Liposome with high coating rate and anti-aging effect as well as preparation method, application and cosmetics thereof

Abstract

The invention belongs to the field of daily chemicals, and discloses a liposome with high coating rate and anti-aging efficacy, which comprises 0.01-3 wt% of oil soluble substance, 1-5 wt% of liquid-solid mixed grease, 1-10 wt% of phospholipid, 1-10 wt% of surfactant, 10-30 wt% of polyol and 45-70 wt% of deionized water, the oil soluble substances comprise tetrahydrocurcumin and pterostilbene; the weight ratio of the tetrahydrocurcumin to the pterostilbene is (0.5-10): 1; the liquid-solid mixed grease consists of liquid grease and solid grease; the weight ratio of the liquid grease to the solid grease is (0.5-3): 1. According to the liposome, the liquid-solid mixed grease is matched with the tetrahydrocurcumin and the pterostilbene for common use, the liquid-solid mixed grease, the tetrahydrocurcumin and the pterostilbene form an amorphous mixture, the crystallinity of oil-soluble substances is reduced, crystallization separation is effectively inhibited, and the tetrahydrocurcumin and the pterostilbene can stably exist in the liposome. Meanwhile, the invention further provides a preparation method, application and cosmetics of the liposome.

Description

Technical Field

[0001] The invention belongs to the field of daily chemicals, and specifically relates to a liposome with high coverage and anti-aging effect, a preparation method thereof, application and cosmetics. Background Art

[0002] At present, most liposome coating technologies use the bilayer vesicle structure of liposomes. The cross section of the vesicle can be regarded as concentric circles. The hydrophilic end of the lipid molecule is located at the edge of the circle, and the hydrophobic end is located between the two circles to form a hydrophobic layer. When using the vesicle to coat an oil-soluble substance, the oil-soluble substance needs to be dissolved in the oil phase and then coated in the hydrophobic layer. For example, patent CN115531319B discloses a high-stability liposome and its preparation method and use, wherein liquid oil, solid oil, phospholipid, gelatin, active ingredient and water are mixed to prepare a liposome composition.

[0003] However, in the bilayer vesicle structure, the volume of the hydrophobic layer accounts for a small proportion, and the central layer of the vesicle is an aqueous phase and cannot encapsulate oil-soluble substances, resulting in a large amount of space waste, which leads to an increase in the demand for lipids when preparing liposomes that encapsulate oil-soluble substances, thereby increasing production costs. In addition, toxic solvents such as chloroform, methanol, ethanol, and ether are mostly required to be added during the preparation of liposome vesicles. The solvent molecules remain inside the vesicles and are difficult to remove, causing potential harm to the human body. The unique bilayer structure of liposome vesicles results in a dense structure of the inner molecular layer with a smaller diameter, while the outer molecular layer with a larger diameter is relatively loose, making it easy for the oil-soluble active substances located between the bilayers to precipitate from the outer molecular layer and undergo oxidative denaturation.

[0004] Tetrahydrocurcumin and pterostilbene are both oil-soluble substances. If these two substances want to exist better in the aqueous phase, either emulsification or liposomes should be used. Based on the characteristics of existing liposomes that oil-soluble substances are easy to precipitate and oxidize, new methods are needed to load tetrahydrocurcumin and pterostilbene.

[0005] The technical problem to be solved in this case is: how to develop a carrier that can enable tetrahydrocurcumin and pterostilbene to exist stably for a long time in the aqueous phase and reliably exert their efficacy. Summary of the invention

[0006] In view of the deficiencies in the prior art, the object of the present invention is to provide a liposome, wherein the liposome adopts a liquid-solid mixed oil in combination with tetrahydrocurcumin and pterostilbene, wherein the liquid-solid mixed oil forms an amorphous mixture with the tetrahydrocurcumin and pterostilbene, reduces the crystallinity of the oil-soluble substance, effectively inhibits crystallization, and enables the tetrahydrocurcumin and pterostilbene to exist stably in the liposome.

[0007] At the same time, the invention also provides a preparation method, application and cosmetics of the liposome.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] A liposome with high encapsulation rate and anti-aging effect, comprising: 0.01-3wt% of oil-soluble substance, 1-5wt% of liquid-solid mixed oil, 1-10wt% of phospholipid, 1-10wt% of surfactant, 10-30wt% of polyol, and 45-70wt% of deionized water;

[0010] The oil-soluble substance includes tetrahydrocurcumin and pterostilbene; the weight ratio of the tetrahydrocurcumin to pterostilbene is 0.5 to 10:1

[0011] The liquid-solid mixed grease is composed of liquid grease and solid grease; the weight ratio of the liquid grease to the solid grease is 0.5-3:1.

[0012] In the above-mentioned liposome with high encapsulation rate and anti-aging effect, the weight ratio of tetrahydrocurcumin to pterostilbene is 8-3:1.

[0013] The above-mentioned liposome with high encapsulation rate and anti-aging effect also contains synergistic ingredients, which are one or more combinations of tranexamic acid, ectoine, and narcissus bulb extract.

[0014] In the above-mentioned liposome with high encapsulation rate and anti-aging effect, the weight ratio of the synergistic component to pterostilbene in the composition is 0.1-0.5:1.

