A whitening and lightening transdermal elastic liposome composition containing saffron and a preparation method thereof

Abstract

This invention discloses a skin-brightening and brightening transdermal elastic liposome composition containing saffron and its preparation method, belonging to the field of daily cosmetic technology. The composition comprises saffron extract, 4-butylresorcinol, Dalbergia odorifera bark extract, niacinamide, Plantago asiatica ferment extract, and Ginkgo biloba ferment extract, supplemented with liposome excipients such as hydrogenated lecithin and deionized water and anhydrous ethanol solvents. Specifically, the Plantago asiatica ferment extract is prepared by co-fermentation of Streptococcus thermophilus and Lactobacillus rhamnosus, and the Ginkgo biloba ferment extract is prepared by co-fermentation of Aspergillus oryzae and Aspergillus cristatus, constructing an elastic liposome structure through a specific process. This composition can effectively inhibit tyrosinase activity in vitro and intracellularly, exhibiting excellent skin-brightening and brightening effects, and is suitable for preparing various skin care products.

Description

Technical Field

[0001] This invention belongs to the technical field of daily cosmetics, and specifically relates to a whitening and brightening transdermal elastin liposome composition containing saffron and its preparation method. Background Technology

[0002] With consumers' increasing demand for skin whitening and skincare, cosmetics with whitening effects have become a market hotspot. Excessive melanin production is the main cause of dull skin and age spots, and tyrosinase is a key rate-limiting enzyme in the melanin synthesis pathway. Inhibiting tyrosinase activity is the core mechanism for achieving skin whitening.

[0003] In existing whitening skincare products, single whitening ingredients often suffer from problems such as limited target area, poor skin penetration, and limited whitening effect; some chemical whitening ingredients may also cause skin irritation, sensitivity, and other safety concerns. Meanwhile, traditional extract preparation methods often use solvent extraction, resulting in low utilization of active ingredients and insufficient synergistic effects between components. Furthermore, in conventional skincare formulas, whitening active ingredients are easily deactivated by external environmental factors and struggle to effectively penetrate the skin barrier to reach their target areas, limiting the full release of whitening efficacy.

[0004] Therefore, developing a whitening composition with strong synergistic effects, high utilization rate of active ingredients, good skin penetration, and gentle safety has become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] The core objective of this invention is to overcome the shortcomings of existing whitening skincare products, such as poor synergy of ingredients, low utilization rate of active ingredients, insufficient skin penetration, and limited whitening effect, and to provide a whitening and brightening transdermal elastin composition containing saffron.

[0006] To achieve the above objectives, the present invention discloses the following technical solutions:

[0007] In a first aspect, the present invention provides a transdermal elastic liposome composition for whitening and brightening, the composition containing saffron extract, 4-butylresorcinol, Dalbergia odorifera bark extract, nicotinamide, Plantago asiatica ferment extract and Ginkgo biloba ferment extract, as well as excipients and solvents for preparing liposomes.

[0008] The mass ratio of the saffron extract, 4-butylresorcinol, Dalbergia odorifera bark extract, nicotinamide, Plantago asiatica ferment extract and Ginkgo biloba ferment extract is (3-5):(0.2-1):(5-10):(0.1-0.3):(5-15):(2-4).

[0009] Preferably, the method for preparing the fermented extract of *Plantago asiatica* leaves includes the following steps:

[0010] Step 1: Take dried Plantago asiatica leaves, crush them through a 60-mesh sieve to obtain Plantago asiatica powder, sterilize for 20 minutes, and after sterilization, add sterile PBS buffer solution containing 2% glucose at a material-to-liquid ratio of 1:20 g / mL, stir evenly to obtain fermentation substrate;

[0011] Step 2: Mix the Streptococcus thermophilus bacterial solution with the Lactobacillus rhamnosus bacterial solution to obtain a composite bacterial solution;

[0012] Step 3: Inoculate the compound bacterial solution into the fermentation substrate at an inoculation rate of 5 v / v%, and ferment at 37℃ and 100 r / min for 72 h. During the fermentation process, the aeration rate is maintained at 0.2 vvm. After the fermentation is completed, the fermentation broth is centrifuged, the supernatant is collected, sterilized by 0.22 μm microfiltration, concentrated under reduced pressure, and freeze-dried to obtain the fermented extract of Plantago asiatica leaves.

[0013] More preferably, the viable count of the thermophilic streptococcal bacterial solution is 1×10⁻⁶. 8 The cFU / mL count of *Streptococcus thermophilus* was determined by the accession number GDMCC 1.1808, and the viable count of the *Lactobacillus rhamnosus* culture was 1 × 10⁻⁶. 8 CFU / mL, the preservation number of Lactobacillus rhamnosus is GDMCC 1.1798;

[0014] The thermophilic streptococcus bacterial solution and the lactobacillus rhamnosus bacterial solution were mixed at a volume ratio of 4:1 to obtain a composite bacterial solution.

[0015] Preferably, the preparation method of the ginkgo fermentation extract includes the following steps:

[0016] Step 1: Take dried ginkgo leaves, crush them through a 30-60 mesh sieve to obtain ginkgo leaf powder. Mix the ginkgo leaf powder with wheat bran powder at a mass ratio of 7:3 to 8:2, adjust the moisture content to 50%-60% with distilled water, sterilize for 30 minutes, and obtain the fermentation substrate.

[0017] Step 2: Mix Aspergillus oryzae spore suspension with Aspergillus cristatus spore suspension to obtain a composite spore suspension. Then, spray the composite spore suspension evenly onto the substrate at an inoculation rate of 20 w / w% of the substrate dry weight. Mix thoroughly and ferment at 28℃ and 70-80% relative humidity for 7-10 days, stirring 1-2 times a day to provide oxygen, dissipate heat, and prevent mycelia from tangling.

[0018] Step 3: After fermentation, the fermented product is mixed with an 80 v / v% ethanol aqueous solution at a ratio of 1:30 g / mL, and refluxed three times for 45 min each time. The extracts are combined and concentrated under reduced pressure to obtain Ginkgo fungal fermentation extract. The extract is further freeze-dried to obtain Ginkgo fermentation extract.

[0019] More preferably, the Aspergillus oryzae spore suspension contains 10 spores. 6 The concentration of CFU / mL was CFU / mL, and the preservation number of *Aspergillus oryzae* was GDMCC 3.236. The spore count of the *Aspergillus cristatus* spore suspension was 10. 6 CFU / mL, the accession number of Aspergillus cristatus is GDMCC3.1025;

[0020] The Aspergillus oryzae spore suspension and Aspergillus triticum spore suspension were mixed at a volume ratio of 4:3 to obtain a composite spore suspension.

[0021] Preferably, the excipients are hydrogenated lecithin, soybean lecithin, cholesterol, and fatty acid sucrose esters;

[0022] The solvents are deionized water and anhydrous ethanol.

