Compound traditional Chinese medicine liposome essence and preparation method thereof

Abstract

The invention belongs to the field of cosmetic preparation, and particularly relates to compound traditional Chinese medicine liposome essence and a preparation method thereof. The essence is prepared from the following components in parts by volume: 0.5 to 1.5 parts of traditional Chinese medicine extracting solution prepared from dendrobium officinale, rhizoma bletillae, semen coicis and lucid ganoderma, 0.1 to 0.5 part of anthocyanin solution with the concentration of 0.25 mg / mL, 70 to 100 parts of egg yolk lecithin, 10 parts of cholesterol, 15 to 25 parts of hyaluronic acid, 8 to 10 parts of yeast extract, 8 to 10 parts of glycerinum, 10 to 20 parts of sodium alginate and 1 to 2 parts of neroli essential oil. The preparation method of the compound traditional Chinese medicine liposome essence comprises two steps of liposome preparation and liposome compounding. The compound traditional Chinese medicine liposome essence prepared by the invention has good transdermal absorption effect and whitening and moisturizing effects, is good in stability and safety, is simple in preparation process, and has wide market application prospects.

Description

Technical Field

[0001] The invention relates to the field of preparation of traditional Chinese medicine cosmetics, and in particular to a compound traditional Chinese medicine liposome essence and a preparation method thereof. Background Art

[0002] As the saying goes, "Everyone has a desire for beauty; the pursuit of beauty has been a trend for thousands of years." Whitening and lightening spots is actually a hot topic that has attracted people's attention since ancient times. With the advancement of science and technology and economy, the disadvantages of chemical products have been continuously exposed, and consumers' consumption patterns have begun to shift towards "advocating nature."

[0003] In recent years, Chinese herbal skin care products have been favored by consumers for their profound Chinese medicine culture and their natural, safe and mild characteristics. The rise of the Chinese herbal cosmetics industry has not only contributed to my country's economic growth, but also satisfied people's pursuit of high-quality life. Therefore, in-depth research on the role of Chinese medicine in cosmetics is of great significance for discovering high-quality raw materials, promoting industrial innovation and meeting consumer needs.

[0004] Currently, commonly used skin care products are traditional forms such as ointments, emulsions, solutions and gels. However, due to the natural barrier function of the skin, it is difficult for skin care products to play a role in skin care. Liposomes are a type of liposome that can encapsulate various active ingredients and have a special structure similar to the phospholipid bilayer of biological cell membranes. They have good physiological compatibility and safety, with a diameter of 10 to 100 nm. They can carry active molecules and penetrate into dermal cells efficiently, better playing a role in skin care, and therefore have potential application prospects in the field of skin care products.

[0005] Based on this, the present invention proposes a compound Chinese medicine liposome essence and a preparation method thereof. Our team deeply explores the "Five Elements Yin and Yang Balance Theory" of Materia Medica, inherits the inspiration of skin care from ancient books, and selects Guizhou medicinal and edible Chinese medicinal materials Bletilla striata, Ganoderma lucidum, Dendrobium officinale and Coix seed with whitening and freckle-removing, anti-oxidation and anti-aging effects as raw materials. At the same time, anthocyanins, neroli essential oil, hyaluronic acid, yeast extract and other whitening and skin care ingredients are compounded, and ultrasonic technology, new freeze-drying technology and high-tech liposome encapsulation technology are used to develop a nano essence with multiple functions such as whitening and moisturizing, fading spots, anti-oxidation and anti-aging. Summary of the invention

[0006] The purpose of the present invention is to provide a compound Chinese medicine liposome essence and a preparation method thereof.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] The compound Chinese medicine liposome essence of the present invention comprises the following components: 0.5-1.5 parts by volume of a Chinese medicine extract prepared from dendrobium officinale, bletilla striata, coix seed and ganoderma lucidum, 0.1-0.5 parts by volume of anthocyanin solution with a concentration of 0.25 mg / mL, 70-100 parts by mass of egg yolk lecithin, 10 parts by mass of cholesterol, 15-25 parts by mass of hyaluronic acid, 8-10 parts by mass of yeast extract, 8-10 parts by mass of glycerol, 10-20 parts by mass of sodium alginate and 1-2 parts by mass of orange blossom essential oil.

[0009] Preferably, the compound Chinese medicine liposome essence of the present invention comprises the following components: 0.8 parts by volume of a Chinese medicine extract prepared from Dendrobium officinale, Bletilla striata, Coix seed and Ganoderma lucidum, 0.2 parts by volume of anthocyanin solution with a concentration of 0.25 mg / mL, 100 parts by mass of egg yolk lecithin, 10 parts by mass of cholesterol, 20 parts by mass of hyaluronic acid, 8 to 10 parts by mass of yeast extract, 8 to 10 parts by mass of glycerol, 15 parts by mass of sodium alginate, and 1 part by mass of orange blossom essential oil.

[0010] The preparation method of the traditional Chinese medicine extract of the present invention is as follows: weigh four traditional Chinese medicines of Dendrobium officinale, Ganoderma lucidum, Bletilla striata and Coix seed in a mass ratio of 1:1:1:1, add them into an extraction solvent at a mass-to-volume ratio of 1g:8-12mL, heat and extract at 60°C for 3h, let stand for 10min, and absorb the supernatant to obtain the extract.

[0011] Preferably, in the method for preparing the traditional Chinese medicine extract of the present invention, the material-liquid ratio is 1 g:10 mL.

[0012] In the method for preparing the traditional Chinese medicine extract of the present invention, the extraction solvent is any one of pure water or 30% to 60% ethanol solution (the volume ratio of anhydrous ethanol to pure water is 30 to 60:100).

[0013] Preferably, in the method for preparing the traditional Chinese medicine extract of the present invention, the extraction solvent is a 60% ethanol solution (the volume ratio of anhydrous ethanol to pure water is 60:100).