[0015] In the above-mentioned liposomes with high encapsulation rate and anti-aging effect, the liquid-solid mixed oil is any combination of glyceryl monostearate / glyceryl monolaurate, acetylated monoglyceride / isononyl isononanoate, triacetin / caprylic capric glyceride, diisopropyl oxalate / acetylated monoglyceride, and isopropyl lanolate / isotridecyl isononanoate.

[0016] In the above-mentioned liposome with high encapsulation rate and anti-aging effect, the phospholipid is one or a mixture of soybean lecithin and egg yolk lecithin;

[0017] The surfactant is a mixture of one or more of Tween-80, Tween-60, decaglycerol monolaurate, decaglycerol monomyristate, hexaglycerol monolaurate, fatty alcohol polyether, and sucrose stearate;

[0018] The polyol is a mixture of one or more of propylene glycol, butylene glycol, pentylene glycol and glycerol.

[0019] At the same time, the present invention also discloses a method for preparing the liposome as described above, comprising the following steps:

[0020] Step 1: Heat the liquid-solid mixed oil to 60-80°C, add phospholipids, and then add the oil-soluble substance to be coated, stir evenly, and obtain a phospholipid mixed liquid;

[0021] Step 2: Mix the surfactant, polyol and water and heat them to the same temperature as the phospholipid mixture to obtain an aqueous phase solution;

[0022] Step 3: Add the phospholipid mixture into the aqueous phase solution, emulsify it evenly, and then perform high-pressure homogenization to obtain liposome micelles.

[0023] In the above-mentioned method for preparing liposomes, in step 3, the pressure of high-pressure homogenization is 80 to 130 MPa, and the number of cycles is 3 to 7 times.

[0024] In addition, the present invention also discloses the use of any of the above liposomes in preparing cosmetics.

[0025] In the above-mentioned use, the cosmetic is an emulsion or a cream.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. The liposome of the present invention adopts a liquid-solid mixed oil to be used together with tetrahydrocurcumin and pterostilbene. The liquid-solid mixed oil forms an amorphous mixture with tetrahydrocurcumin and pterostilbene, reduces the crystallinity of the oil-soluble substance, effectively inhibits crystallization, and enables tetrahydrocurcumin and pterostilbene to exist stably in the liposome.

[0028] 2. The combination of tetrahydrocurcumin and pterostilbene of the present invention has been verified to have very excellent anti-aging, antioxidant, anti-inflammatory and whitening effects; by analyzing the anti-aging properties of these two raw materials, UVA was used to irradiate fibroblasts, and the anti-wrinkle effect of the sample to be tested was evaluated by detecting the changes in the content of 8-hydroxydeoxyguanosine (8-OHdG), type I collagen (Collagen I), and γ-H2AX. It was found that when the two exist at the same time, they have extremely strong anti-aging effects. At the same time, through the verification of antioxidant, anti-inflammatory and whitening effects, it was also found that the two have strong synergy. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is a particle size distribution curve of the liposomes of Liposome Example 1;

[0030] Figure 2 is a particle size distribution curve of the liposomes of liposome Example 8;

[0031] Figure 3 This is a particle size distribution curve of the liposomes of Liposome Example 2. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0033] Source of reagents:

[0034] Tetrahydrocurcumin: purchased from Sami-Sabinsa Group Limited, USA, product model: CURCUMALONGA ROOT EXTRACT MICRONIZED CH;

[0035] Pterostilbene: purchased from Sami-Sabinsa Group Limited, USA. The product model is PTEROCARPUSMARSUPIUM BARK EXTRACT (PTERO WHITE 90%) CH.

[0036] The above two raw materials are both oil-soluble and water-soluble products.

[0037] Part I. Preparation of liposomes

[0038] Liposome Example 1

[0039] 1. By mass, heat 1 part of glyceryl monostearate and 2 parts of glyceryl monolaurate to 65°C, add 1.5 parts of soybean lecithin, then add 0.75 parts of tetrahydrocurcumin and 0.25 parts of pterostilbene, and stir slowly to obtain a phospholipid mixture;

[0040] 2. Add 3 parts of Tween-80 to 30 parts of glycerol and 60 parts of water, and heat to 65°C to obtain an aqueous phase solution;

[0041] 3. Add the phospholipid mixture into the aqueous phase, and perform high-pressure homogenization after uniform emulsification. The high-pressure homogenization pressure is 100 MPa, and the number of cycles is 5 to obtain liposome micelles.

[0042] The liposome micelle prepared in the present application is a single-layer structure, and the soybean lecithin molecules and Tween-80 molecules are combined to form a single-layer micelle that encapsulates an amorphous mixture formed by monostearate glyceryl / monolaurin, tetrahydrocurcumin and pterostilbene.

[0043] The liposome micelles prepared in Example 1 were left at room temperature (25° C.) for 5 months, and no crystals were observed to be precipitated, and no obvious changes in appearance were observed.

[0044] The particle size distribution of the liposomes in this liposome example can be referred to Figure 1; About 82.2% of the liposomes have an average particle size of 72.2nm; about 17.8% of the liposomes have an average particle size of 13.35nm.