[0023] Saffron extract is obtained through supercritical CO2 extraction to obtain high-purity active ingredients, which can inhibit melanocyte activity, reduce melanin production, and also have antioxidant effects, which can eliminate free radicals and brighten skin tone.

[0024] 4-Butylresorcinol can effectively inhibit tyrosinase activity, block the melanin synthesis pathway, and specifically improve skin dullness and pigmentation problems, resulting in rapid whitening effects.

[0025] The extract of Dalbergia odorifera bark was prepared by ultrasonic-assisted reflux extraction. It is rich in polyphenolic active substances and has both antioxidant and tyrosinase inhibitory effects, which can enhance the whitening synergistic effect of the composition and soothe the skin.

[0026] Niacinamide can regulate melanin transport, reduce melanin deposition on the skin surface, brighten skin tone and even out skin tone, while improving skin barrier function and enhancing the skin's absorption efficiency of other active ingredients.

[0027] Plantago asiatica fermentation extract is prepared by co-fermentation of Streptococcus thermophilus and Lactobacillus rhamnosus. During the fermentation process, it is transformed into small molecule active peptides, polysaccharides and other substances. Not only does it have whitening activity, but it can also act as an active carrier to enhance the synergistic effect of other ingredients and improve the overall whitening effect.

[0028] Ginkgo fermentation extract is optimized through a combined fermentation process with Aspergillus oryzae and Aspergillus cristatus, enriching it with active ingredients such as flavonoids and terpenes. It can inhibit tyrosinase activity and also has antioxidant and skin metabolism-promoting effects, further enhancing its whitening and brightening effects.

[0029] Secondly, the present invention provides the application of the elastin liposome composition described in the first aspect in the preparation of skin care products with whitening and brightening effects.

[0030] Thirdly, the present invention provides a method for preparing the elastic liposome composition described in the first aspect, the method comprising the following steps:

[0031] Step 1: Mix hydrogenated lecithin, soybean lecithin, cholesterol, and fatty acid sucrose esters in a mass ratio of 5:2:1:3 to obtain mixture A. Mix saffron extract and 4-butylresorcinol in a mass ratio to obtain mixture B. Mix mixture A and mixture B in a mass ratio of 11:1 to obtain liposome wall material. Heat the liposome wall material with anhydrous ethanol in a water bath at 50-55℃ until completely dissolved in a mass-volume ratio of 1:7 (g / mL). Then remove the ethanol by vacuum distillation to obtain a transparent oil film.

[0032] Step 2: Combine the extracts of Dalbergia odorifera bark, nicotinamide, fermented extract of Plantago asiatica leaf and fermented extract of Ginkgo biloba to obtain liposome core material. Mix the liposome core material with deionized water at a mass-volume ratio of 1:150 (g / mL) to completely dissolve it and obtain the core material solution.

[0033] Step 3: Add the core material solution to the transparent oil film and stir at 700-800 r / min for 10 min to obtain an emulsion. Transfer the emulsion to a high-pressure homogenizer and cycle it 3 times at a pressure of 16000-18000 psi to obtain the composition.

[0034] The beneficial effects of this invention are:

[0035] 1. The present invention provides a scientifically formulated blend of six active ingredients: saffron extract, 4-butylresorcinol, Dalbergia odorifera bark extract, nicotinamide, Plantago asiatica ferment extract, and Ginkgo biloba ferment extract. The fermented extracts complement traditional whitening ingredients, effectively inhibiting tyrosinase activity and blocking melanin synthesis and transport pathways at multiple targets, resulting in excellent whitening effects.

[0036] 2. This invention constructs an elastic liposome carrier using lipid components such as hydrogenated lecithin and soybean lecithin, and combines it with active ingredients such as saffron extract, 4-butylresorcinol, and rosewood bark extract. This not only protects the active ingredients from the influence of the external environment, but also enhances the skin barrier permeability and improves the efficiency of ingredient action. Attached Figure Description

[0037] Figure 1 Raman spectroscopy of human skin in vivo;

[0038] Figure 2 The image shows the Raman spectrum of the sample.

[0039] Figure 3 A line graph showing the relative permeability of samples in the skin at different time points;

[0040] Figure 4This is a distribution map of the content of samples at different depths in the skin at different times. Detailed Implementation

[0041] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0042] Unless otherwise specified, the test methods used in the examples and comparative examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified; and the percentages mentioned in the examples and comparative examples are mass percentages unless otherwise specified.

[0043] In this invention

[0044] Saffron: also known as crocus (Crocus sativus L.), commercially available;

[0045] Dried bark of Pterocarpus marsupium (Fabaceae family): Dried bark of the Pterocarpus marsupium plant, commercially available;

[0046] Plantago major leaves: Dried leaves of Plantago major (Plantago macrocarpa), a plant belonging to the Plantaginaceae family and the Plantago genus, are commercially available.

[0047] Ginkgo leaves: Dried leaves of Ginkgo biloba (Ginkgo genus) in the Ginkgoaceae family, commercially available;

[0048] Streptococcus thermophilus: accession number GDMCC 1.1808, purchased from Guangdong Provincial Microbial Culture Collection Center;

[0049] Lactobacillus rhamnosus: accession number GDMCC 1.1798, purchased from Guangdong Provincial Center for Microbial Culture Collection;

[0050] Aspergillus oryzae: accession number GDMCC 3.236, purchased from Guangdong Provincial Center for Microbial Culture Collection;

[0051] Aspergillus cristatus: accession number GDMCC 3.1025, purchased from Guangdong Provincial Center for Microbial Culture Collection;

[0052] Lactobacillus plantarum: accession number GDMCC 1.191, purchased from Guangdong Provincial Center for Microbial Culture Collection;

[0053] Aspergillus niger: accession number GDMCC 3.452, purchased from Guangdong Provincial Center for Microbial Culture Collection;

[0054] All raw materials not mentioned in the other embodiments are commercially available.

[0055] Preparation of saffron extract

[0056] Step 1: Take dried saffron and grind it with a high-speed grinder, then pass it through a 60-mesh sieve to obtain saffron powder;

[0057] Step 2: Weigh 500g of saffron powder and place it in a supercritical CO2 extraction vessel. Set the extraction parameters as follows: extraction temperature 35-45℃, extraction pressure 20-30MPa, CO2 flow rate 10-20L / h, and extraction time 4h. Start the CO2 circulation system. Liquefied CO2 is pumped into the extraction vessel through a high-pressure pump, where it comes into full contact with the saffron powder. After dissolving the active ingredients, a supercritical fluid mixture is formed and enters the separation system. The extracted extract is obtained through separation.

[0058] Step 3: Place the extracted extract in a vacuum drying oven and dry it at 35℃ and -0.09MPa for 1-2 hours to obtain saffron extract.