[0014] The preparation method of the compound Chinese medicine liposome essence of the present invention comprises the following steps:

[0015] S1 Preparation of liposomes:

[0016] Weigh egg yolk lecithin and cholesterol according to the formula, add 2 parts by volume of anhydrous ethanol, and perform rotary evaporation to form a film of the liquid; put it in an oven, vacuum dry it for 8 hours, and take it out; add Chinese herbal medicine extract, anthocyanin solution and 10 parts by volume of PBS phosphate buffer with a pH of 7.45, and perform ultrasonic treatment for 25 minutes to disperse it into liposomes; filter, and perform rotary evaporation at 75°C to remove ethanol to obtain the product;

[0017] S2 liposome compound:

[0018] Add the formulated amounts of hyaluronic acid, yeast extract, glycerin, sodium alginate and neroli essential oil to obtain the essence.

[0019] In the method for preparing the compound Chinese medicine liposome essence of the present invention, the rotary evaporation conditions in step S1 are: rotary evaporation at 50-65° C. for 15-30 min.

[0020] Preferably, in the method for preparing the compound Chinese medicine liposome essence of the present invention, the rotary evaporation conditions in step S1 are: rotary evaporation at 55° C. for 20 min.

[0021] In the method for preparing the compound Chinese medicine liposome essence of the present invention, the filtering in step S1 is specifically: first filtering with a 0.45 μm PES microporous filter membrane, and then filtering with a 0.22 μm PES microporous filter membrane.

[0022] The unit of mass parts in the present invention is mg, and the unit of volume parts is mL.

[0023] Beneficial effects of the present invention:

[0024] The present invention studies Yimu Bencao, inherits the wisdom of skin care in ancient books, follows the principle of Chinese medicine compatibility, adopts the medicinal and edible Chinese medicines Bletilla striata, Ganoderma lucidum, Dendrobium candidum and Coix seeds with whitening, anti-oxidation and anti-aging effects as raw materials, and compound anthocyanins, hyaluronic acid and other auxiliary materials with whitening and moisturizing effects to prepare a compound Chinese medicine liposome essence. Through transdermal absorption test, human moisturizing test and in vitro tyrosinase inhibition test, it is confirmed that the essence prepared by the present invention has a transdermal penetration promoting effect, good moisturizing effect, and good whitening effect. It is confirmed by high temperature stability, low temperature stability and long-term stability tests that the essence prepared by the present invention has no stratification, coagulation, mildew and deterioration under the above conditions, proving that the essence has good stability. It is confirmed by pH determination and safety evaluation test of the essence that the essence of the present invention is safe, reliable, non-irritating, and suitable for skin care.

[0025] The preparation process of the compound Chinese medicine liposome essence of the present invention is simple. The inventors have determined the optimal preparation process conditions of the compound Chinese medicine liposome essence through a series of experiments, which are as follows: through antioxidant experiments, it is determined that 60% ethanol solution is the best extraction solvent for four Chinese medicinal materials, namely, Dendrobium officinale, Ganoderma lucidum, Bletilla striata and Coix seed; through investigation of different solid-liquid ratios, it is determined that the best solid-liquid ratio of the above four medicinal materials to 60% ethanol solution is 1:10; through single factor dosage investigation and prescription optimization experiments, it is determined that the best mass ratio of egg yolk lecithin to cholesterol is 10:1, 55°C is the best rotary evaporation temperature, and 20 minutes is the best rotary evaporation time. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 They are Chinese medicinal materials (a: Dendrobium officinale; b: Ganoderma lucidum; c: Bletilla striata; d: Coix lachryma-jobi);

[0027] Figure 2 It is anthocyanin solution;

[0028] Figure 3 are Chinese herbal medicine extracts with different alcohol-water ratios (in the figure: a is extracted with 90% ethanol solution; b is extracted with 60% ethanol solution; c is extracted with 30% ethanol solution);

[0029] Figure 4 The extracts of Chinese medicine with different alcohol-water ratios after being placed at 4°C for 24 hours (in the figure: a is extracted with 60% ethanol solution; b is extracted with 30% ethanol solution; c is extracted with pure water);

[0030] Figure 5 The film formation of liposomes at different ratios of egg yolk phosphatidylcholine to cholesterol (in the figure: a is 6:1; b is 7:1; c is 8:1; d is 9:1; e is 10:1);

[0031] Figure 6 The clarity of blank liposomes after passing through a microporous filter membrane at different ratios of egg yolk phosphatidylcholine to cholesterol (in the figure: 1 is 6:1; 2 is 7:1; 3 is 8:1; 4 is 9:1; 5 is 10:1);

[0032] Figure 7 The Tyndall effect after blank liposome filtration at different ratios of egg yolk phosphatidylcholine to cholesterol (in the figure: 1 is 6:1; 2 is 7:1; 3 is 8:1; 4 is 9:1; 5 is 10:1);

[0033] Figure 8 The film formation of liposomes at different rotary evaporation temperatures (in the figure: a is 45°C; b is 50°C; c is 55°C; d is 60°C; e is 65°C);

[0034] Fig. 9 The film formation of liposomes at different rotary evaporation times (in the figure: a is 10 min; b is 15 min; c is 20 min; d is 25 min; e is 30 min);

[0035] Fig.10 is the glucose standard curve;

[0036] Fig.11 The effect of different material-liquid ratios on the extraction rate of traditional Chinese medicine;

[0037] Fig.12 This is the moisturizing test result of liposome essence;

[0038] Fig.13 This is the standard curve of the penetration enhancement test of compound Chinese medicine liposome essence;

[0039] Fig.14 It is the cumulative penetration amount of liposome essence in effective area;

[0040] Fig.15 This is the experimental result of the inhibition of tyrosinase by the Chinese herbal compound liposome essence;

[0041] Fig.16 This is the result of the high temperature resistance test of liposome essence;

[0042] Fig.17 This is the result of the low temperature resistance test of liposome essence;

[0043] Fig.18 This is the result of long-term stability test of liposome essence;

[0044] Fig.19 This is the result of measuring the pH value of the essence;

[0045] Fig. 20 This is a transmission electron microscopy image of the liposome essence;

[0046] Fig.21 The results of the safety test. DETAILED DESCRIPTION

[0047] The technical solution of the present invention is described in detail below in conjunction with specific embodiments. The following embodiments are only used for explanation and illustration, and do not constitute a limitation on the technical solution of the present invention.

