prepared by ergothioneine powder;

[0049] Figure 7 The ratio of mouse skin collagen in the essence water prepared by the liposome vesicles prepared in Example 1 and the essence water prepared by ergothioneine powder. DETAILED DESCRIPTION

[0050] The centrifuge used in the following examples is the Centrifuge 5810-microcentrifuge (Eppendorf); dry heat donor temperature-controlled transdermal diffusion instrument (LOGAN, USA); vacuum freeze dryer (LYO-0.5 / HZ-05A); high performance liquid chromatograph (HPLC, Agilent); gas chromatograph (GC, Agilent); cell disruptor (Ningbo Xinzhi Biotechnology Co., Ltd.); ergothioneine was from Shenzhen Zhongke Xinyang Biotechnology Co., Ltd.; polyvinyl alcohol (PVA) was purchased from Aladdin Biochemical Technology Co., Ltd.; polyvinyl pyrrolidone K30 (PVPk30) was purchased from Shenggong Bioengineering (Shanghai) Co., Ltd.; egg yolk lecithin PC70 was purchased from Xi'an Tianzheng Pharmaceutical Excipients Co., Ltd.; soybean lecithin PC23 and soybean lecithin PC75 were purchased from Shaanxi Zelang Biotechnology Co., Ltd.; phospholipids were purchased from Shandong Siyang Biotechnology Co., Ltd.; stearyl phosphate was purchased from Hubei Svituo New Materials Technology Co., Ltd.; cetearyl phosphate was purchased from Hangzhou Dingyan Chemical Co., Ltd.; tristearyl phosphate was purchased from Guoli Chemical.

[0051] Embodiment 1 The preparation method of the liposome vesicle which suppresses the degradation of ergothioneine

[0052] like Figure 1 , the liposome vesicle for suppressing the degradation of thioneine in the present embodiment includes inner water phase 1, phospholipid bilayer 2, and high molecular polymer wrapped shell 3. Ergothioneine is loaded in the inner water phase, and thioneine can be degraded into trimethylamine under strong light such as UV. The substrate used in the phospholipid bilayer 2 can produce ion action with trimethylamine, so that a small amount of trimethylamine produced can be stably wrapped in the liposome vesicle and combined with the phospholipid bilayer substrate, and will not leak outside the liposome vesicle.

[0053] like Figure 2 , a physical diagram of the liposome vesicle used to inhibit the degradation of thioneine in the present embodiment.

[0054] A method for preparing a liposome vesicle that inhibits thioneine degradation of the present embodiment comprises the following steps:

[0055] (1) Weigh 80 mg of phospholipid bilayer matrix (stearyl phosphate, 110 mg of egg yolk lecithin PC70) and 20 mg of cholesterol, add 5 mL of anhydrous ethanol and stir until completely dissolved to prepare oil phase A;

[0056] (2) the oil phase A was subjected to rotary evaporation of most of the anhydrous ethanol at 55° C. and then vacuum dried to prepare a liposome film;

[0057] (3) 8 g of 10% ergothioneine aqueous solution was added to the above film, and then an equal mass of glycerol was added, and the film was rotated until it was completely dissolved, and 20% ultrasonic power ice bath ultrasound was used for 5 min to prepare liposome B;

[0058] (4) filtering the liposome B through a 0.22 μm filter membrane to prepare liposome C;

[0059] (5) 30 mg of PVA was added to 10 mL of PBS (pH = 7.4), stirred at 70 ° C until completely dissolved, and then allowed to stand at room temperature and 20 mg of PVPk30 was added to prepare solvent D with a concentration of 3 g / LPVA and 2 g / LPVPk30;

[0060] (6) Slowly dropwise add an equal volume of solvent D to liposome C, stir at 4° C. and 800 rpm for 1 h, and then let stand at 4° C. for 1 h to prepare ergothioneine liposome vesicles.

[0061] Example 2-6 Optimization of liposome vesicle components for inhibiting thioneine degradation

[0062] In this example, the components of the phospholipid bilayer substrate in step (1) were screened by trimethylamine content and odor evaluation. The specific method is as follows: liposome vesicles were prepared according to the method for preparing liposome vesicles in Example 1, except that the phospholipid bilayer substrate was replaced with the components in Table 1, and the prepared liposome vesicles were placed under light conditions for 1 month to detect the trimethylamine content and evaluate the odor.