[0059] Preparation of Dalbergia odorifera bark extract

[0060] Step 1: Take dried rosewood bark and crush it with a high-speed pulverizer, then pass it through a 60-mesh sieve to obtain rosewood bark powder;

[0061] Step 2: Add 70 v / v% ethanol aqueous solution to the Dalbergia odorifera bark powder at a material-to-liquid ratio of 1:20 (g / mL), sonicate at 30 kHz and 60 ℃ for 30 min, then transfer to a reflux extraction device and reflux extract at 60 ℃ 3 times, 30 min each time, filter and collect the combined extracts.

[0062] Step 3: Concentrate the extract under reduced pressure using a rotary evaporator to obtain a concentrated extract, and then freeze-dry it to obtain the extract of Dalbergia odorifera bark.

[0063] Preparation of fermented extract of Plantago asiatica

[0064] Step 1: Take dried Plantago asiatica leaves and crush them through a 60-mesh sieve using a high-speed pulverizer to obtain Plantago asiatica powder. Then, steam sterilize at 121℃ for 20 minutes. Add the sterilized raw material to a sterile PBS buffer solution containing 2% glucose at a material-to-liquid ratio of 1:20 g / mL, stir evenly, and obtain the fermentation substrate.

[0065] Step 2: Take 1×10⁻⁶ live bacteria. 8 CFU / mL Streptococcus thermophilus (GDMCC 1.1808) bacterial suspension with a viable count of 1×10⁻⁶ 8 A composite bacterial solution was obtained by mixing CFU / mL Lactobacillus rhamnosus (GDMCC 1.1798) bacterial suspension at a volume ratio of 4:1.

[0066] Step 3: Transfer the fermentation substrate to the fermenter, inoculate the compound bacterial solution into the fermentation substrate at an inoculation rate of 5v / v%, and ferment at 37℃ and 100r / min for 72h. During the fermentation process, maintain the aeration rate at 0.2vvm. After the fermentation is completed, centrifuge the fermentation broth, collect the supernatant, and sterilize it by passing it through a 0.22μm microfiltration filter.

[0067] Step 4: Concentrate the sterilized fermentation broth under reduced pressure and freeze-dry it to obtain the fermented extract of Plantago asiatica leaves.

[0068] Preparation of Ginkgo Fermentation Extract

[0069] Step 1: Take dried ginkgo leaves and crush them through a 60-mesh sieve using a high-speed pulverizer to obtain ginkgo leaf powder. Mix the ginkgo leaf powder with wheat bran powder at a mass ratio of 7:3 to 8:2, adjust the moisture content to 50%-60% with distilled water, and sterilize by steaming at 121℃ for 30 minutes to obtain the fermentation substrate for later use.

[0070] Step 2, spore count 10 6 CFU / mL Aspergillus oryzae (GDMCC 3.236) suspension and spore count 10 6 A CFU / mL Aspergillus cristatus (GDMCC 3.1025) suspension was mixed at a volume ratio of 4:3 to obtain a composite spore suspension. The composite spore suspension was then evenly sprayed onto the substrate at an inoculation rate of 20 w / w% of the substrate dry weight. The mixture was thoroughly stirred and fermented at 28℃ and 70-80% relative humidity for 7-10 days, stirring 1-2 times a day to provide oxygen, dissipate heat, and prevent the mycelium from tangling.

[0071] Step 3: After fermentation, the fermented product is mixed with an 80 v / v% ethanol aqueous solution at a ratio of 1:30 g / mL, and refluxed three times for 45 min each time. The extracts are combined and concentrated under reduced pressure to obtain Ginkgo fungal fermentation extract. The extract is further freeze-dried to obtain Ginkgo fermentation extract.

[0072] Preparation of elastoliposome compositions

[0073] 1. Composition 1-3

[0074] Step 1: Mix hydrogenated lecithin, soybean lecithin, cholesterol, and fatty acid sucrose esters in a mass ratio of 5:2:1:3 to obtain mixture A. Mix saffron extract and 4-butylresorcinol in the mass ratio of Table 1 to obtain mixture B. Mix mixture A and mixture B in a mass ratio of 11:1 to obtain liposome wall material. Heat the liposome wall material with anhydrous ethanol in a water bath at 50-55℃ until completely dissolved in a mass-volume ratio of 1:7 (g / mL). Then remove the ethanol by vacuum distillation to obtain a transparent oil film.

[0075] Step 2: Combine the extracts of Dalbergia odorifera bark, nicotinamide, fermented extract of Plantago asiatica leaves, and fermented extract of Ginkgo biloba to obtain liposome core material. Mix the liposome core material with deionized water at a mass-to-volume ratio of 1:150 (g / mL) until completely dissolved. Add an appropriate amount of pH adjuster to adjust the pH to 6.5±0.2 to obtain the core material solution for later use.

[0076] Step 3: Add the core material solution to the transparent oil film and stir at 700-800 r / min for 10 min to obtain an emulsion. Transfer the emulsion to a high-pressure homogenizer and cycle it 3 times at a pressure of 16000-18000 psi to obtain the elastic liposome composition.

[0077] The mass ratios of saffron extract, 4-butylresorcinol, Dalbergia odorifera bark extract, nicotinamide, Plantago asiatica ferment extract, and Ginkgo biloba ferment extract in the above preparation method are shown in Table 1.

[0078] Table 1

[0079] Raw material name Composition 1 Composition 2 Composition 3 Saffron extract 3 4 5 4-Butylresorcinol 0.2 0.5 1 Palm bark extract 5 8 10 Niacinamide 0.1 0.2 0.3 Plantago asiatica ferment extract 5 10 15 Ginkgo Fermentation Extract 2 3 4

[0080] 2. Composition 4-9

[0081] Based on the formulation of composition 2, missing components are supplemented with the remaining components according to the proportions. The preparation methods are the same as those for compositions 1-3, resulting in compositions 4-9, as detailed in Table 2.

[0082] Table 2

[0083] Raw material name Composition 4 Composition 5 Composition 6 Composition 7 Composition 8 Composition 9 Saffron extract / 4 4 4 4 4 4-Butylresorcinol 0.5 / 0.5 0.5 0.5 0.5 Palm bark extract 8 8 / 8 8 8 Niacinamide 0.2 0.2 0.2 / 0.2 0.2 Plantago asiatica ferment extract 10 10 10 10 / 10 Ginkgo Fermentation Extract 3 3 3 3 3 /

[0084] 3. Composition 10-13

[0085] 3.1 Composition 10: Based on the formulation of Composition 2, the fermented extract of Plantago asiatica leaves is replaced with fermented extract of Plantago asiatica leaves ① to obtain Composition 10. The preparation method of fermented extract of Plantago asiatica leaves ① is the same as that of fermented extract of Plantago asiatica leaves, except that Lactobacillus rhamnosus in step 2 is replaced with Lactobacillus plantarum (GDMCC1.191).