[0048] Example 1

[0049] A compound Chinese medicine liposome essence, the formula is as follows:

[0050] 0.8mL of traditional Chinese medicine extract made from Dendrobium officinale, Bletilla striata, Coix seed and Ganoderma lucidum, 0.2mL of 0.25mg / mL anthocyanin solution, 100mg egg yolk lecithin, 10mg cholesterol, 20mg hyaluronic acid, 9mg yeast extract, 9mg glycerin, 15mg sodium alginate, and 1mg orange blossom essential oil.

[0051] The preparation method of the above-mentioned compound Chinese medicine liposome essence comprises the following steps:

[0052] S1 Preparation of liposomes

[0053] Weigh egg yolk lecithin and cholesterol according to the formula, add 2 mL of anhydrous ethanol, and perform rotary evaporation at 55°C for 20 min to form a film of the liquid; put it into an oven, vacuum dry it for 8 h, and take it out; add Chinese herbal medicine extract, anthocyanin solution and 10 mL of PBS phosphate buffer with a pH of 7.45, and disperse it into liposomes by ultrasound; the obtained liposomes are first filtered with a 0.45 μm PES microporous filter membrane, and then filtered with a 0.22 μm PES microporous filter membrane, and rotary evaporated at 75°C to remove ethanol, and the liposomes are obtained;

[0054] S2 liposome complex

[0055] Add the formulated amounts of hyaluronic acid, yeast extract, glycerin, sodium alginate and neroli essential oil to obtain the essence.

[0056] Example 2

[0057] A compound Chinese medicine liposome essence, the formula is as follows:

[0058] 0.5mL of traditional Chinese medicine extract made from Dendrobium officinale, Bletilla striata, Coix seed and Ganoderma lucidum, 0.1mL of 0.25mg / mL anthocyanin solution, 70mg egg yolk lecithin, 10mg cholesterol, 15mg hyaluronic acid, 8mg yeast extract, 8mg glycerin, 10mg sodium alginate, and 1mg orange blossom essential oil.

[0059] The preparation method of the above-mentioned compound Chinese medicine liposome essence comprises the following steps:

[0060] S1 Preparation of liposomes

[0061] Weigh egg yolk lecithin and cholesterol according to the formula, add 2 mL of anhydrous ethanol, and steam at 50°C for 15 minutes to form a film; put it in an oven, vacuum dry it for 8 hours, and take it out; add Chinese herbal medicine extract, anthocyanin solution and 10 mL of PBS phosphate buffer with a pH of 7.45, and disperse it into liposomes by ultrasound; the obtained liposomes are first filtered with a 0.45 μm PES microporous filter membrane, and then filtered with a 0.22 μm PES microporous filter membrane, and steamed at 75°C to remove ethanol, and the liposomes are obtained;

[0062] S2 liposome complex

[0063] Add the formulated amounts of hyaluronic acid, yeast extract, glycerin, sodium alginate and neroli essential oil to obtain the essence.

[0064] Example 3

[0065] A compound Chinese medicine liposome essence, the formula is as follows:

[0066] 1.5mL of traditional Chinese medicine extract made from Dendrobium officinale, Bletilla striata, Coix seed and Ganoderma lucidum, 0.5mL of 0.25mg / mL anthocyanin solution, 100mg egg yolk lecithin, 10mg cholesterol, 25mg hyaluronic acid, 10mg yeast extract, 10mg glycerin, 20mg sodium alginate, and 2mg orange blossom essential oil.

[0067] The preparation method of the above-mentioned compound Chinese medicine liposome essence comprises the following steps:

[0068] S1 Preparation of liposomes

[0069] Weigh egg yolk lecithin and cholesterol according to the formula, add 2 mL of anhydrous ethanol, and steam at 65°C for 30 minutes to form a film; put it in an oven, vacuum dry it for 8 hours, and take it out; add Chinese herbal medicine extract, anthocyanin solution and 10 mL of PBS phosphate buffer with a pH of 7.45, and disperse it into liposomes by ultrasound; the obtained liposomes are first filtered with a 0.45 μm PES microporous filter membrane, and then filtered with a 0.22 μm PES microporous filter membrane, and steamed at 75°C to remove ethanol, and the liposomes are obtained;

[0070] S2 liposome complex

[0071] Add the formulated amounts of hyaluronic acid, yeast extract, glycerin, sodium alginate and neroli essential oil to obtain the essence.

[0072] In order to further verify the reliability of the present invention and screen out the best solution, the inventor conducted a series of experiments, as follows:

[0073] 1. Materials and instruments

[0074] The main materials and reagents are shown in Table 1, and the main instruments are shown in Table 2.

[0075] Table 1 Main materials and reagents

[0076]

[0077] Table 2 Main instruments

[0078]

[0079]

[0080] 2. Experimental methods and results

[0081] 2.1 Selection of solvents for Chinese herbal medicine extracts

[0082] In view of the low toxicity of ethanol to the human body, distilled water and ethanol of various concentrations were selected as solvents in this experiment to evaluate their solubility properties.

[0083] Weigh 1g of each of the four Chinese medicinal materials, Dendrobium officinale, Ganoderma lucidum, Bletilla striata, and Coix seed, in a mass ratio of 1:1:1:1. Figure 1 Weigh 5 mg of anthocyanin and add 20 mL of pure water to prepare an anthocyanin solution with a concentration of 0.25 mg / mL, as shown in Figure 2 As shown. Use anhydrous ethanol to prepare pure water, 30%, 60%, and 90% ethanol solutions as solvents for Chinese medicine extracts. The detailed ratios are shown in Table 3. The solid-liquid ratio is 1:10g / mL. Heat the extract in a constant temperature water bath at 60°C for 3h. After stopping the heating, let it stand for ten minutes, absorb the supernatant, and save it for later use. Figure 3 shown.

[0084] Table 3 Solvent ratio of Chinese herbal medicine extracts

[0085]

[0086] The supernatant was placed at 4°C for 24 h. Figure 4 A large amount of precipitation was observed in the 90% ethanol solution extract, which was discarded and the DPPH clearance rate of the remaining three groups was determined.