[0045] Liposome Example 2

[0046] 1. By weight, heat 2 parts of triacetin and 2 parts of caprylic capric glyceride to 70°C, add 3 parts of soybean lecithin, then add 0.01 parts of tetrahydrocurcumin and pterostilbene (weight ratio 3:1), slowly stir evenly to obtain a phospholipid mixture;

[0047] 2. By mass, add 4 parts of hexaglycerol monolaurate to 25 parts of propylene glycol and 61 parts of water, and heat to 70°C to obtain an aqueous solution;

[0048] 3. Add the phospholipid mixture into the aqueous phase solution, and perform high-pressure homogenization after uniform emulsification. The high-pressure homogenization pressure is 110 MPa, and the number of cycles is 5 to obtain liposome micelles.

[0049] The liposome micelles prepared in Example 2 were left at room temperature for 4 months, and no crystals were observed to be precipitated, and no obvious changes in appearance were observed.

[0050] The particle size distribution of the liposomes in this liposome example can be referred to Figure 3 ; About 73.1% of the liposomes have an average particle size of 86.39 nm; about 26.9% of the liposomes have an average particle size of 12.78 nm.

[0051] Liposome Example 3

[0052] 1. By weight, heat 3 parts of acetylated monoglyceride and 2 parts of isononyl isononanoate to 60°C, add 2 parts of egg yolk lecithin, then add 1.5 parts of tetrahydrocurcumin and pterostilbene (weight ratio 3:1), slowly stir evenly to obtain a phospholipid mixture;

[0053] 2. Add 6 parts of Tween-60 to 15 parts of pentanediol and 70.5 parts of water, and heat to 60°C to obtain an aqueous solution;

[0054] 3. Add the phospholipid mixture into the aqueous solution, emulsify evenly, and then perform high-pressure homogenization operation. The high-pressure homogenization pressure is 90 MPa and the number of cycles is 3 to obtain liposome micelles.

[0055] The liposome micelles prepared in Liposome Example 3 were left at room temperature for 6 months, and no crystals were observed to precipitate, and no obvious changes in appearance were observed.

[0056] Liposome Example 4

[0057] 1. By weight, heat 1 part of acetylated monoglyceride and 1 part of diisopropyl oxalate to 70°C, add 3 parts of soybean lecithin, then add 2.5 parts of tetrahydrocurcumin and pterostilbene (weight ratio 3:1), slowly stir evenly to obtain a phospholipid mixture;

[0058] 2. Add 6 parts of decamer monomyristate glyceride to 22 parts of pentanediol and 64.5 parts of water, and heat to 70°C to obtain an aqueous solution;

[0059] 3. Add the phospholipid mixture into the aqueous solution, emulsify evenly, and then perform high-pressure homogenization operation. The high-pressure homogenization pressure is 120 MPa and the number of cycles is 4 to obtain liposome micelles.

[0060] The liposome micelles prepared in Liposome Example 4 were left at room temperature for 5 months, and no crystals were observed to be precipitated, and no obvious changes in appearance were observed.

[0061] Liposome Example 5

[0062] 1. By weight, heat 1.5 parts of isopropyl lanolin and 2 parts of diisopropyl oxalate / acetylated monoglyceride to 72°C, add 2.5 parts of soybean lecithin, then add 2 parts of tetrahydrocurcumin and pterostilbene (weight ratio 3:1), slowly stir evenly to obtain a phospholipid mixture;

[0063] 2. Add 5 parts of sucrose stearate to 12 parts of glycerol and 77 parts of water, and heat to 72°C to obtain an aqueous solution;

[0064] 3. Add the phospholipid mixture into the aqueous solution, emulsify evenly, and then perform high-pressure homogenization operation. The high-pressure homogenization pressure is 110 MPa and the number of cycles is 6 to obtain liposome micelles.

[0065] The liposome micelles prepared in Liposome Example 5 were left at room temperature for 6 months without crystal precipitation and without obvious changes in appearance.

[0066] Liposome Example 6

[0067] 1. According to the mass ratio, heat 2 parts of diisopropyl oxalate and 1 part of acetylated monoglyceride to 65°C, add 1 part of egg yolk lecithin, and then add 1 part of tetrahydrocurcumin and pterostilbene (weight ratio 3:1), slowly stir evenly to obtain a phospholipid mixture;

[0068] 2. Add 6 parts of fatty alcohol polyether to 18 parts of butanediol and 71 parts of water, and heat to 65°C to obtain an aqueous solution;

[0069] 3. Add the phospholipid mixture into the aqueous solution, emulsify evenly, and then perform high-pressure homogenization operation. The high-pressure homogenization pressure is 120 MPa and the number of cycles is 7 to obtain liposome micelles.

[0070] The liposome micelles prepared in Liposome Example 6 were left at room temperature for 6 months, and no crystals were observed to precipitate, and no significant changes in appearance were observed.

[0071] Liposome Example 7

[0072] 1. By weight, heat 0.5 parts of acetylated monoglyceride and 1 part of isononyl isononanoate to 75°C, add 2.5 parts of soybean lecithin, then add 2.2 parts of tetrahydrocurcumin and pterostilbene (weight ratio 3:1), slowly stir evenly to obtain a phospholipid mixture;

[0073] 2. By mass, add 4 parts of hexaglycerol monolaurate to 24 parts of propylene glycol and 65.8 parts of water, and heat to 75°C to obtain an aqueous phase solution;

[0074] 3. Add the phospholipid mixture into the aqueous solution, emulsify evenly, and then perform high-pressure homogenization operation. The high-pressure homogenization pressure is 130 MPa and the number of cycles is 6 to obtain liposome micelles.