[0063] Odor evaluation method: 7 subjects were selected to apply the sample on the back of their hands, smearing in circles, smelling whether there was an amine smell during the process of applying until it was completely absorbed, and scoring. Odor level description: 0 no amine smell; 1 slight amine smell; 2 amine smell; 3 obvious amine smell; 4 extremely strong amine smell. Result judgment: The average score of the 7 people was taken. If the result was less than 1 point, it was evaluated as no amine smell was found, and if the result was greater than 1 point, it was evaluated as having an amine smell.

[0064] Trimethylamine content detection method: Take 0.4 mL of ergothioneine liposome vesicles and add them to an ultrafiltration centrifuge tube. Collect the filtrate after centrifugation at 12000 rpm for 30 min. Use GC to detect the actual content of trimethylamine in the sample. Repeat the test three times for each sample and take the average value.

[0065] Experimental results: According to the trimethylamine content and odor evaluation results, the trimethylamine content and odor evaluation scores detected in stearyl alcohol phosphate were the lowest, so the selected stearyl alcohol phosphate was the most suitable for preparing liposome vesicles.

[0066] Table 1 Component optimization results

[0067] Egg yolk lecithin PC70 added amount Components and dosage Trimethylamine content Odor evaluation Example 1 110mg 80mg stearyl phosphate 0mg / L 0 Example 2 110mg 80mg soy lecithin PC-23 15mg / L 1.2 Example 3 110mg 80mg soy lecithin PC-75 8mg / L 0.6 Example 4 110mg 80mg cetearyl phosphate 5mg / L 0.4 Example 5 110mg 80mg phospholipids 27mg / L 2.1 Example 6 110mg 80mg tristearyl phosphate 1056mg / L 4

[0068] Example 7-9 Optimization of the amount of stearyl alcohol phosphate added in liposome vesicles for inhibiting the degradation of ergothioneine

[0069] The present embodiment optimizes the addition amount of stearyl alcohol phosphate by encapsulation efficiency, particle diameter and PDI, and the specific method is as follows, prepares liposome vesicle according to the preparation method of liposome vesicle in Example 1, and the difference is that the addition amount of stearyl alcohol phosphate in step (1) is replaced with the addition amount of table 2. The smaller the value of PDI, the more uniform the particle size distribution; On the contrary, the larger the value of PDI, the more uneven the particle size distribution. Therefore, PDI is an important indicator for measuring the uniformity of particle size distribution. The encapsulation efficiency detection method is as follows, takes 0.4mL of thioneine liposome vesicle and adds it to an ultrafiltration centrifuge tube, collects the filtrate after centrifugation for 30min at 12000rpm, and uses HPLC to detect thioneine content, and is recorded as W 游离 .

[0070]

[0071] The results are shown in Table 2. When the added amount of stearyl alcohol phosphate is 80 mg, the obtained liposome vesicle encapsulation efficiency is the highest and the PDI value is the smallest. Therefore, adding 80 mg of stearyl alcohol phosphate to prepare liposome vesicles is most appropriate.

[0072] Table 2 Optimization results of stearyl alcohol phosphate addition

[0073] Stearyl Phosphate Encapsulation efficiency Particle size PDI Example 1 80mg 90.35％ 175nm 0.177 Example 7 40mg 74.66％ 205nm 0.342 Example 8 60mg 80.93％ 189nm 0.289 Example 9 100mg 60.24％ 176nm 0.422

[0074] Example 10-12 Optimization of cholesterol addition in liposome vesicles for inhibiting degradation of ergothioneine

[0075] In this example, the amount of cholesterol added was optimized by encapsulation efficiency, particle size and PDI. The specific method is as follows: liposome vesicles were prepared according to the method for preparing liposome vesicles in Example 1, except that the amount of cholesterol added in step (1) was replaced with the amount in Table 3. As shown in Table 3, adding cholesterol to liposome vesicles can increase the stability of the membrane, but too high a concentration of cholesterol will lead to difficulty in forming or hydrating the liposome vesicle membrane. When the amount of cholesterol added is 20 mg, the liposome vesicle obtained is optimal, so adding 20 mg of cholesterol to prepare liposome vesicles is most appropriate.