[0086] 3.2 Composition 11: Based on the formulation of Composition 2, the fermented extract of Plantago asiatica leaves is replaced with Plantago asiatica leaf extract to obtain Composition 11. The preparation method of Plantago asiatica leaf extract ② is as follows: first, take dried Plantago asiatica leaves and crush them through a 60-mesh sieve using a high-speed pulverizer to obtain Plantago asiatica powder. Mix the Plantago asiatica powder with distilled water at a mass-volume ratio of 1:20 g / mL, sonicate at 28 kHz and 30℃ for 30 min, then heat to 80℃ for 5 h for extraction, filter and collect the extract and filter residue, extract the filter residue again at 80℃ for 5 h, filter and collect the extract, finally combine the extracts, concentrate under reduced pressure, freeze dry to obtain Plantago asiatica leaf extract;

[0087] 3.3 Composition 12: Based on the formulation of Composition 2, Ginkgo fermentation extract is replaced with Ginkgo fermentation extract ① to obtain Composition 11. The preparation method of Ginkgo fermentation extract ① is the same as that of Ginkgo fermentation extract, except that Aspergillus cristatus in step 2 is replaced with Aspergillus niger (GDMCC 3.452).

[0088] 3.4 Composition 13: Based on the formulation of Composition 2, the fermented ginkgo extract is replaced with ginkgo extract to obtain Composition 13. The preparation method of ginkgo extract is as follows: dried ginkgo leaves are pulverized through a 60-mesh sieve using a high-speed pulverizer to obtain ginkgo leaf powder. The ginkgo leaf powder is mixed with an 80% v / v% ethanol aqueous solution at a ratio of 1:30 g / mL and extracted by reflux three times for 45 min each time. The extracts are combined and concentrated under reduced pressure to obtain ginkgo extract. The extract is further freeze-dried to obtain ginkgo extract.

[0089] Performance testing

[0090] I. In vitro testing

[0091] 1. Experimental Materials and Instruments

[0092] 1.1 Experimental Samples

[0093] 1.1.1 Test Samples: The compositions 1-13 prepared above were diluted with PBS buffer to a concentration of 300 μL. .

[0094] 1.1.2. Control Sample:

[0095] ① Blank liposome control group: A blank preparation containing only hydrogenated lecithin, soybean lecithin, and other liposome wall materials, without active ingredients such as saffron extract, 4-butylresorcinol, Dalbergia odorifera bark extract, nicotinamide, Plantago asiatica ferment extract, and Ginkgo biloba ferment extract, and diluted with PBS buffer to the corresponding concentration.

[0096] ② Negative control: PBS buffer;

[0097] ③Enzyme reaction blank group: containing only PBS buffer and L-DOPA solution, without tyrosinase (used for baseline calibration of inhibition rate calculation);

[0098] ④ Positive control: 0.5 mmol·L -1 Arbutin solution (solvent PBS)

[0099] 1.2 Reagents and Cells

[0100] Reagents: Tyrosinase (purity ≥98%, diluted to 1kU / mL tyrosinase solution), L-DOPA (purity ≥98%, diluted to 0.5g / L DOPA solution), PBS buffer (pH 6.8), Triton X-100, methanol (analytical grade).

[0101] Cells: Mouse melanoma cells B16-F10, cultured in DMEM medium containing 10% fetal bovine serum.

[0102] 2. Experimental Methods

[0103] 2.1 In vitro tyrosinase inhibitory activity experiment

[0104] Take a 96-well plate, set 3 duplicate wells in each group, and add samples according to the following system:

[0105] Test sample group / positive control group / blank liposome control group: 40 μL test sample + 120 μL L-DOPA solution + 40 μL tyrosinase solution;

[0106] Enzyme reaction blank group: 40 μL PBS buffer + 120 μL L-DOPA solution + 40 μL PBS buffer (PBS was used instead of tyrosinase to calibrate background absorbance);

[0107] Negative control group: 40 μL PBS buffer + 120 μL L-DOPA solution + 40 μL tyrosinase solution.

[0108] After incubation at 37°C for 30 min, the absorbance (A value) of each well was measured at 475 nm using an ELISA reader.

[0109] Calculate the inhibition rate using the formula: Tyrosinase inhibition rate = [(A] / (Tyrosinase inhibition rate = [ ... 阴性 -A 酶空白 )-(A 样品 -A 酶空白 )] / (A 阴性 -A 酶空白 )×100%, where A 阴性 The absorbance of the negative control group, A 酶空白 A represents the absorbance of the blank group in the enzyme reaction. 样品 The absorbance of the test sample group / positive control group / blank liposome control group was measured.

[0110] 2.2 Intracellular tyrosinase inhibitory activity experiment

[0111] B16-F10 cells in logarithmic growth phase were seeded in 96-well plates (5 × 10⁶ cells / well). 3 (Number of cells / well), incubate at 37℃ for 24 h. Discard the supernatant, and add the corresponding solutions to the groups as follows:

[0112] Test sample group / positive control group / blank liposome control group: Add the corresponding concentration of sample solution;

[0113] Cell blank group: Add an equal volume of serum-free DMEM medium;

[0114] Negative control group: Add an equal volume of PBS buffer;

[0115] Each group was divided into 6 replicates, and cultured for another 24 hours. The supernatant was discarded, the cells were washed twice with PBS, and 100 μL of Triton X-100 solution was added. The cells were then frozen at -80°C for 1 hour and thawed at room temperature to lyse them. 100 μL of 0.1% L-DOPA solution was added to each well, and the cells were incubated at 37°C for 2 hours. The absorbance (OD value) was measured at 490 nm using a microplate reader.

[0116] Intracellular tyrosinase activity is calculated using the formula: Intracellular tyrosinase activity = (OD 给药组 -OD 细胞空白 ) / (OD 阴性组 -OD 细胞空白 )×100%, Inhibition rate = 100% - Cellular tyrosinase activity.

[0117] 3. Experimental Data

[0118] 3.1 In vitro tyrosinase inhibition rate data

[0119] The results of the in vitro tyrosinase inhibitory activity assay are shown in Table 3;

[0120] Table 3 Results of in vitro tyrosinase inhibition rate

[0121] Sample Name Inhibition rate / % Composition 1 75.6 Composition 2 81.5 Composition 3 79.8 Composition 4 55.7 Composition 5 58.9 Composition 6 56.8 Composition 7 63.9 Composition 8 46.3 Composition 9 51.4 Composition 10 66.4 Composition 11 42.7 Composition 12 71.2 Composition 13 44.3 Positive control 26.5 Blank liposome control group 2.4

[0122] 3.2 Intracellular tyrosinase inhibition rate data

[0123] The results of the intracellular tyrosinase inhibitory activity assay are shown in Table 4;

[0124] Table 4 Tyrosinase inhibition rate in melanoma cells

[0125] Sample Name Inhibition rate / % Composition 1 31.5 Composition 2 35.2 Composition 3 33.1 Composition 4 21.3 Composition 5 23.6 Composition 6 22.1 Composition 7 24.3 Composition 8 18.4 Composition 9 20.8 Composition 10 26.8 Composition 11 17.5 Composition 12 25.7 Composition 13 20.1 Positive control 10.5 Blank liposome control group 1.8

[0126] 4. Results Analysis

[0127] Experimental results showed that all test samples (compositions 1-13) inhibited tyrosinase in vitro and tyrosinase in B16-F10 cells. Among them, composition 2 had the best whitening activity, with an in vitro tyrosinase inhibition rate of 81.5% and an intracellular tyrosinase inhibition rate of 35.2%, indicating that the elastic liposome composition of the present invention has excellent whitening activity.