[0075] The liposome micelles prepared in Liposome Example 7 were left at room temperature for 5 months, and no crystals were observed to precipitate, and no obvious changes in appearance were observed.

[0076] Liposome Example 8

[0077] 1. By weight, heat 1 part of triacetin and 1.5 parts of caprylic capric glyceride to 80°C, add 2 parts of soybean lecithin, then add 3 parts of tetrahydrocurcumin and pterostilbene (weight ratio 3:1), slowly stir evenly to obtain a phospholipid mixture;

[0078] 2. By mass, add 4 parts of hexaglycerol monolaurate to 18.5 parts of glycerol and 71 parts of water, and heat to 80°C to obtain an aqueous phase solution;

[0079] 3. Add the phospholipid mixture into the aqueous solution, emulsify evenly, and then perform high-pressure homogenization operation. The high-pressure homogenization pressure is 110 MPa and the number of cycles is 5 to obtain liposome micelles.

[0080] The liposome micelles prepared in Liposome Example 8 were left at room temperature for 4 months, and no crystals were observed to precipitate, and no significant changes in appearance were observed.

[0081] The particle size distribution of the liposomes in this liposome example can be referred to Figure 2; About 75.4% of the liposomes have an average particle size of 83.6nm; about 24.6% of the liposomes have an average particle size of 12.13nm.

[0082] In the above-mentioned liposome examples 1 to 8, liposome solutions are obtained; the purpose of preparing liposomes in the present invention is to achieve the encapsulation, delivery and sustained release of high-concentration oil-soluble components through liposomes.

[0083] It can be inferred from the above results that the liposomes of the present invention are single-layer liposomes, which are different from traditional double-layer liposomes and can accommodate more fats. Through the combination of solid fats and liquid fats, the oil-soluble substances can be prevented from precipitating during long-term storage.

[0084] Due to the sustained-release characteristics of liposomes, the laboratory method of the applicant cannot accurately verify the efficacy of liposomes. In order to further accurately reflect the efficacy of the oil-soluble substance of the present invention, through the second part, the active ingredient with both oil and water solubility is placed in the water phase for relevant functional verification.

[0085] Liposome safety testing

[0086] Safety test was performed using liposome example 1, liposome example 2, and liposome example 8

[0087] 1. Materials and Methods

[0088] 1. Test substances: Liposome Example 1, Liposome Example 2, Liposome Example 8

[0089] 2. Negative control: blank control.

[0090] 3. Subjects: 30 people, male 9 People, Female 21 People, Age 21 to 52 years old, meeting the volunteer inclusion criteria.

[0091] 4. Test site: flexed forearm

[0092] 5. Spot test method: Select qualified spot test equipment, weigh 0.020mL~0.025mL of the test substance and put it into the small chamber of the spot tester. Apply the spot tester with the test substance to the test area with low-allergenic tape, and gently press it with the palm of your hand to evenly apply it to the skin for 24 hours. Observe the skin reaction according to the standard 30 minutes (after the indentation disappears), 24 hours and 48 hours after removing the test substance spot tester, and record the observation results. The rating standards can be seen in Table 7;

[0093] Table 7 Human skin patch test skin reaction grading standard

[0094]

[0095] 2. Test results

[0096] The test results refer to Table 8;

[0097] Table 8 Summary of cosmetics human skin patch test results

[0098]

[0099] Note: The skin reactions of 30 subjects at different observation times are shown in the attachment.

[0100] The results of human skin patch tests showed that among 30 people 0 cases Appear Level 1 and above Reaction. For details, please see Annex 1, Annex 2 and Annex 3;

[0101] Attachment 1:

[0102]

[0103]

[0104] Attachment 2:

[0105]

[0106]

[0107] Attachment 3:

[0108]

[0109]

[0110] Through the above tests, it can be found that the composition of the present invention is safe and non-irritating.

[0111] Part II Screening of the optimal ratio of active ingredients

[0112] In the embodiments and comparative examples, the compositions were prepared according to the concentrations in Table 1 and stirred to form a uniform phase.

[0113] Table 1 Formula table Unit: g

[0114]

[0115]

[0116] Part III Performance Test

[0117] The performance test is divided into three parts, namely whitening performance test, anti-aging performance test, and antioxidant performance test. The following describes them one by one.

[0118] Test 1 Whitening performance test

[0119] In the biosynthesis of melanin in the skin, tyrosinase is a key enzyme that acts on DOPA to form DOPAQUINONE, which spontaneously undergoes a series of reactions to finally form melanin. In a phosphoric acid solution at pH 6.8, tyrosinase can catalyze the conversion of DOPA into DOPAQUINONE, and the absorbance value can be measured at 475nm on a spectrophotometer. Cosmetics that inhibit tyrosinase activity can reduce the conversion of DOPA into DOPAQUINONE, thereby reducing the absorbance value. Based on the change in absorbance value, the inhibitory effect of cosmetics on tyrosinase activity can be evaluated.

[0120] Test Basis

[0121] The test was carried out according to the “Melanin content and tyrosinase activity detection method based on melanocytes”.

[0122] Purpose of the test

[0123] This test is based on melanocytes and evaluates the whitening effect of the sample by detecting changes in tyrosinase activity.