[0076] Table 3 Optimization results of cholesterol addition

[0077] Cholesterol Addition Encapsulation efficiency Particle size PDI Example 1 20mg 90.35％ 175nm 0.177 Example 10 10mg 80.02％ 148nm 0.195 Embodiment 11 40mg 74.65％ 211nm 0.252 Example 12 80mg 63.63％ 267nm 0.342

[0078] Example 13-14 Optimization of the amount of glycerol added in the liposome vesicles that inhibits the degradation of ergothioneine

[0079] In this example, the amount of glycerol added was optimized by encapsulation efficiency, particle size and PDI. The specific method is as follows: liposome vesicles were prepared according to the method for preparing liposome vesicles in Example 1, except that the amount of glycerol added in step (3) was replaced with the amount in Table 4. As shown in Table 4, the liposome vesicles were optimal when the amount of glycerol added was 8 g, and 8 g of glycerol was finally selected to prepare liposome vesicles.

[0080] Comparative Example 1

[0081] Liposomal vesicles were prepared according to the preparation method of liposomal vesicles in Example 1, except that glycerol was not added, and thioneine did not enter the inner aqueous phase, resulting in extremely low encapsulation efficiency.

[0082] Table 4 Optimization results of glycerol addition

[0083] Glycerol addition Encapsulation efficiency Particle size PDI Example 1 8g 90.35％ 175nm 0.177 Comparative Example 1 0 0 / / Embodiment 13 4g 46.76％ 127nm 0.386 Embodiment 14 16g 85.66％ 151nm 0.248

[0084] Example 15-17 Optimization of PVA concentration in liposome vesicles for inhibiting degradation of ergothioneine

[0085] In this example, the PVA concentration was optimized by encapsulation efficiency, particle size and PDI. The specific method is as follows: liposome vesicles were prepared according to the method for preparing liposome vesicles in Example 1, except that the PVA concentration in step (5) was replaced with the concentration in Table 5. As shown in Table 5, the particle size increased with the increase of PVA concentration, and the encapsulation efficiency and PDI increased first and then decreased with the increase of PVA concentration. When the PVA concentration was 3 g / L, the encapsulation efficiency was the highest, and finally 3 g / L of PVA was selected for the preparation of liposome vesicles.

[0086] Comparative Example 2

[0087] Liposome vesicles were prepared according to the method for preparing liposome vesicles in Example 1, except that PVA was not added in step (5).

[0088] Table 5 Optimization results of PVA concentration

[0089] PVA concentration (g / L) Encapsulation efficiency Particle size PDI Example 1 3 90.35％ 175nm 0.177 Comparative Example 2 0 86.87％ 137nm 0.175 Embodiment 15 1 85.24％ 153nm 0.174 Example 16 2 86.94％ 162nm 0.169 Embodiment 17 4 64.85％ 189nm 0.243

[0090] Example 18-19 Optimization of PVPk30 concentration in liposome vesicles for inhibiting degradation of ergothioneine

[0091] In this example, the concentration of PVPk30 is optimized by encapsulation efficiency, particle size and PDI. The specific method is as follows: liposome vesicles are prepared according to the method for preparing liposome vesicles in Example 1, except that the concentration of PVPk30 in step (5) is replaced with the concentration in Table 6. As shown in Table 6, the particle size increases with the increase of the concentration of PVPk30, the encapsulation efficiency increases first and then decreases with the increase of the concentration of PVPk30, and the PDI increases. When the concentration of PVPk30 is 2 g / L, the encapsulation efficiency is the highest, and finally the preparation of liposome vesicles of 2 g / L PVPk30 is selected.

[0092] Comparative Example 3

[0093] Liposome vesicles were prepared according to the method for preparing liposome vesicles in Example 1, except that PVPk30 was not added in step (5).

[0094] Table 6 Optimization results of PVPk30 concentration

[0095] PVPk30 concentration (g / L) Encapsulation efficiency Particle size PDI Example 1 2 90.35％ 175nm 0.177 Comparative Example 3 0 82.87％ 165nm 0.174 Embodiment 18 1 89.54％ 176nm 0.177 Embodiment 19 3 84.35％ 178nm 0.203

[0096] Example 20 Optimization of stirring temperature in step (6) in liposome vesicles for inhibiting degradation of ergothioneine

[0097] In this example, the stirring temperature in step (6) was optimized by encapsulation efficiency, particle size and PDI. The specific method is as follows: liposome vesicles were prepared according to the method for preparing liposome vesicles in Example 1, except that the stirring temperature in step (6) was replaced with the temperature in Table 7. As shown in Table 7, the encapsulation efficiency increased with decreasing temperature, and the PDI decreased with decreasing temperature. When the stirring temperature was 4°C, the encapsulation efficiency was the highest and the PDI was the lowest. Finally, 4°C was selected for the preparation of liposome vesicles.