[0128] Compositions 4-9 are formulations of composition 2 lacking a single component, and their inhibition rates are all lower than those of composition 2. Among them, composition 8, which lacks Plantago asiatica ferment extract, has the lowest inhibition rate, indicating that Plantago asiatica ferment extract is a key whitening active carrier in the composition and can enhance the synergistic effect of other components.

[0129] Compositions 10 and 12 are formulations that replace the fermentation bacteria, and their inhibition rates are both lower than that of composition 2. Specifically, composition 10 (replaced with Lactobacillus plantarum for fermentation) has an in vitro inhibition rate of 66.4% and an intracellular inhibition rate of 26.8%; composition 12 (replaced with Aspergillus niger for fermentation) has an in vitro inhibition rate of 71.2% and an intracellular inhibition rate of 25.7%. This indicates that the combination of thermophilic streptococci with Lactobacillus rhamnosus and Aspergillus oryzae with Aspergillus cristatus selected in this invention can more efficiently transform the active substances in the raw materials to generate fermentation products with stronger whitening activity, which is a key process parameter to ensure the performance of the composition.

[0130] Compositions 11 and 13 respectively replace the fermented extracts of Plantago asiatica and Ginkgo biloba with conventional water extraction or alcohol extraction processes, resulting in a decrease in the in vitro and intracellular tyrosinase inhibition rate of the formulation. This indicates that the fermentation treatment followed by extraction process provided by the present invention is superior to conventional extraction processes and can effectively improve the performance of the formulation.

[0131] II. Human Efficacy Experiments

[0132] 1. Transdermal test

[0133] 1.1 Purpose of Testing

[0134] By using test sample composition 2 on one subject, the penetration effect of the sample in human skin at 0h, 0.5h, 1h, 2h and 4h after using the sample by different methods was studied using a human (in vivo) Raman non-invasive optical test method.

[0135] 1.2 Detection Method

[0136] A 1cm×1cm area was selected on the forearm of the human body for testing: test sample composition 2 was tested on the skin of the test area at time points of 0h, 0.5h, 1h, 2h and 4h, and the relative permeability of the sample at each time point after using composition 2 was calculated.

[0137] 1.3. Test Samples

[0138] Composition 2.

[0139] 1.4 Testing Instruments

[0140] LabRAM Odyssey High-Speed ​​High-Resolution Microconfocal Raman Spectrometer (HORIBA).

[0141] 1.5 Test Environment

[0142] Environmental requirements: Temperature: 22℃±2℃; Humidity: 50.00%RH±10.00%RH.

[0143] 1.6 Testing Process

[0144] The subjects were instructed to clean the test area on the inside of their arms with water after their visit. After cleaning, they sat quietly in a constant temperature and humidity room for 30 minutes. After 30 minutes, the subjects used the product according to the test requirements. In vivo Raman tests were conducted on the human body 20h, 0.5h, 1h, 2h and 4h after using the composition.

[0145] 1.7 Sample Completion Status

[0146] Planned enrollment: 1 person

[0147] The actual number of participants was 1.

[0148] One person completed the task.

[0149] 1.8 Test Results and Statistical Methods

[0150] Raman spectroscopy imaging data processing includes spectral preprocessing and data analysis. Spectral preprocessing includes several steps such as cosmic ray removal, spectral smoothing, background noise removal, baseline calibration, and spectral normalization. Univariate data analysis primarily focuses on analyzing the Raman spectral data of biochemical substances corresponding to specific peak positions to reveal the distribution of these substances within human skin.

[0151] Data analysis employed Labspec software to perform baseline calibration and confirm the positions of characteristic peaks in the Raman spectra. Calculations were performed on the obtained Raman spectra, including peak intensity, peak shift, peak area, and full width at half maximum (FWHM). Simultaneously, Labspec software was used to perform numerical analysis of peak intensities corresponding to different depths and to plot their spatial distribution.

[0152] The product's penetration behavior was determined by using Raman signals, which distinguish the product from the intrinsic signals of the skin, to confirm its distribution at different skin depths.

[0153] The data were tested for normality using the Shapiro-Wilk Test with ORIGIN software. The comparison before and after the data showed a normal distribution. If the data was normally distributed, a paired t-test was used; otherwise, a rank-sum test of two related samples was used.

[0154] Relative penetration rate (%) = Normalized (change in Raman characteristic peaks caused by use of the test product) * 100.00%.

[0155] 1.9 Test Results

[0156] 1.9.1 Analysis of human skin results before using samples

[0157] The laser power used in the experiment was 2.68 mW, and the integration time at a single point was 0.5 s. Depth Raman imaging was performed in the XZ direction, using a point-by-point scanning method. The longitudinal step interval for acquiring spectral data was 10 μm, and the scanning area was 20 μm × 120 μm. The entire spectral measurement process was completed within 5 minutes. Figure 1 This is the intrinsic Raman spectrum of human skin in vivo, including the 943cm². -1 1275cm -1 1455cm -1 1655cm -1 2846cm -1 2883cm -1 2934cm -1 3226cm -1 The peaks exhibit Raman characteristics.

[0158] Table 5. Assignment of Raman characteristic peaks and their representative components in skin

[0159] <![CDATA[Peak position (cm -1 )]]> Vibration modes Main representative components 943 N(CC) skeleton, collagen skeleton proline, hydroxyproline 1275 CN absorption band (amide III band) Glycine backbone, proline, nucleic acid 1455 <![CDATA[C-H bending mode of protein (CH2 stretching / CH3 asymmetric deformation)]]> Structural proteins, elastin 1655 νC=O stretching vibration (amide I band, including α-folding, β-folding and random coiling) Actin, collagen, keratin 2846 <![CDATA[Asymmetric stretching of CH2]]> lipids 2883 <![CDATA[Symmetric stretching of CH2]]> lipids 2934 <![CDATA[Asymmetric stretching of CH3]]> lipids and proteins

[0160] Interpretation of results:

[0161] The vibrational modes and substances corresponding to the characteristic peaks are shown in Table 5. The Raman spectrum clearly shows that the skin contains proline, hydroxyproline, glycine, nucleic acids, as well as structural proteins, elastin, actin, collagen, keratin and other proteins and intercellular lipids.