[0087] 2.1.1 [DPPH-] ​​Antioxidant Experiment Screening for the Optimal Solvent Ratio

[0088] The DPPH free radical scavenging ability test kit was used to perform the DPPH antioxidant test according to the instructions. Blank solution, test solution and control solution were prepared respectively. The absorbance values ​​of different samples at a wavelength of 515 nm were measured, and the free radical scavenging rate was calculated according to the following formula: 空白 represents the absorbance of the blank solution, A 测定 Absorbance of representative sample set:

[0089] Clearance rate (%) = [(A 空白 -A 测定 ) / A 空白 ]×100%

[0090] 2.1.2 Results and Analysis

[0091] The absorbance of different samples at 515 nm is shown in Table 4, and the DPPH free radical scavenging test results of the water extract, 30% ethanol solution, and 60% ethanol solution are shown in Tables 5-7.

[0092] Table 4 Absorbance of samples at 515nm

[0093]

[0094] Table 5 DPPH free radical scavenging test results of water extract

[0095]

[0096] Table 6 DPPH free radical scavenging test results of 30% ethanol solution extract

[0097]

[0098] Table 7 DPPH free radical scavenging test results of 60% ethanol solution extract

[0099]

[0100] From the results in the above table, it can be seen that 60% ethanol solution is selected as the extraction solvent for the four Chinese medicinal materials of Dendrobium officinale, Ganoderma lucidum, Bletilla striata and Coix seed.

[0101] 2.2 Single factor dosage investigation and prescription optimization

[0102] As the most basic and widely used thin film dispersion method for preparing liposomes, the process is simple and easy to operate. This method was used in this experiment.

[0103] 2.2.1 Selection of the ratio of egg yolk lecithin and cholesterol

[0104] Egg yolk lecithin contains higher phosphatidylcholine than soybean lecithin, so egg yolk lecithin and cholesterol were selected for the experiment. First, accurately weigh the egg yolk lecithin and cholesterol with a mass ratio of 6:1, 7:1, 8:1, 9:1, and 10:1, and put them into 2mL EP tubes respectively, add 2mL of anhydrous ethanol, and sonicate completely. The egg yolk lecithin and cholesterol with different ratios are shown in Table 8.

[0105] Table 8 Different ratios of egg yolk lecithin and cholesterol

[0106]

[0107] 2.2.2 Preparation of blank liposomes by thin film dispersion method

[0108] Rotary evaporation was performed at 65°C for 15 minutes to form a film on the wall. Then, the container was sealed with filter paper and rubber band to prevent contamination by impurities, and the container was placed in an oven for vacuum drying for 8 hours. Then, 10 mL of PBS phosphate buffer with a pH value of 7.45 was taken out with a pipette and added to the container where the film had been formed. The container was ultrasonically dispersed for about 25 minutes to form liposomes.

[0109] To ensure pure liposomes, first filter with a 0.45 μm PES microporous membrane, then filter with a 0.22 μm PES microporous membrane, and observe the phenomenon.

[0110] 2.2.3 Screening of matrix ratio of blank liposomes

[0111] The liposome film formation of different egg yolk phosphatidylcholine and cholesterol ratios after rotary evaporation is shown in Figure 5 , Table 9. After two microporous membrane filtrations, the clarity of the liposomes is as follows Figure 6 As shown in Figure 2. According to the classification of dispersed systems, liposomes are a thermodynamically unstable colloidal system. When irradiated with a laser pen, the sample produces the Tyndall effect as shown in Figure 2. Figure 7 By observing the three indicators of wall residue, glossiness and clarity after hydration for 25 minutes, the liposomes with different ratios were evaluated, and the results of the investigation on the dosage of egg yolk lecithin and cholesterol were obtained, as shown in Table 10.

[0112] Table 9 Liposome film formation with different ratios of egg yolk phosphatidylcholine and cholesterol

[0113]

[0114] Table 10 Results of the investigation of the dosage of egg yolk lecithin and cholesterol

[0115]

[0116] Combined with the above table, it can be seen that when the ratio of egg yolk lecithin to cholesterol is 10:1, the prepared liposomes are optimal, so 10:1 is selected as the optimal ratio.

[0117] 2.2.4 Screening of rotary evaporation temperature of blank liposomes

[0118] The mass ratio of egg yolk lecithin and cholesterol was selected to be 10:1 to prepare blank liposomes. The rotary evaporation time was fixed at 15 minutes, and the film formation of liposomes was observed at rotary evaporation temperatures of 45°C, 50°C, 55°C, 60°C, and 65°C. The three indicators of film formation, film thickness, and film uniformity were used to investigate the results. Figure 8 , as shown in Table 11.

[0119] Table 11 Effect of different rotary evaporation temperatures on liposome film formation

[0120]

[0121] After film formation, seal with filter paper and rubber band to prevent impurities from falling in, put into oven, vacuum dry for 8 hours, and take out. Use a pipette to draw 10mL PBS phosphate buffer with a pH value of 7.45, add it to the container where the film has been formed, and ultrasonicate for about 25 minutes to disperse it to form liposomes. The effects of different rotary evaporation temperatures on liposomes were evaluated using three indicators: wall residue, solid residue, and clarity. The results are shown in Table 12.

[0122] Table 12 Results of the investigation on the effect of different rotary evaporation temperatures on the preparation of liposomes

[0123]

[0124]

[0125] Comprehensive analysis of Tables 11 and 12 shows that when the rotary evaporation temperature is 55°C, the film-forming effect is better, the prepared liposomes have less residue on the wall and solid residue, and the liquid clarity is good, so 55°C is selected as the optimal rotary evaporation temperature.

[0126] 2.2.5 Blank liposome rotary evaporation time screening

[0127] The mass ratio of egg yolk lecithin to cholesterol was 10:1 to prepare blank liposomes. The rotary evaporation temperature was fixed at 55°C, and the film formation of the liposomes was observed at rotary evaporation times of 10 min, 15 min, 20 min, 25 min, and 30 min. The three indicators of film formation, film thickness, and film uniformity were used to investigate the results. Fig. 9 , as shown in Table 13.