[0098] Comparative Example 4

[0099] Liposome vesicles were prepared according to the method for preparing liposome vesicles in Example 1, except that the stirring temperature in step (6) was changed to 50°C.

[0100] Table 7 Optimization results of stirring temperature in step (6)

[0101] Stirring temperature Encapsulation efficiency Particle size PDI Example 1 4℃ 90.35％ 175nm 0.177 Embodiment 20 25℃ 82.86％ 178nm 0.184 Comparative Example 4 50℃ 48.94％ 110nm 0.535

[0102] Optimization of stirring speed in step (6) in liposome vesicles for inhibiting degradation of ergothioneine in Example 21-22

[0103] In this example, the stirring speed in step (6) was optimized by encapsulation efficiency, particle size and PDI. The specific method is as follows: liposome vesicles were prepared according to the method for preparing liposome vesicles in Example 1, except that the stirring speed in step (6) was replaced with the speed in Table 8. As shown in Table 8, the encapsulation efficiency first increased and then decreased with the increase of the speed. When the stirring speed was 800 rpm, the encapsulation efficiency was the highest and the PDI was the lowest. Finally, 800 rpm was selected for the preparation of liposome vesicles.

[0104] Comparative Example 5

[0105] Liposome vesicles were prepared according to the method for preparing liposome vesicles in Example 1, except that the stirring speed in step (6) was changed to 3200 rpm.

[0106] Table 8 Optimization results of stirring speed in step (6)

[0107] Stirring speed Encapsulation efficiency Particle size PDI Example 1 800rpm 90.35％ 175nm 0.177 Embodiment 21 400rpm 89.42％ 178nm 0.247 Embodiment 22 1600rpm 68.93％ 110nm 0.289 Comparative Example 5 3200rpm 38.23％ 99nm 0.369

[0108] Example 23 Optimization of the concentration of thioneine aqueous solution in liposome vesicles for inhibiting thioneine degradation

[0109] The present embodiment screens the concentration of thioneine aqueous solution by encapsulation efficiency, particle diameter, PDI, odor evaluation, trimethylamine concentration, and the specific grammar is as follows, prepare liposome vesicles according to the preparation method of liposome vesicles in Example 1, except that the concentration of thioneine aqueous solution in step (3) is replaced with the concentration of Table 9, measure encapsulation efficiency, particle diameter, PDI, and place under illumination conditions for 3 months to carry out trimethylamine content detection and odor evaluation. Trimethylamine content detection and odor evaluation method: the same as evaluation method in Example 2-6.

[0110] Comparative Examples 6-7

[0111] Liposome vesicles were prepared according to the method for preparing liposome vesicles in Example 1, except that the concentration of the ergothioneine aqueous solution was replaced with 12% and 14%, respectively.

[0112] As shown in Table 9, when the concentration of thioneine aqueous solution is below 12%, the encapsulation efficiency is basically unchanged and can reach a higher level. When the concentration of thioneine aqueous solution reaches 14%, the encapsulation efficiency begins to decline. Therefore, the concentration of thioneine aqueous solution can not be higher than 12%. When the concentration of thioneine aqueous solution is below 10%, the odor evaluation result is better, and the trimethylamine content is lower. When the concentration of thioneine aqueous solution is 12% and above, the odor evaluation and trimethylamine content begin to increase. Based on the above results, the best thioneine aqueous solution concentration is 10%.

[0113] Table 9 Optimization results of thioneine aqueous solution concentration

[0114] Concentration of ergothioneine aqueous solution Encapsulation efficiency Particle size PDI Odor evaluation Trimethylamine content Example 1 10％ 90.35％ 175nm 0.177 0.2 3mg / L Embodiment 23 8％ 90.43％ 172nm 0.179 0.1 1mg / L Comparative Example 6 12％ 89.45％ 178nm 0.172 4 56mg / L Comparative Example 7 14％ 69.40％ 174nm 0.173 4 224mg / L

[0115] Example 24 Comparison of the formula stability of thioneine powder and thioneine liposome vesicles

[0116] The liposome vesicles and ergothioneine powder prepared in Example 1 were respectively configured into essence water to make the final concentration of ergothioneine 0.1%. The essence water preparation method was as follows: 1,2-hexanediol, p-hydroxyacetophenone, and butanediol were configured as phase A, ORBII and GTCC were configured as phase B, EG, 305, ergothioneine, glycerol, and water were configured as phase C, phases A, B, and C were heated to 85° C., phase C was added to phase B, and homogenized for 2 minutes. When the temperature dropped to 40° C., phase A was added, and homogenization was continued for 10 minutes.