[0124] Test Materials

[0125] 1Test system

[0126] The cells used in this test are melanocytes, batch number: MC23122801

[0127] Main reagents

[0128] DF-12 culture medium (Gibco), PBS (Solabo), kojic acid (Yuanye Biotechnology), melanin standard (Sigma), DMSO (Sigma), sodium hydroxide (Sinopharm Group), anhydrous ethanol (Sinopharm Group), and ether (Sinopharm Group).

[0129] 2 Main Equipment

[0130] CO2 incubator (Thermo, 150I), clean bench (Sujing Antai, SW-CJ-1F), and microplate reader (BioTek, Epoch).

[0131] 4 Test Methods

[0132] Tyrosinase activity inhibition rate test method

[0133] 1) Cell inoculation: After the cells are revived, when the plating rate reaches about 60%, the cells are inoculated into a 6-well plate and incubated overnight in a CO2 incubator (37°C, 5% CO2).

[0134] (2) Treatment of control and test samples

[0135] Active composition: Dilute with 95% ethanol to a sample concentration of 10%;

[0136] Positive control (kojic acid, purity ≥96%): dilute with 95% ethanol to a positive control concentration of 0.1%;

[0137] Negative control: pure water

[0138] 3) Administration: According to the test grouping, when the cell plating rate in the 6-well plate reaches 40% to 60%, group administration is carried out, and 3 replicate wells are set up for each group. 2 mL of culture medium is added to each well of the blank control group, 2 mL of culture medium containing kojic acid is added to each well of the positive control group, and 2 mL of culture medium containing the corresponding concentration of the sample to be tested is added to each well of the sample group. After the administration is completed, the 6-well plate is placed in a CO2 incubator (37°C, 5% CO2) and cultured for 24 hours. After 24 hours, the culture medium is discarded and washed once with PBS.

[0139] 4) Collect cells: add 500 μL PBS to each well, transfer the cells into a 1.5 mL centrifuge tube using a cell scraper, and centrifuge at 6000 rpm for 5 min.

[0140] 5) Lysing cells: discard PBS, add 500 μL of 0.5% sodium deoxycholate to each well to lyse cells, lyse at 0°C for 1 h, centrifuge at 12000 rpm, 4°C for 20 min, and transfer the supernatant to a 1.5 mL centrifuge tube.

[0141] 6) Protein quantification: Protein quantification was performed on all samples according to the BCA kit instructions.

[0142] 7) L-DOPA reaction: 90 μL of the sample was added to a centrifuge tube, and 10 μL of 0.1% L-DOPA was added to each tube.

[0143] 8) OD value reading: At 0 min and 60 min, read the absorbance of each sample at a wavelength of 475 nm using an ELISA reader.

[0144] 9) Tyrosinase inhibition rate calculation formula: inhibition rate (%) = 1-[(sample 60 min OD value-sample 0 min OD value) / (control group 60 min OD value-control group 0 min OD value)*100%.

[0145] The test results can be seen in Table 3;

[0146] Table 3 Whitening performance test results

[0147]

[0148]

[0149] Results Analysis

[0150] 1. From Example 1 to Example 11, when the total amount of tetrahydrocurcumin and pterostilbene is controlled at 500 μg / ml, the performance reaches a peak when the ratio of the two is 3 to 4:1; the value of Example 10 fluctuates. In the early experiments, we believe that it is an experimental error. After repeated experiments and comparison of the results of subsequent free radical scavenging experiments, it is found that when the ratio of the two is 1:4, there is indeed a certain performance improvement.

[0151] 2. From Example 12 to Example 14, according to the ratio of 3:1, when the sum of the concentrations of the two is between 500 and 1000 μg / ml, the performance is optimal, that is, at 1000 μg / ml, the performance is close to the top.

[0152] Test 2 Antioxidant performance test

[0153] Excessive production of free radicals can lead to natural aging and photoaging of the skin, thus causing wrinkles. Therefore, whether a product has the ability to scavenge free radicals is one of the important indicators for evaluating anti-aging and anti-wrinkle cosmetics (raw materials).

[0154] This test refers to the laboratory method (HMC-WI-030 DPPH free radical scavenging rate) and compares the DPPH free radical scavenging rate test results of the test sample with that of the negative control. If the scavenging rate of the test sample is higher than that of the negative control and there is a significant difference (P<0.05), it can be considered that the test sample has certain anti-wrinkle and antioxidant effects.

[0155] Test methods

[0156] (1) Treatment of control and test samples

[0157] Active composition: Dilute with 95% ethanol to a sample concentration of 0.1%;

[0158] Positive control (vitamin E, purity ≥96%): dilute with 95% ethanol to a positive control concentration of 0.1%;

[0159] Negative control: pure water.

[0160] (2) Test operation steps

[0161] Set up sample tubes, sample background tubes, DPPH tubes and solvent background tubes. Each group needs to set up 3 parallel tubes. Add different

[0162] Gently shake the reagent solution and let it stand at room temperature for 5 minutes. Transfer each group of reaction solutions into a 1 cm cuvette and measure the absorbance at 517 nm.

[0163] (3) Calculation formula

[0164] The calculation formula of free radical scavenging rate (%) is:

[0165]

[0166] Where: T—absorbance of sample tube, i.e. absorbance of solution after sample reacts with DPPH;

[0167] T0—sample background absorbance;

[0168] C—the average value of the 3 absorbance values ​​of the DPPH tube, i.e., the absorbance value of the DPPH solution when no sample is added; C0—the background absorbance value of the solvent;

[0169] The test results can be seen in Table 5;

[0170] Table 5 Antioxidant performance test results

[0171]

[0172]

[0173] Note: The DPPH free radical scavenging rate is rounded to three decimal places. P<0.05 indicates that there are significant differences between the sample and the positive control and the negative control.