[0162] 1.9.2. Examination of the transdermal permeability of the test samples

[0163] The Raman spectra of the sample were measured experimentally; the Raman spectra are shown below. Figure 2 .

[0164] Interpretation of results:

[0165] like Figure 2The image shows the Raman characteristic peaks of the sample. These peaks are numerous and complex, and their overall intensity is higher than that of the intrinsic Raman spectroscopy of skin. The characteristic peak is located at 643 cm⁻¹. -1 746cm -1 839cm -1 994cm -1 1038cm -1 1149cm -1 1171cm -1 1299cm -1 14545cm -1 1593cm -1 1634cm -1 and 2900cm -1 .

[0166] The full spectrum correlation characteristic peaks of the sample were used to track the penetration of the sample into the human skin.

[0167] Based on the data processing results, the relative permeability of samples in the skin at different time points is as follows:

[0168] Table 6. Relative permeability of samples in skin at different time points

[0169] sample 0h 0.5h 1h 2h 4h Composition 2 0.00% 0.00% 1.93% 3.28% 5.31%

[0170] The relative permeability of samples in the skin at different time points is shown in the dotted line graph. Figure 3 .

[0171] Images showing the content distribution of the sample at different depths in the skin at different times after sample application are shown below. Figure 4 .

[0172] Interpretation of results:

[0173] Through the Figure 4 Depth analysis of Raman images can reveal the distribution of the sample at different depths in human skin, as shown in Figure 4. It can be seen that: within 0h to 0.5h, the sample does not penetrate; within 1h, the sample penetrates into the active epidermis, reaching a penetration depth of 70μm; within 2h to 4h, the sample continues to penetrate the active epidermis, reaching a penetration depth of 60μm to 70μm.

[0174] 2. Whitening Test

[0175] 2.1 Inspection Basis

[0176] JYT-SOP-ZB-12 "Testing Methods for Skin L-value, Red Zone a* Value, B-value, and ITA° Value"

[0177] JYT-SOP-ZB-05 "Test Method for Red Zone a* Value in Skin Red Area"

[0178] JYT-SOP-ZB-13 "Test Methods for Skin Melanin Index (MI) and Erythrocyte Index (EI)"

[0179] JYT-SOP-ZB-03 "Test Method for Skin Glossiness"

[0180] JYT-SOP-ZB-19 "Testing Method for Visual Skin Color Grading"

[0181] 2.2 Experimental Objective

[0182] Subjects who meet the selection criteria are selected for this trial. Instrumental testing is used to observe the subjects' skin data before and after using the product (composition 2), thereby evaluating the skin improvement and safety after using the product and providing a basis for the whitening efficacy claims of cosmetics.

[0183] 2.3 Adverse Reactions in Experiments

[0184] No adverse reactions occurred during this test.

[0185] 2.4 Evaluation Indicators and Principles

[0186] 2.4.1 Data Acquisition of Red Zone a* Value and ITA°

[0187] The Colorimeter CL400 skin color testing probe is used to collect the red zone a* value and ITA°. The CK skin tester uses a probe that emits white LED light, with the LEDs distributed in a ring to ensure uniform skin illumination. The emitted light is scattered in all directions; some penetrates the skin surface, while some disperses outwards. The probe detects the light reflected back from the skin and displays the corresponding value. The probe's raw data is calibrated using a special color matrix to make it as close as possible to standard values. The measured skin color is represented by XYZ values ​​(tri-color excitation) and calculated into corresponding L*a*b values. L* represents the black-and-white axis value—luminance, and a* and b* represent chromaticity coordinates. a* represents the degree of color shift from green to red, and b* represents the degree of color shift from blue to yellow. The index ITA° is an evaluation index calculated from the combined luminance L, A, and B values.

[0188] 2.4.2, Red pigment index EI

[0189] The redness of skin tone was measured using the cutometeMPA580 multi-functional skin analyzer manufactured by Courage-Khazaka (CK GmbH, Germany). A higher value indicates a redder skin tone, while a lower value is better.

[0190] 2.4.3 Melanin Index (MI) Collection

[0191] Skin melanin is collected using the Mexameter MX18 skin melanin testing probe. The measurement is based on the principle of spectral absorption, determining the amount of skin melanin by measuring the amount of light reflected from human skin at a specific wavelength.

[0192] 2.4.4 Skin Glossiness Data Collection

[0193] Skin gloss was measured using the Deflin skin gloss meter. The principle behind this method is that skin gloss is reflected by the direct reflection and scattering of light incident on the skin surface. Because skin varies not only in structure and brightness but also in color, the gloss of different skin types can be accurately and conveniently measured by testing the reflected and scattered light from the skin surface.

[0194] 2.4.5 Skin Tone Grading Data Collection

[0195] Skin tone grading is based on subjective evaluation by dermatologists / test supervisors using dermoscopy combined with standard color charts. It is divided into 10 levels, with the lower the value, the lighter the skin tone.

[0196] 2.4.6. Adverse reaction records of subjects

[0197] If any adverse reaction occurs on the subject's skin during the entire testing process, the test should be terminated and recorded. Adverse reactions are also one of the test indicators.

[0198] 2.5 Experimental Design

[0199] 2.5.1 Testing period: 28 days

[0200] 2.5.2 Test environment temperature: 20℃~22℃, humidity: 40%~60%.

[0201] 2.5.3 Test period: 2025 / 12 / 03-2025 / 12 / 31

[0202] 2.5.4. Protocol Requirements: Single-blind, randomized enrollment, and before-and-after comparison of the product itself.

[0203] 2.5.5 Test site: Face

[0204] 2.5.6 Test Procedure

[0205] 2.5.6.1 D0 (Initial value, before product use): Qualified subjects, after screening, cleaned their faces under the guidance of staff, and then wiped clean with lint-free absorbent paper towels. They sat quietly in a suitable room for at least 30 minutes, during which time they could not drink water or beverages. The test area was exposed, and they remained relaxed, avoiding touching the test area. After 30 minutes, researchers conducted instrumental tests on the evaluation items.

[0206] 2.5.6.2 After the initial value is determined, test samples are distributed to the subjects, and product instructions are provided to clarify the method of use (including application site, method of use, frequency of use, precautions, etc.) to ensure that the subjects can use the product correctly.

[0207] 2.5.6.3 After using the product continuously for 28 days as required, the subjects visited for testing. The testing requirements and procedures were consistent with the initial value determination. Researchers conducted instrument tests and data analysis on the evaluation items, recorded adverse reactions, and the subjects answered a questionnaire.

[0208] 2.6 Subject Screening

[0209] 2.6.1 Number of subjects: No fewer than 30 valid cases.

[0210] 2.6.2 Basic information of the subjects: 30 subjects were recruited and enrolled in this trial, with 0 dropouts, and 30 valid subjects. Among them, there were 14 males and 16 females. The youngest age was 18 years old, the oldest age was 38 years old, and the average age was 25.30±6.21 years old.