[0117] Table 10 Method for preparing essence water

[0118]

[0119] The essence water prepared by respectively configuring the liposome vesicles and thioneine powder prepared in Example 1 was placed at 50° C. under light conditions for 3 months for odor evaluation, thioneine content detection, and trimethylamine content detection.

[0120] Evaluation 1 Odor Evaluation

[0121] Odor evaluation method: the same as the evaluation method in Example 2-6.

[0122] According to the results of Table 11, under illumination conditions, after the experiment started for 30 days, the test subject in the essence water obtained by ergothioneine powder configuration could smell strong amines, and the test subject of the essence water obtained by the liposome vesicle configuration prepared by Example 1 had no amine smell, and after 90 days of the experiment, the test subject in the essence water obtained by ergothioneine powder configuration could smell strong amines, and the test subject of the essence water obtained by the liposome vesicle configuration prepared by Example 1 had no amine smell. Under 50 ° C conditions, the odor evaluation results were basically consistent with the respective result change trends under illumination conditions, and were better than the respective results under illumination conditions.

[0123] Table 11 Evaluation results of the odor of the essence water prepared by the liposome vesicles and ergothioneine powder prepared in Example 1

[0124]

[0125]

[0126] Evaluation 2 Ergothioneine Content

[0127] Evaluation method:Utilize the real content of thioneine in HPLC detection sample, each sample is repeated and tested three times, finally averaged. According to table 12 results shown, under illumination conditions, after the experiment starts 90 days, thioneine content in the essence water obtained by thioneine powder configuration decreases by 25.05%, and thioneine content in the essence water obtained by the liposome vesicle configuration prepared by embodiment 1 decreases by 1.89%, under 50 DEG C of conditions, thioneine content result is basically consistent with each result change trend under illumination conditions, and is better than each result under illumination conditions.

[0128] Table 12 Liposomal vesicles prepared in Example 1, thioneine powder respectively configured to obtain thioneine content test results of the essence water

[0129]

[0130] Evaluation 3 Trimethylamine content

[0131] Evaluation method:Utilize the real content of trimethylamine in GC detection sample, each sample is repeated and tested three times, finally averaged. According to the results shown in Table 13, under illumination conditions, after the experiment starts 90 days, the essence water trimethylamine content obtained by ergothioneine powder configuration is 102mg / L, and the essence water trimethylamine content obtained by the liposome vesicle configuration prepared by embodiment 1 is 4mg / L, under 50 DEG C of conditions, trimethylamine content result is basically consistent with the result change trend of each illumination condition, and content is lower than the result of each illumination condition.

[0132] Table 13 The liposome vesicles prepared in Example 1 and the thioneine powder were respectively configured to obtain the essence water trimethylamine content test results

[0133]

[0134] In summary, first, thioneine is prepared into liposome vesicles, and the light stability and thermal stability of thioneine are affected, compared to thioneine powder, thioneine liposome vesicles are significantly lower than thioneine powder in smell score, thioneine content is significantly higher than thioneine powder, and trimethylamine content is significantly lower than thioneine powder. Therefore, thioneine is prepared into liposome vesicles, and thioneine degradation can be suppressed, and the light stability and thermal stability of thioneine are improved.

[0135] Example 25 Comparison of transdermal penetration of essence water prepared from thioneine powder and thioneine liposome vesicles

[0136] The essence water 1 and 2 obtained by configuring in Example 24 were subjected to transdermal experiment. The essence water (blank essence water) without thioneine was configured according to the method in Table 10 in Example 24 as a control, and the corresponding sample was diluted to thioneine concentration of 1 mM with a reference substance.