[0174] Results Analysis

[0175] 1. From Example 1 to Example 11, when the total amount of tetrahydrocurcumin and pterostilbene is controlled at 500 μg / ml and the ratio of the two is 2 to 4:1, the performance reaches the peak.

[0176] 2. From Example 12 to Example 14, according to the ratio of 3:1, when the sum of the concentrations of the two is between 500 and 1000 μg / ml, the performance is optimal, that is, at 1000 μg / ml, the performance is close to the top.

[0177] Test 3 Anti-aging performance test

[0178] Collagen is the most abundant extracellular matrix protein in the human body, and type I collagen is the most abundant protein in the skin. The distribution of type I collagen in zebrafish is the same as that in humans and shows a high degree of conservation with humans. Their innate expression decreases significantly at 6 days after fertilization and becomes very low between 10 and 12 days after fertilization. Testing the expression of zebrafish type I collagen genes (col1a1a, col1a1b and col1a2), comparing the relative expression changes of zebrafish type I collagen genes in the treatment group and the blank control group, and calculating the promotion rate of type I collagen gene expression can evaluate the anti-wrinkle and firming effects of raw materials or products.

[0179] 1. Instruments and equipment

[0180] 1Zebrafish breeding equipment

[0181] The equipment must be equipped with temperature control devices, water circulation and filtration devices, and the breeding container should be made of glass or common food-grade PC materials. You can use general zebrafish breeding equipment or equip the fish tank according to laboratory needs (such as 45cm×45cm×30cm in length, width and height).

[0182] 2 spawning boxes / tanks

[0183] Made of glass, stainless steel or other inert materials, equipped with a mesh screen (mesh size is 2mm×2mm, made of stainless steel or multiple materials to protect fish eggs).

[0184] 3. Water quality monitoring equipment: pH meter, dissolved oxygen meter, salinity meter (conductivity meter).

[0185] 4 Analytical balance: accuracy 0.1mg.

[0186] 5. Microscope: A stereo microscope equipped with a camera system and a maximum magnification greater than 80 times.

[0187] 6 Test container: glass or polystyrene test container (such as 6-well plate, culture dish). If the test substance may be adsorbed to the polystyrene container (such as non-polar chemicals), inert materials (such as glass) should be selected to reduce adsorption.

[0188] 7 Thermostat: Accuracy ±1°C.

[0189] 8. Brine shrimp incubator: The equipment needs to be equipped with a hatching bucket, air pipe, air volume regulating valve, air pump cut-off switch and check valve device.

[0190] 9 Ultra-micro UV spectrophotometer: The wavelength range includes 260nm and 280nm.

[0191] 10PCR amplifier.

[0192] 11Real-time PCR (qPCR) instrument.

[0193] 12Other conventional testing instruments and equipment

[0194] Pipettes, centrifuge tubes, glass containers (such as beakers, volumetric flasks, etc.), vortex mixers, ultrasonic water baths, centrifuges, low-temperature refrigerators, adjustable pipettes and tips.

[0195] 2 Main reagents and consumables

[0196] 1 Water complies with GB / T 6682.

[0197] 2. Methanol, dimethyl sulfoxide, ethanol and other co-solvents are of analytical grade.

[0198] 3. Water for broodstock breeding

[0199] Weigh 4g of sea salt and dissolve it in 10L of water to prepare a solution with a salinity of 0.25‰ to 0.50‰, a conductivity of 500μS / cm to 800μS / cm, a dissolved oxygen saturation of 80%, a pH value of 6.5 to 8.5, and a hardness of 30mg / L to 300mg / L (calculated as calcium carbonate).

[0200] 4 Brine shrimp (Artemia salina) eggs

[0201] The dormant eggs of brine shrimp need to be kept dry and dark. Weigh about 15g of sea salt and dissolve it in 1L of water to prepare the brine shrimp egg hatching water. The density should not exceed 4g / L brine shrimp eggs.

[0202] 5Fish embryo culture medium

[0203] Weigh 2940 mg of anhydrous calcium chloride, 1233 mg of magnesium sulfate heptahydrate, 630 mg of sodium bicarbonate, and 55 mg of potassium chloride and dissolve them in 10 L of water. The pH value is 6.5 to 8.5. All chemicals are of analytical grade.

[0204] 6 Phosphate buffered saline (PBS) solution

[0205] 7. Acetyl hexapeptide-8 solution: Prepare 0.8 g / L acetyl hexapeptide-8 solution with water.

[0206] 8RNAlater solution (Invitrogen; AM7020) was stored frozen.

[0207] 9TRIzol reagent (Invitrogen; 15596018) was stored in a refrigerator.

[0208] 10 Chloroform

[0209] 11 Isopropyl alcohol is stored frozen.

[0210] 12 Ethanol Weigh 37.5 mL of ethanol and dissolve it in 12.5 mL of ultrapure distilled water and store it frozen.

[0211] 13gDNA Eraser PrimerScript RT Kit (Takara; Cat no.RR047A)

[0212] 14 Real-time PCR kit: SYBR Premix Ex Taq (Takara; Cat no.RR420A)

[0213] 3 Experimental methods

[0214] The test needs to set up a blank control group (fish embryo culture medium), a positive control group (acetyl hexapeptide-8) and a test group (test substance). If an organic solvent is used to assist dissolution, the solvent concentration in each group of solutions must be the same.