[0211] 2.6.3 Selection Criteria:

[0212] 1) Age between 18 and 60 years old;

[0213] 2) Healthy Chinese male and female subjects, with no allergic diseases and no history of allergies to cosmetics or other topical preparations;

[0214] 3) The subjects had no skin lesions, eczema, etc.;

[0215] 4) The subjects did not have serious chronic wasting diseases (asthma, diabetes, etc.);

[0216] 5) The facial skin should be free of birthmarks, scratches, white spots, pigmented nevi, or other conditions that could affect the test;

[0217] 6) Keep the test area dry during the test and do not use any liquid substances, other cosmetics, drugs and health products that may affect the results;

[0218] 7) Can read Chinese and understand it accurately, and has signed the informed consent form for the experimental research.

[0219] 2.6.4 Exclusion criteria:

[0220] 1) Currently pregnant or breastfeeding;

[0221] 2) Has a history of skin cancer within the past 5 years;

[0222] 3) Subjects with severe bacterial, viral, or fungal infections on their skin;

[0223] 4) The test subject's skin has obvious sunburn, scars, pigmented nevi, hirsutism, or other conditions that may affect the test measurement results;

[0224] 5) History of immunosuppression or immunodeficiency (including HIV or AIDS), or current use of immunosuppressive drugs or radiation.

[0225] 6) Suffering from uncontrollable diseases such as asthma, epilepsy, diabetes, hypertension, hyperthyroidism or hypothyroidism;

[0226] 7) Those who have participated in other clinical trials (such as hormone replacement therapy HRT) or taken oral hormones that may affect the results within 3 months, or those who plan to undergo HRT treatment during the trial period;

[0227] 8) Currently using any medications that may affect skin condition or reaction, such as antihistamines, antibiotics, insulin, anti-inflammatory drugs, steroids, corticosteroids, aspirin, thyroid medications, etc.

[0228] 9) Have undergone chemotherapy or other radiation therapy, or received any prescription medications or treatments that may affect skin condition;

[0229] 10) Has been diagnosed with an allergic reaction or other known or suspected allergic reaction (systemic, inhalation, or local); has a history of partial allergic reactions.

[0230] 11) Individuals currently receiving dermatological treatment, or subjects who have taken hydroxy acids, whitening agents, or anti-aging drugs within the past month;

[0231] 12) Other diseases or conditions that reduce the likelihood of enrollment or complicate enrollment, as determined by the researcher, such as frequent changes in work environment or unstable living environment that may lead to loss to follow-up; alcoholism and / or psychoactive substances, drug abuse and dependence.

[0232] 2.6.5 Exit Criteria:

[0233] If a subject experiences adverse reactions, is lost to follow-up without cause, violates the study protocol (such as using other cosmetics or drugs that may affect the study results), or has other special circumstances, and is deemed unsuitable to continue the study after assessment by the study leader, the subject will be asked to withdraw.

[0234] 2.7 Data Statistics

[0235] 2.7.1 Descriptive statistics: Use statistical analysis software to perform descriptive statistics on each measurement value, including quantity, mean, standard deviation, minimum value, maximum value, etc.

[0236] 2.7.2 Difference Analysis: Before-and-after comparison: For the significance test of the normal distribution of the improvement values ​​in the experimental data, if Sig. (two-tailed) > 0.01, then it is normally distributed, and a paired t-test is performed; otherwise, a two-sample rank-sum test is used. For ordinal data and other data before and after, a two-sample rank-sum test is used. All statistical analyses use two-tailed tests, with a significance factor α = 0.05.

[0237] 2.8 Calculation Formula

[0238] Initial value D0: refers to the base value before any products are used.

[0239] DX (Test Value at Any Follow-up Time): This refers to the test data value after the subject has used the product at any follow-up time.

[0240] n: Number of subjects

[0241] Improvement at any follow-up time: refers to the difference between the subject's DX after using the product and the initial value D0 at any follow-up time.

[0242] Improvement rate of any follow-up time: refers to the ratio between the improvement value of any follow-up time and the initial value D0.

[0243] 2.9 Result Judgment

[0244] If the data at any test time point before and after product use during the test period, including instrument test results, doctor evaluation results, and relevant experimental test results, show improvement compared to the data before use or the blank control area, and there is a significant difference (p < 0.05) under the statistical analysis method and test level conditions, the product is considered to have the corresponding efficacy effect during the test period.

[0245] 2.10 Experimental Results

[0246] 2.10.1 Individual Type Angle (ITA°)

[0247] 2.10.1.1 Descriptive statistical results of the Individual Type Angle (ITA°)

[0248] Table 7. Descriptive statistics of the Individual Type Angle (ITA°)

[0249] Time point Statistical quantity average value Median Standard deviation Minimum value Maximum value D0 30 25.97 25.87 3.35 19.73 32.58 D28 30 34.04 34.6 3.95 25.85 39.5

[0250] 2.10.1.2 Improvement value and statistical results of individual type angle (ITA°)

[0251] Table 8. Statistical results of improvement values ​​and differences in the Individual Type Angle (ITA°).

[0252] Comparison of time points Average improvement Average improvement rate Maximum improvement value Maximum improvement rate Median improvement Median improvement rate p-value Significance D28VS.D0 8.07 31.07% 13.85 53.33% 7.72 29.73% ＜0.001 ***

[0253] Note: "ns" means p > 0.05, "*" means 0.01 ≤ p < 0.05; "**" means 0.001 ≤ p < 0.01, and "***" means p < 0.001.

[0254] 2.10.1.3 Analysis of Individual Type Angle (ITA°) Index

[0255] The skin color probe test provides feedback on the subject's skin luster and brightness index, ITA° (an evaluation index calculated by combining brightness L, redness A, and yellowness B). Before and after product use, the ITA° index of the subject's skin is analyzed to determine if there is a significant improvement.

[0256] 2.10.1.4 Individual Type Angle (ITA°) Test Results

[0257] The results of this trial showed that after 28 days of continuous use of the product, the subjects' individual type angle (ITA°) improved by an average of 31.07%, and there were significant differences from the initial values ​​(P < 0.05).

[0258] 2.10.2, Red Zone a* Value

[0259] 2.10.2.1 Descriptive statistical results of a* value in the red zone

[0260] Table 9. Descriptive statistics of a* values ​​in the red zone.

[0261] Time point Statistical quantity average value Median Standard deviation Minimum value Maximum value D0 30 24.02 24.39 2.42 17.83 27.79 D28 30 16.39 16.83 1.61 12.3 18.77

[0262] 2.10.2.2 Improvement values ​​and statistical results of red zone a* values

[0263] Table 10 Statistical results of improvement and difference of a* value in the red zone

[0264] Comparison of time points Average improvement Average improvement rate Maximum improvement value Maximum improvement rate Median improvement Median improvement rate p-value Significance D28VS.D0 7.63 31.77% 9.17 38.18% 7.58 31.56% ＜0.001 ***

[0265] Note: "ns" means p > 0.05, "*" means 0.01 ≤ p < 0.05; "**" means 0.001 ≤ p < 0.01, and "***" means p < 0.001.