[0128] Table 13 Effect of different rotary evaporation time on liposome film formation

[0129]

[0130] After film formation, seal with filter paper and rubber band to prevent impurities from falling in, put into oven, vacuum dry for 8 hours, and take out. Use a pipette to draw 10mL of PBS phosphate buffer with a pH value of 7.45, add it to the container where the film has been formed, and ultrasonicate for about 30 minutes to disperse it to form liposomes. The effects of different rotary evaporation times on liposomes were evaluated using three indicators: wall residue, solid residue, and clarity. The results are shown in Table 14.

[0131] Table 14 Results of the investigation on the effect of different rotary evaporation temperatures on the preparation of liposomes

[0132]

[0133] Comprehensive analysis of Tables 13 and 14 shows that when the rotary evaporation time is 20 min, the film-forming effect is better, the prepared liposomes have less residue on the wall and solid residue, and the liquid clarity is good, so 20 min is selected as the optimal rotary evaporation time.

[0134] In summary, egg yolk lecithin and cholesterol were used to prepare blank liposomes in a mass ratio of 10:1, and the optimal rotary evaporation process conditions were: 55°C, 20min.

[0135] 2.2.6 Effect of different material-liquid ratios on the extraction rate of traditional Chinese medicine

[0136] Accurately weigh 1 g each of four Chinese medicinal materials, including Dendrobium officinale, Ganoderma lucidum, Bletilla striata, and Coix seed, with a mass ratio of 1:1:1:1, and then mix them according to the solid-liquid ratio of 1:8, 1:9, 1:10, 1:11, and 1:12, respectively. Heat and extract in a constant temperature water bath at 60°C for 3 h. After stopping the heating, let it stand for 10 min, aspirate the supernatant, and determine the polysaccharide extraction rate.

[0137] The polysaccharide extraction rate was determined by the phenol-sulfuric acid method, as follows: Take an appropriate amount of Chinese herbal polysaccharide extract, add 1 mL of 5% phenol aqueous solution and 5 mL of concentrated sulfuric acid in sequence, add water to make up to 15 mL, mix well, react in a 35°C water bath for 15 min, then measure the absorbance at 485 nm and substitute it into the glucose standard curve (glucose standard curve see Fig.10 ), the polysaccharide concentration in the extract was obtained, and the polysaccharide extraction rate was calculated according to the following formula:

[0138] Polysaccharide extraction rate = (actual amount of polysaccharides obtained / total amount of polysaccharides in the raw material) × 100%

[0139] The different feed-liquid ratios are shown in Table 15, and the effects of different feed-liquids on the polysaccharide extraction rate are shown in Table 16. Fig.11 .

[0140] Table 15 Different feed-liquid ratios

[0141]

[0142] Table 16 Data analysis of the effect of solid-liquid ratio on polysaccharide extraction rate

[0143]

[0144] The results show that when the solid-liquid ratio is 1:10, the polysaccharide extraction rate is the highest. Therefore, 1:10 is selected as the optimal solid-liquid ratio of Dendrobium officinale, Ganoderma lucidum, Bletilla striata, Coix seed and extraction solvent.

[0145] 2.3 Liposome drug delivery

[0146] The mass ratio of egg yolk lecithin and cholesterol was fixed at 10:1 (a total of 110 mg), the rotary evaporation temperature was 55 ° C, and the rotary evaporation time was 20 min. The reduced pressure rotation formed a thin film on the wall of the vessel. Seal with filter paper and rubber band to prevent impurities from entering and contaminating, put into the oven, vacuum dry for 8 hours, and take out. Then use a pipette to absorb 0.8 mL of traditional Chinese medicine extract and 0.2 mg of anthocyanin solution. Then absorb 10 mL of PBS phosphate buffer with a pH of 7.45, add it to the container where the film has been formed, and then perform ultrasound for about 25 minutes to disperse it to form liposomes.

[0147] To ensure pure liposomes, first filter with a 0.45 μm PES microporous membrane and then filter with a 0.22 μm PES microporous membrane. To ensure safety, adjust the rotary evaporation temperature to 75°C to evaporate the ethanol.

[0148] The drug encapsulation efficiency of the compound Chinese medicine liposome essence was determined by ultrafiltration centrifugation method as follows: 0.5 mL of the compound Chinese medicine liposome essence was placed in a centrifugal ultrafiltration tube, centrifuged at 8000 r / min for 30 min, and then the ultrafiltrate was filtered and sampled, and the free drug content was determined by HPLC to obtain W 游 ; Take another 1.0 mL of compound Chinese medicine liposome essence, sonicate, dilute to 10 mL with mobile phase, centrifuge for 10 min, filter the supernatant and inject to determine W 测 Calculate the liposome encapsulation efficiency, encapsulation efficiency = (W 总 -W 游 ) / W 总 ×100%.W 总 =W 测 ×10 / 2,W 总 is the total dosage, W 游 The encapsulation efficiency of the compound Chinese medicine liposome essence was determined to be 92.7±3.6%.

[0149] 2.4 Liposome Essence Compound

[0150] Liposomes were prepared according to item "2.3", and hyaluronic acid, yeast extract, glycerin, sodium alginate, and orange blossom essential oil were added in the formula to obtain an essence. The composition of the prepared essence is shown in Table 17.

[0151] Table 17 Essence ingredients

[0152]

[0153] 2.5 Liposome Essence Viscosity Test and Evaluation

[0154] Weigh 10 mg of sodium alginate and add 200 mL of pure water to fully swell for 10 hours.

[0155] The effect of the amount of sodium alginate added on the viscosity of this product was investigated. Liposome liquid was added according to different amounts of sodium alginate. A small amount of liquid was applied to the back of the hand to feel its ductility and viscosity. The results are shown in Table 18.

[0156] Table 18 Evaluation results of skin viscosity perception of different addition amounts of sodium alginate

[0157]

[0158] It can be seen from Table 18 that the optimum addition amount of sodium alginate is 15 parts.

[0159] 2.6 Moisturizing test of Chinese herbal compound liposome essence

[0160] After the liposomes were prepared, an appropriate amount of glycerol was added to measure the moisturizing properties.