[0215] 1.1 Blank control group setting

[0216] 36 zebrafish were randomly selected and evenly distributed into a 24-well plate with 3 wells, each well containing 12 zebrafish and 2.5 mL of fish embryo culture medium.

[0217] 1.2 Positive control group setting

[0218] 36 zebrafish were randomly selected and evenly distributed into a 24-well plate with 3 wells, each well containing 12 zebrafish and 2.5 mL of acetyl hexapeptide-8 working solution.

[0219] 1.3 Test substance treatment

[0220] 36 zebrafish were randomly selected and evenly distributed into 24-well plates with 3 wells, each containing 12 zebrafish and 2.5 mL of the test solution. They were placed in a 28°C±1°C incubator for 24 h±1 h. The 12 zebrafish in each well were collected into a 1.5 mL tube, the solution was removed, 0.5 mL of RNAlater solution was added, and the tubes were frozen for storage.

[0221] 2 RNA extraction

[0222] Remove RNAlater solution and rinse with PBS solution three times. Place in ice bath, remove PBS solution, and add 500μL TRIzol reagent. Homogenize embryos with a pellet pestle and place in ice bath for 10 minutes. Add 100μL chloroform, vortex for 1 minute, and place in ice bath for 5 minutes. Centrifuge samples for 20 minutes, transfer the upper layer solution to a 1.5mL centrifuge tube, add 250μL isopropanol, vortex for 1 minute, place in ice bath for 10 minutes, and centrifuge samples for 20 minutes. Remove all supernatant, add 500μL ethanol, vortex for 1 minute, centrifuge samples for 5 minutes, and place in ice bath for 10 minutes. Repeat this step 3 times. Remove ethanol, centrifuge samples for 5 minutes, and air-dry samples in a fume hood with the lid open for 10 minutes. Add 10μL DNase / RNase-free ultrapure distilled water and heat at 55℃ for 15 minutes.

[0223] 3cDNA synthesis

[0224] The RNA sample was diluted to 1000 ng / 7 μL with DNase / RNase-free ultrapure distilled water. The RNA was synthesized into cDNA using the PrimerScript RT Kit with gDNA Eraser (Takara; Cat no.RR047A) and stored frozen.

[0225] 4. Real-time RT-PCR

[0226] Prepare primer mix and real-time PCR master mix for each primer. Dilute cDNA sample 10-fold with DNase / RNase-free ultrapure distilled water. Add 9 μL PCR master mix and 1 μL diluted cDNA sample to each well of 96-well PCR plate. Seal the 96-well PCR plate with optical film, centrifuge the plate for 5 minutes, and perform real-time PCR amplification.

[0227] 5 Data and result calculation

[0228] Real-time PCR data were collected. Ct was used as the amplification result, the amplification amount of β-actin gene was used as the housekeeping gene, and the relative expression amount of each gene col1a1a was calculated as the test result.

[0229] Note: If a co-solvent is used in the preparation of the test substance, the blank control group value in the formula shall be replaced by the solvent control group value.

[0230] The mean and standard error of each test group were calculated, and the statistical results were expressed as mean ± standard error. The data were analyzed by variance analysis using statistical software, and the relative expression of the target gene between the test group and the blank control group (or solvent control group) was tested by two-tailed T test to obtain the p value. p<0.05 indicated a significant difference.

[0231] Results Report

[0232] 1. Collagen gene expression promotion rate

[0233] The promotion rate of collagen gene expression by the test substance at the corresponding test concentration.

[0234] 2 reviews

[0235] Evaluate the ability of the test substance to promote collagen gene expression, and analyze whether the test values ​​of the test substance treatment group and the blank control group are

[0236] There is statistical difference (p<0.05).

[0237] 3. Interpretation of the results

[0238] If the type I collagen gene expression promotion rate is positive and statistically significant (p<0.05), the test substance is judged to have anti-wrinkle and firming effects and can be used to support anti-wrinkle and firming effect claims. The results of the type I collagen gene expression promotion rate can be referred to Table 6;

[0239] Table 6 Promotion rate of zebrafish type I collagen-based col1a1a expression

[0240]

[0241]

[0242] Results Analysis

[0243] 1. From Example 1 to Example 11, when the total amount of tetrahydrocurcumin and pterostilbene is controlled at 500 μg / ml and the ratio of the two is 2 to 5.6:1, the performance reaches the peak value;

[0244] 2. From Example 12 to Example 14, according to the ratio of 3:1, when the sum of the concentrations of the two is between 500 and 1000 μg / ml, the performance is optimal, that is, at 1000 μg / ml, the performance is close to the top, and this trend is consistent with the above-mentioned tyrosinase inhibition rate and free radical scavenging.

[0245] Part III External Inspection Test

[0246] We sent the liposome Example 1 to Shaanxi Boxi General Testing Technology Co., Ltd. for testing;

[0247] The test report numbers are: G1276-2401260, G1276-2401266, G1276-2401270, G1276-2312213;

[0248] The conclusions are:

[0249] In vitro antioxidant effect test (G1276-2401260): The superoxide dismutase (SOD) activity and the nuclear transcription factor NRf2 content of the sample-encapsulated active ingredient A2 (tetrahydrocurcumin, pterostilbene) at a concentration of 0.0156% (v / v) were significantly increased, with the increase rates being 212.87% and 176.60%, respectively, indicating that the sample can enhance the superoxide dismutase (SOD) activity and the nuclear transcription factor NRf2 content at this concentration, and has an antioxidant effect.