[0137] The specific method of transdermal experiment is as follows:

[0138] After KM mice were killed by cervical dislocation, the dorsal hair was removed, the dorsal skin was separated, and the subcutaneous vascular fascia and adipose tissue were removed. The skin was repeatedly rinsed with saline until the wash solution was clear and free of turbidity. The skin was trimmed into 2cm×2cm pieces and stored on ice for later use. The transdermal diffusion instrument was loaded into the dry heating pool according to the label, the temperature control probe was inserted, and the stirring bar was placed in the diffusion pool. The treated skin was taken and fixed between the dosing chamber and the diffusion pool, with the dermis layer close to the inside of the diffusion pool and the epidermis layer facing the dosing chamber. The effective transdermal diffusion area of ​​the skin is 1.74cm 2 . Add 1mL of essence water 1, essence water 2 or blank essence water to the dosing chamber, and the volume of the receiving pool is 3mL. The receiving solution is normal saline (preheated to 32°C), the temperature is set to 32°C, and the speed is 250r / min. Set the sampling point time, and sample at 0h, 2h, 4h, 8h, 12h, 18h, and 24h respectively. The sampling volume is 3mL, and 3mL of the receiving solution is added again. Repeat the operation at the next sampling point. After sampling, HPLC is used to detect the content of ergothioneine in the receiving solution. The results are shown in Table 14 and Figure 3 As shown in the figure, the cumulative transdermal amount of ergothioneine Q (μg / cm 2 ) and the sampling time t were plotted and fitted with a formula to obtain a linear regression line and a linear regression equation. The slope of the line was the permeation rate constant J (μg / (cm 2 h), since the control was blank skin without any experimental samples added, no curve fitting was performed.

[0139]

[0140] Q—Cumulative transdermal dose (μg / cm 2 );C i—The sample concentration (μg / mL) measured at the i-th sampling time point; n-1—The sample concentration (μg / mL) measured at the n-1-th sampling interval; V i — sampling volume 3mL; A— is the effective diffusion surface area 1.74cm 2 .

[0141] Table 14 The results of transdermal penetration of the essence water prepared by the liposome vesicles and thioneine powder prepared in Example 1

[0142] Group Fitting equation <![CDATA[J(μg / (cm 2 ·h))]]> <![CDATA[Q 24 (μg / cm 2 )]]> Essence water 1 Q=0.387t-0.1436 0.387 8.52 Essence Water 2 Q=0.4684t-0.326 0.4684 10.68

[0143] From Table 14 and Figure 3 As shown in the results, the cumulative transdermal amount of ergothioneine in the essence water prepared by ergothioneine powder and the essence water prepared by liposome vesicles prepared in Example 1 was 8.52 μg / cm 2 , 10.68μg / cm 2 Compared with the essence water prepared by ergothioneine powder, the 24h cumulative transdermal amount of ergothioneine in the essence water prepared by the liposome vesicles prepared in Example 1 was increased by 25.35%. It shows that preparing ergothioneine into liposome vesicles can improve the transdermal rate of ergothioneine.

[0144] Example 26 Evaluation of the anti-aging ability of thioneine liposome vesicles in vivo

[0145] 44 ordinary female KM mice, weighing (32±2) g, and 6 weeks old, were randomly divided into 4 groups, 11 mice in each group, namely, blank group, modeling group, experimental group, and control group. The hair on the back of the mice was removed before administration.

[0146] The blank group was routinely fed, and the other groups were subcutaneously injected with 40 mg / mouse / day D-galactose until the end of the experiment. After 2 weeks of modeling, the experimental group was smeared with the essence water 2 prepared in Example 24, the control group was smeared with the essence water 1 prepared in Example 24, and the modeling group was smeared with the essence water (blank essence water) without ergothioneine, once a day, 300 μL each time, and a total of 35 times of administration. After the end of the administration, the skin on the back of the mouse was taken for Massons staining, malondialdehyde MDA content detection and hydroxyproline HYP content detection. 。

[0147] Experimental results: Figure 4-5As shown in the figure, injecting mice with D-galactose will cause lipid peroxidation in the skin, which will lead to a decrease in HYP content and indirectly reduce the collagen content in the skin. Compared with the modeling group, the MDA level in the skin of the experimental group and the control group was significantly lower than that of the modeling group (p<0.01), and the HYP content was significantly increased (p<0.01, p<0.05). In particular, the mice that were smeared with liposome vesicles configured as essence water had lower MDA content in the skin (p<0.01) and higher HYP content (p<0.01) than the mice that were smeared with ergothioneine powder configured as essence water, indicating that ergothioneine liposome vesicles have a better anti-aging effect at the same concentration.