[0161] 2.6.1 In vitro moisturizing test

[0162] The compound liposome essence prepared in this study and four common brands of essences on the market were used for in vitro moisturizing tests. In this study, a 2cm×3cm device was prepared using medical slides and breathable tape, which resembled the stratum corneum and epidermis of human skin. This simulation device provides an effective tool for skin research. One drop of sample was added and placed at room temperature. Fig.12 The mass of the sample before placement (M1) and after placement (M2) of the simulation device was precisely weighed, with a weighing interval of 5 minutes, for a total of 4 times, and the moisture retention rate was calculated according to the following formula. The results are shown in Table 19.

[0163]

[0164] Where: M1 is the sample weight, g; M2 is the weight of the sample after standing at room temperature, g.

[0165] Table 19 In vitro moisturizing rate of common essences on the market

[0166]

[0167] According to the data in Table 19, within 20 minutes, the moisturizing rates of the five essences are in the following order: sample 1 > compound liposome essence > sample 3 > sample 4 > sample 2, indicating that the moisturizing rate of this product is good.

[0168] 2.6.2 Human moisturizing test

[0169] The skin water and oil changes of three randomly selected testers were tested before and after using the liposome essence (immediately, 10 minutes, and 30 minutes), and the moisture value was used to evaluate the moisturizing property of the compound Chinese medicine liposome essence. The difference in skin moisture value before and after using the liposome essence reflects the instant hydration ability of the essence, and the skin moisture value after 30 minutes reflects the continuous hydration ability of the compound Chinese medicine liposome essence. The results are shown in Table 20.

[0170] Table 20 Changes in water and oil content of users at different time periods after using the essence

[0171]

[0172] As can be seen from the above table, the essence has a good moisturizing effect.

[0173] 2.7 Penetration test of compound Chinese medicine liposome essence

[0174] 2.7.1 Determination of standard curve

[0175] 2.7.1.1 Preparation of reference solution

[0176] Accurately weigh 10 mg of dry anhydrous glucose and place it in a 50 mL volumetric flask. Add water to the scale to obtain a 0.2 mg / mL glucose solution. This process strictly follows the experimental specifications to ensure the accuracy and repeatability of the solution.

[0177] 2.7.1.2 Drawing of standard curve

[0178] Respectively pipette 0.1mL, 1.25mL, 1.5mL, 1.75mL, 2.0mL, 2.25mL, 2.5mL, and 2.75mL of the reference solution into a 10mL volumetric flask, and add pure water to the scale line. Use concentrated sulfuric acid to hydrolyze the polysaccharide into monosaccharides, and quickly dehydrate them to form aldehyde derivatives, which are then reacted with phenol to form an orange-yellow compound, and its content is determined by colorimetry. Take 1mL of the solution into test tubes, add 5% phenol solution and concentrated sulfuric acid, shake evenly, keep a constant temperature water bath for 30 minutes, and then ice bath for 5 minutes. Measure the absorbance value A at 485nm respectively, use A as the ordinate and the concentration as the abscissa to draw a standard curve, see Fig.13 .

[0179] 2.7.2 Treatment of mouse skin

[0180] The rats were depilated, the skin on the back was peeled, and the subcutaneous fat was scraped clean. The stratum corneum of the skin should be avoided during the process. After being fully washed with freshly prepared physiological saline, the skin was placed under a microscope for observation, and undamaged rat skin with uniform thickness was selected for subsequent experiments. The surface moisture of the rat skin was absorbed by filter paper and spread on a glass plate, and then wrapped with plastic wrap and tin foil and stored in a -20℃ freezer.

[0181] 2.7.3 Transdermal absorption test

[0182] First, prepare the skin sample, rinse it with saline, put it at room temperature, absorb the residual liquid, and confirm the integrity of the skin again. The skin is sandwiched between the two chambers of the permeation pool, and the Chinese medicine compound liposome essence is added to the donor chamber; the receiving chamber is filled with PBS buffer with a pH of 7.45. The mouse skin is in close contact with the two solutions, and the bubbles are exhausted. The temperature is set to (37+0.5)℃ to simulate the actual skin environment, and the magnetic stirring speed is set to 350r / min. Regular sampling (time points are 0h, 0.5h, 1h, 2h, 4h, 6h, 8h, 10h, 24h) is used to measure the absorbance and analyze the drug concentration in the receiving chamber. The transdermal absorption kinetics of the drug are evaluated by calculating the change of the cumulative penetration of the drug over time. The measured absorbance is shown in the table below. The absorbance is substituted into the equation to obtain the polysaccharide concentration, and the cumulative transdermal amount per unit area (Q) is calculated according to the following formula:

[0183]

[0184] This study used the linear regression method to evaluate the drug transdermal diffusion rate. By measuring the volume of the V receiving chamber, the C sampling concentration and the skin exposure area, the time-cumulative permeation curve was drawn and regression analysis was performed to obtain the drug permeation rate per unit time per unit area. Fig.14 As can be seen from the figure, liposome essence has a transdermal penetration-enhancing effect, which is beneficial to the skin's absorption and moisturizing.

[0185] 2.8 Whitening efficacy evaluation - in vitro tyrosinase inhibition test

[0186] Take PBS solution with pH=6.8 as blank control, take appropriate amount of Chinese medicine compound liposome essence (0.5mg / mL), blank liposome, drug extract (1.5mg / mL) and arbutin (150μg / mL) 1mL each in a centrifuge tube; add 500μL tyrosinase solution (100UmL-1), and place in a water bath at 37℃ in the dark for 10min; add 2mL of 1.0mg / mL 3,4-dihydroxyphenylalanine (L-dopa) solution to each tube in turn, and react in the dark for 5min, and measure the absorbance at a wavelength of 475nm. Calculate the inhibition rate of tyrosinase activity:

[0187]

[0188] A is the absorbance value of the enzyme-containing system with sample, B is the absorbance value of the enzyme-free system with sample, C is the absorbance value of the enzyme-containing system without sample, and D is the absorbance value of the enzyme-free system without sample.