[0250] In vitro anti-wrinkle efficacy test (G1276-2401266): The contents of 8-hydroxydeoxyguanosine (8-OHdG) and γ-H2AX in the sample-encapsulated active ingredient A2 (tetrahydrocurcumin, pterostilbene) at a concentration of 0.12% (v / v) were significantly decreased, with inhibition rates of 55.34% and 22.30%, respectively. The content of type I collagen (Collagen I) was significantly increased, with an increase rate of 31.74%, indicating that the sample can reduce the contents of 8-hydroxydeoxyguanosine (8-OHdG) and γ-H2AX, and increase the content of type I collagen (Collagen I) at this concentration, and has anti-wrinkle efficacy.

[0251] In vitro whitening efficacy test (G1276-2401270): The melanin content of the sample-encapsulated active substance A2 (tetrahydrocurcumin, pterostilbene) at a concentration of 0.058% (v / v) was significantly reduced, with an inhibition rate of 30.73%, and a tyrosinase activity inhibition rate of 28.28%, indicating that the sample can reduce the melanin content and inhibit tyrosinase activity at this concentration, and has a whitening effect.

[0252] The permeation behavior of the sample was studied based on the suckling pig skin-Franz cell system (G1276-2312213): After 8 hours of surface exposure, the cumulative permeation amount of the active substance encapsulated in the sample was 12.62 μg, the diffusion percentage was 7.01%, and the recovery rate was 99.57%, indicating that the active substance A2 encapsulated in the sample had undergone permeation behavior.

[0253] Part 4 Application Formulation

[0254] The product of the present invention can be applied to various application formulas. The following lists essence milk and essence cream as examples, and the formulas are as follows:

[0255] The formula of the essence milk can be referred to Table 7;

[0256] Table 7 Recipe

[0257]

[0258]

[0259] The formula of the essence cream can be found in Table 8;

[0260] Table 8 Recipe

[0261]

[0262]

[0263] The applicant declares that the present invention illustrates the process method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned process steps, and it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A liposome with high encapsulation rate and anti-aging effect, characterized in that: include: 0.01-3wt% of oil-soluble substances, 1-5wt% of liquid-solid mixed oil, 1-10wt% of phospholipids, 1-10wt% of surfactants, 10-30wt% of polyols, and 45-70wt% of deionized water; The oil-soluble substance includes tetrahydrocurcumin and pterostilbene; the weight ratio of the tetrahydrocurcumin to pterostilbene is 0.5 to 10:1 The liquid-solid mixed grease is composed of liquid grease and solid grease; the weight ratio of the liquid grease to the solid grease is 0.5-3:

1.

2. The liposome with high encapsulation rate and anti-aging effect according to claim 1, characterized in that: The weight ratio of tetrahydrocurcumin to pterostilbene is 8-3:

1.

3. The liposome with high encapsulation rate and anti-aging effect according to claim 1, characterized in that: The invention also contains synergistic ingredients, which are one or more combinations of tranexamic acid, ectoine, and narcissus bulb extract.

4. The liposome with high encapsulation rate and anti-aging effect according to claim 3, characterized in that: The weight ratio of the synergistic component to pterostilbene in the composition is 0.1-0.5:

1.

5. The liposome with high encapsulation rate and anti-aging effect according to claim 1, characterized in that: The liquid-solid mixed oil is any combination of glyceryl monostearate / glyceryl monolaurate, acetylated monoglyceride / isononyl isononanoate, triacetin / caprylic capric glyceride, diisopropyl oxalate / acetylated monoglyceride, isopropyl lanolate / isotridecyl isononanoate.

6. The liposome with high encapsulation rate and anti-aging effect according to claim 1, characterized in that: The phospholipid is one of soybean lecithin and egg yolk lecithin, or a mixture of both; The surfactant is a mixture of one or more of Tween-80, Tween-60, decaglycerol monolaurate, decaglycerol monomyristate, hexaglycerol monolaurate, fatty alcohol polyether, and sucrose stearate; The polyol is a mixture of one or more of propylene glycol, butylene glycol, pentylene glycol and glycerol.

7. A method for preparing liposomes according to any one of claims 1 to 6, characterized in that: The steps include: Step 1: Heat the liquid-solid mixed oil to 60-80°C, add phospholipids, and then add the oil-soluble substance to be coated, stir evenly, and obtain a phospholipid mixed liquid; Step 2: Mix the surfactant, polyol and water and heat them to the same temperature as the phospholipid mixture to obtain an aqueous phase solution; Step 3: Add the phospholipid mixture into the aqueous phase solution, emulsify it evenly, and then perform high-pressure homogenization to obtain liposome micelles.

8. The method for preparing liposomes according to claim 7, characterized in that: In step 3, the pressure of high-pressure homogenization is 80 to 130 MPa, and the number of cycles is 3 to 7 times.

9. Use of the liposome according to any one of claims 1 to 6 in preparing cosmetics.

10. The use according to claim 9, characterized in that The cosmetics are lotions and creams.

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