[0148] To further examine the collagen fiber content in aged skin, Massons staining was used for evaluation. The stained tissue sections were photographed using an inverted microscope and processed using NIH Image J to calculate the collagen content (the percentage of the collagen-positive blue area to the total tissue area). Figure 6-7 , collagen was stained blue. Compared with the blank group ( Figure 6 -A), the collagen fibers in the model group showed a loose and irregular reticular state ( Figure 6 -B), which shows that the collagen in skin tissue is lost after aging, and the proportion of collagen decreases significantly. Figure 6 -B), control group ( Figure 6 -C), experimental group ( Figure 6 -D) Collagen fibers were arranged neatly, and the percentage of collagen in mouse skin was significantly higher than that in the modeling group (p<0.01), especially in mice smeared with liposome vesicles configured as essence water, the collagen fibers were arranged regularly and densely, and the percentage of collagen in mouse skin was significantly higher than that in mice smeared with ergothioneine powder configured as essence water. In summary, these results show that ergothioneine can prevent collagen fiber degradation caused by skin aging, and the anti-aging effect is better when ergothioneine is encapsulated in liposome vesicles.

[0149] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A liposome vesicle that inhibits the degradation of ergothioneine, characterized in that, The liposome vesicle embeds ergothioneine in the interior of the phospholipid bilayer of the liposome vesicle by constructing an embedding structure, and the outer layer of the phospholipid bilayer is also wrapped with a biocompatible high molecular polymer shell; Wherein, the substrate used for the phospholipid bilayer is one or more of stearyl alcohol phosphate, soybean lecithin, egg yolk lecithin, cetearyl alcohol phosphate, tristearyl alcohol phosphate, and phospholipids; The biocompatible high molecular polymer is one or more of maltose, cellulose and its derivatives, starch and its derivatives, polyvinyl alcohol, polyamino acids, dextran, sodium alginate, polyvinyl pyrrolidone, sodium hyaluronate, chitosan, and polyethylene glycol.

2. a liposome vesicle suppressing the degradation of thioneine according to claim 1, is characterized in that, The substrates used for the phospholipid bilayer are stearyl alcohol phosphate and egg yolk lecithin, and the mass ratio of stearyl alcohol phosphate to egg yolk lecithin is 4-10:

11.

3. a liposome vesicle suppressing the degradation of thioneine according to claim 1, is characterized in that, The mass ratio of the substrate used in the phospholipid bilayer to ergothioneine is 15-21:64-112.

4. a liposome vesicle suppressing the degradation of thioneine according to claim 1, is characterized in that, The biocompatible high molecular polymer is polyvinyl alcohol and polyvinyl pyrrolidone, and the mass ratio of polyvinyl alcohol to polyvinyl pyrrolidone is 1-4:1-3.

5. a method for preparing a liposome vesicle that suppresses the degradation of thioneine according to claim 1, characterized in that, The specific steps include: (1) adding anhydrous ethanol to the phospholipid bilayer substrate and cholesterol and stirring until completely dissolved to prepare oil phase A; (2) removing anhydrous ethanol from the oil phase A to prepare a liposome film; (3) adding thioneine aqueous solution and glycerol into the above-mentioned film and rotating until the film is completely dissolved and ice bath ultrasound is prepared into liposome B; (4) filtering the liposome B to prepare liposome C; (5) adding a biocompatible polymer into PBS to prepare solvent D; (6) dripping an equal volume of solvent D into liposome C, stirring, leaving standstill, and preparing thioneine liposome vesicle.

6. a method for preparing a liposome vesicle that suppresses the degradation of thioneine according to claim 5, is characterized in that, In step (1), the mass ratio of the phospholipid bilayer substrate to cholesterol is 15-21:1-8.

7. a method for preparing a liposome vesicle that suppresses thioneine degradation according to claim 5, is characterized in that, In step (3), the mass concentration of the thioneine aqueous solution is 8-10%.

8. a method for preparing a liposome vesicle that suppresses thioneine degradation according to claim 5, is characterized in that, In step (3), the mass ratio of thioneine aqueous solution and glycerol is 2:1-4.

9. a method for preparing a liposome vesicle that suppresses thioneine degradation according to claim 5, is characterized in that, In step (6), the stirring speed is 400-1600 rpm; the stirring temperature is 4-25°C.

10. Application of the liposome vesicle for suppressing the degradation of thioneine according to any one of claims 1 to 4 in the field of cosmetics.

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