[0189] Tyrosinase is a key enzyme in the synthesis of melanin in organisms. Inhibiting tyrosinase activity is one of the main strategies to reduce melanin production. The experimental results of the inhibition of tyrosinase by the Chinese herbal compound liposome essence are as follows: Fig.15 shown.

[0190] 2.9 Stability evaluation

[0191] 2.9.1 High temperature test

[0192] The compound liposome essence of Chinese medicine may delaminate, change color or chemical properties under high temperature. In this experiment, the liposome essence was exposed to a high temperature environment of 40℃ for about 30 minutes, and the liquid state and properties were compared with those before the test after it was taken out and restored to room temperature. Whether there is a significant difference observed by the naked eye is used to evaluate the stability of the essence under high temperature conditions, such as Fig.16 shown.

[0193] 2.9.2 Low temperature test

[0194] The compound liposome essence of Chinese medicine may become more viscous or solidify at low temperature, or undergo changes in physical properties. In this test, the liposome essence is exposed to a low temperature environment of 0℃ for about 30 minutes to simulate the stability under cold conditions. The essence is taken out and restored to room temperature. The gel state and properties are compared with those before the test. Whether there is a significant difference that can be observed with the naked eye is used to evaluate the stability of the essence under low temperature conditions, such as Fig.17 The high temperature test and low temperature test results are shown in Table 21.

[0195] Table 21 Liposome high temperature / low temperature stability evaluation results

[0196]

[0197] 2.9.3 Long-term stability test of liposome serum

[0198] Take an appropriate amount of this product in a 20mL dry glass bottle and place it at room temperature. Observe the clarity of this product and whether it is moldy or deteriorated on the 1st, 7th and 14th days. The results are shown in Table 22. Fig.18 As shown, it can be seen from the results that the compound Chinese medicine liposome essence has good stability.

[0199] Table 22 Analysis and evaluation of long-term stability test results

[0200]

[0201] 2.10 Determination of pH of Liposome Essence

[0202] Use high-quality test paper and lay it flat on the glass slide. Make sure the glass rod is dry and then dip it into the Chinese medicine compound liposome essence and apply it to the test paper. After waiting for 30 seconds, compare the color change with the standard colorimetric card to evaluate the experimental effect and judge its acidity and alkalinity. The result is as follows: Fig.19 shown.

[0203] 2.11 Transmission electron microscopy observation of properties

[0204] The prepared liposome essence was observed by transmission electron microscopy. The results are as follows Fig. 20 shown.

[0205] 2.12 Safety evaluation of serum

[0206] 10 volunteers were randomly selected to conduct the serum application test, and the subjects were required to apply the serum every 48 hours to evaluate its effect on the skin. The skin condition was observed at 0h, 0.5h, and 1h after application, and the application time was 5 days. According to the skin reactions of the subjects in the follow-up visit, the safety of the serum was evaluated according to the safety scoring criteria in Table 23. Fig.21 The results of the skin safety test of the compound Chinese medicine liposome essence showed that the subjects had no adverse reactions such as erythema and edema on their skin, proving that the essence is safe and reliable and suitable for skin care.

[0207] Table 23 Safety scoring criteria

[0208]

[0209] 3. Discussion and Conclusion

[0210] 3.1 Discussion

[0211] 3.1.1 Screening of liposome blank matrix

[0212] Liposome blank matrix screening is a key step in the preparation of liposome drug delivery systems. The purpose is to optimize the composition and preparation conditions of liposomes. First, select the appropriate lipid material. In this experiment, cholesterol and egg yolk lecithin are selected, which will form the bilayer of liposomes. Then, liposomes are prepared by thin film dispersion method and other techniques. Next, the morphology and clarity of the liposomes are evaluated. Finally, the composition and preparation method of the liposomes are adjusted according to the experimental results until the ideal blank matrix is ​​obtained, which lays the foundation for the subsequent loading of drugs.

[0213] 3.1.2 Selection of solvents for drug extracts

[0214] The preliminary experimental method was used to extract four traditional Chinese medicinal materials, namely Dendrobium officinale, Ganoderma lucidum, Bletilla striata and Coix seed. Anhydrous ethanol was used for extraction in this experiment. After screening, 60% ethanol solution was determined to be the best extraction solvent for the four drugs. To ensure safety, the rotary evaporation temperature was adjusted to 75℃ to evaporate the ethanol.

[0215] 3.1.3 Single factor test of liposome preparation conditions

[0216] This preparation used the uniformity, glossiness, clarity, and solid residue of the compound Chinese medicine liposome essence as test indicators, and investigated the amount of egg yolk lecithin, cholesterol, rotary evaporation temperature, rotary evaporation time, and hyaluronic acid. After comprehensive evaluation, it was preliminarily determined that the mass ratio of egg yolk lecithin to cholesterol was 10:1 as the best ratio for preparing liposomes, 55°C as the best rotary evaporation temperature for liposomes, and 20 minutes as the best rotary evaporation time for liposomes.

[0217] 3.1.4 Antioxidant test

[0218] [DPPH-] ​​solution is purple in color and has strong absorption at 515nm. The DPPH free radical scavenging ability detection kit was used to calculate the free radical scavenging rate of the samples, and it was found that 60% ethanol solution was the best extraction solvent for four Chinese medicinal materials: Dendrobium officinale, Ganoderma lucidum, Bletilla striata, and Coix seed.

[0219] 3.1.5 Essence transdermal penetration test

[0220] Prepare the reference solution, draw the standard curve, take the mice for hair removal, select the undamaged and uniform thickness mouse skin for subsequent experiments, and then conduct the transdermal absorption test, calculate the penetration rate of the liposome essence, that is, the permeation amount of the liposome essence per unit area per unit time, and draw the cumulative permeation amount of the effective area. Compared with the blank, it proves that the liposome essence has a good transdermal effect.

[0221] 3.1.6 Moisture retention

[0222] In order to evaluate the moisturizing effect of the compound Chinese medicine liposome essence, this study randomly selected three volunteers and measured their skin moisture values ​​before and after use (immediately, 10 minutes, and 30 minutes). The results showed that the skin moisture increased significantly after use, proving that the essence has a good moisturizing effect.

[0223] 3.1.7 Evaluation of the whitening effect of compound Chinese herbal liposome essence

[0224] Since tyrosinase is the key to the synthesis of melanin in organisms, inhibiting tyrosinase activity is an important way to reduce the production of melanin. In order to evaluate the whitening effect of the compound Chinese herbal liposome essence, an in vitro tyrosinase inhibition experiment was used for evaluation, and a control group was designed to calculate the tyrosinase activity inhibition rate. The experiment found that the compound Chinese herbal liposome essence has a high tyrosinase inhibition rate, which helps to inhibit the production of melanin and improve the whitening effect, thus proving that this essence has good whitening effect.

[0225] 3.1.8 Stability of compound Chinese medicine liposome extract

[0226] Stability is a key indicator for the quality evaluation of compound Chinese medicine liposome essence. This study comprehensively evaluated the prepared liposome essence by examining its pH, high temperature stability, low temperature stability and long-term stability. The product is light yellow, uniform and transparent, has no sense of stratification, and is smooth to apply. The pH is close to neutral and is harmless to the skin. It has good heat and cold resistance, and no adverse reactions were observed in the long-term stability test. In summary, the compound Chinese medicine liposome essence has excellent stability and practicality.

[0227] 3.2 Summary

[0228] This study shows that the compound Chinese medicine liposome essence is prepared by using four Chinese medicines, namely, Dendrobium officinale, Ganoderma lucidum, Bletilla striata and Coix seed, as the active ingredients and blank liposomes prepared with egg yolk lecithin and cholesterol in a mass ratio of 10:1 as the carrier, which has the effect of promoting penetration. Anthocyanins are added for dual antioxidant effect, sodium alginate is used as the main component of the matrix, glycerin is added as a moisturizer, rose essential oil is used for flavor correction, and microporous filter membrane is used to prepare the compound Chinese medicine liposome essence. The obtained compound Chinese medicine liposome essence is uniform and delicate, easy to absorb and spread, has certain antioxidant and moisturizing effects, has less raw materials, and has a simple preparation process. The use of Chinese medicine for both medicinal and edible purposes is safe and environmentally friendly, and can be developed and utilized as a popular skin care cosmetic. This article has certain reference value for the development and utilization of compound Chinese medicine liposome essence skin care products.

[0229] Although the present invention has been described in detail above by means of general description, specific implementation methods and experiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A compound Chinese medicine liposome essence, characterized in that: The essence comprises the following components: 0.5 to 1.5 parts by volume of a traditional Chinese medicine extract prepared from Dendrobium officinale, Bletilla striata, Coix seed and Ganoderma lucidum, 0.1 to 0.5 parts by volume of anthocyanin solution with a concentration of 0.25 mg / mL, 70 to 100 parts by mass of egg yolk lecithin, 10 parts by mass of cholesterol, 15 to 25 parts by mass of hyaluronic acid, 8 to 10 parts by mass of yeast extract, 8 to 10 parts by mass of glycerol, 10 to 20 parts by mass of sodium alginate and 1 to 2 parts by mass of orange blossom essential oil.

2. The compound Chinese medicine liposome essence according to claim 1, characterized in that: The essence comprises the following components: 0.8 parts by volume of a traditional Chinese medicine extract prepared from Dendrobium officinale, Bletilla striata, Coix seed and Ganoderma lucidum, 0.2 parts by volume of anthocyanin solution with a concentration of 0.25 mg / mL, 100 parts by mass of egg yolk lecithin, 10 parts by mass of cholesterol, 20 parts by mass of hyaluronic acid, 8 to 10 parts by mass of yeast extract, 8 to 10 parts by mass of glycerol, 15 parts by mass of sodium alginate and 1 part by mass of orange blossom essential oil.

3. The compound Chinese medicine liposome essence according to claim 1, characterized in that: The preparation method of the traditional Chinese medicine extract is as follows: weigh four traditional Chinese medicines of Dendrobium officinale, Ganoderma lucidum, Bletilla striata and Coix seed in a mass ratio of 1:1:1:1, add them into an extraction solvent in a mass-to-volume ratio of 1g:8-12mL, heat and extract at 60°C for 3h, let stand for 10min, and absorb the supernatant to obtain the extract.

4. The compound Chinese medicine liposome essence according to claim 3, characterized in that: The material-liquid ratio in the preparation method of the traditional Chinese medicine extract is: 1g:10mL.

5. The compound Chinese medicine liposome essence according to claim 3, characterized in that: The extraction solvent in the preparation method of the traditional Chinese medicine extract is any one of pure water and 30% to 60% ethanol solution.

6. The compound Chinese medicine liposome essence according to claim 5, characterized in that: The extraction solvent in the preparation method of the traditional Chinese medicine extract is 60% ethanol solution.

7. The method for preparing the compound Chinese medicine liposome essence according to claim 1, characterized in that: The preparation method steps are as follows: S1 Preparation of liposomes: Weigh egg yolk lecithin and cholesterol according to the formula, add 2 parts by volume of anhydrous ethanol, and perform rotary evaporation to form a film of the liquid; put it into an oven, vacuum dry it for 8 hours, and take it out; add the Chinese medicine extract, anthocyanin solution and 10 parts by volume of PBS phosphate buffer with a pH of 7.45, and perform ultrasonication for 25 minutes to disperse it into liposomes; filter, and perform rotary evaporation at 75°C to remove ethanol to obtain the product; S2 liposome compound: Add the formulated amounts of hyaluronic acid, yeast extract, glycerin, sodium alginate and neroli essential oil to obtain the essence.

8. The method for preparing the compound Chinese medicine liposome essence according to claim 7, characterized in that: The rotary evaporation conditions in step S1 are: rotary evaporation at 50-65° C. for 15-30 min.

9. The method for preparing the compound Chinese medicine liposome essence according to claim 8, characterized in that: The rotary evaporation conditions in step S1 are: rotary evaporation at 55° C. for 20 min.

10. The method for preparing the compound Chinese medicine liposome essence according to claim 7, characterized in that: The filtration in step S1 is specifically as follows: first filtering with a 0.45 μm PES microporous filter membrane, and then filtering with a 0.22 μm PES microporous filter membrane.

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