[0266] 2.10.2.3 Analysis of the a* value index in the red zone

[0267] VISIA analysis provides feedback on the red zone a* value data of the test area. A comparison before and after product use is conducted to analyze whether there is a significant decrease in the red zone a* value of the test area.

[0268] 2.10.2.4 Test results of red zone a* value

[0269] The results of this trial showed that after 28 days of continuous use of the product, the red zone a* value of the subjects improved by an average of 31.77%, and there were significant differences from the initial value (P<0.05).

[0270] 2.10.3, Red pigment index EI

[0271] 2.10.3.1 Descriptive Statistical Results of the Red Pigment Index (EI)

[0272] Table 11 Descriptive statistics of the red pigment index (EI)

[0273] Time point Statistical quantity average value Median Standard deviation Minimum value Maximum value D0 30 281.86 279.46 22.20 233.05 336.12 D28 30 195.97 195.6 14.19 163.14 221.92

[0274] 2.10.3.2 Improvement values ​​and statistical results of the red pigment index (EI)

[0275] Table 12 Statistical results of improvement values ​​and differences in the EI index (red pigment).

[0276] Comparison of time points Average improvement Average improvement rate Maximum improvement value Maximum improvement rate Median improvement Median improvement rate p-value Significance D28VS.D0 85.89 30.47% 114.2 40.52% 84.4 29.94% ＜0.001 ***

[0277] Note: "ns" means p > 0.05, "*" means 0.01 ≤ p < 0.05; "**" means 0.001 ≤ p < 0.01, and "***" means p < 0.001.

[0278] 2.10.3.3 Analysis of the Red Pigment Index (EI)

[0279] The skin color probe is used to test and provide feedback on the red pigment index of the subject's skin. A comparison before and after product use is analyzed to determine if there is a significant decrease in the subject's skin red pigment index.

[0280] 2.10.3.4, Results of EI test for red pigment index

[0281] The results of this trial showed that after 28 days of continuous use of the product, the subjects' red pigment index (EI) improved by an average of 30.47%, and there were significant differences from the initial values ​​(P < 0.05).

[0282] 2.10.4 Melanin Index (MI)

[0283] 2.10.4.1 Descriptive Statistical Results of Melanin Index (MI)

[0284] Table 13 Descriptive statistics of melanin index (MI)

[0285] Time point Statistical quantity average value Median Standard deviation Minimum value Maximum value D0 30 262.30 264.69 23.90 208.79 315.35 D28 30 183.09 184.41 17.31 144.07 219.5

[0286] 2.10.4.2 Statistical Results of Improvement Values ​​and Differences in Melanin Index (MI)

[0287] Table 14 Statistical Results of Improvement Values ​​and Differences in Melanin Index (MI)

[0288] Comparison of time points Average improvement Average improvement rate Maximum improvement value Maximum improvement rate Median improvement Median improvement rate p-value Significance D28VS.D0 79.21 30.20% 104.66 39.90% 79.51 30.31% ＜0.001 ***

[0289] Note: "ns" means p > 0.05, "*" means 0.01 ≤ p < 0.05; "**" means 0.001 ≤ p < 0.01, and "***" means p < 0.001.

[0290] 2.10.4.3 Analysis of the Melanin Index (MI)

[0291] The skin color probe is used to test and provide feedback on the subject's skin melanin levels. A comparison before and after product use is analyzed to determine if there is a significant decrease in the subject's skin melanin levels.

[0292] 2.10.4.4 Melanin Index (MI) Test Results

[0293] The results of this trial showed that after 28 days of continuous use of the product, the melanin index (MI) of the subjects improved by an average of 30.20%, and there were significant differences from the initial values ​​(P < 0.05).

[0294] 2.10.5 Skin Gloss

[0295] 2.10.5.1 Descriptive statistical results of skin glossiness

[0296] Table 15 Descriptive statistical results of skin glossiness

[0297] Time point Statistical quantity average value Median Standard deviation Minimum value Maximum value D0 30 6.52 6.66 0.89 4.28 8.28 D28 30 8.61 8.79 1.18 5.61 11.01

[0298] 2.10.5.2 Statistical Results of Improvement Values ​​and Differences in Skin Glossiness

[0299] Table 16. Statistical results of improvement values ​​and differences in skin radiance.

[0300] Comparison of time points Average improvement Average improvement rate Maximum improvement value Maximum improvement rate Median improvement Median improvement rate p-value Significance D28VS.D0 2.09 32.06% 2.73 41.87% 2.12 32.52% ＜0.001 ***

[0301] 2.10.5.3 Analysis of Skin Gloss Index

[0302] The skin smoothness and brightness probes were used to test and provide feedback on the subjects' skin radiance. Before and after product use, the results were analyzed to determine if there was a significant improvement in skin radiance.

[0303] 2.10.5.4 Skin Glossiness Test Results

[0304] The results of this trial showed that after 28 days of continuous use of the product, the subjects' skin radiance improved by an average of 32.06%, and there were significant differences from the initial values ​​(P < 0.05).

[0305] 2.10.6 Visual Skin Tone Scale

[0306] 2.10.6.1 Descriptive Statistical Results of Visual Skin Color Scales

[0307] Table 17 Descriptive Statistical Results of Visual Skin Color Scales

[0308] Time point Statistical quantity average value Median Standard deviation Minimum value Maximum value D0 30 6.43 6.50 0.94 4.50 8.50 D28 30 4.22 4.00 0.85 3.00 5.50

[0309] 2.10.6.2 Visual Skin Color Grading Improvement Values ​​and Statistical Results

[0310] Table 18. Statistical results of improvement values ​​and differences in visual skin tone grades.

[0311] Comparison of time points Average improvement Average improvement rate Maximum improvement value Maximum improvement rate Median improvement Median improvement rate p-value Significance D28VS.D0 2.21 34.37% 3.50 54.69% 2.00 31.25% ＜0.001 ***

[0312] 2.10.6.3 Analysis of Visual Skin Color Grading Indicators

[0313] The participants' visual skin tone levels were evaluated and analyzed by dermatologists. Before and after product use, the participants' visual skin tone levels were compared to determine if there was a significant improvement.

[0314] 2.10.6.4 Visual Skin Tone Rating Test Results

[0315] The results of this experiment showed that after 28 days of continuous use of the product, the subjects' visual skin color grade improved by an average of 34.37%, and there were significant differences from the initial values ​​(P < 0.05).

[0316] 2.11 Adverse Reactions in Subjects

[0317] 2.11.1 Adverse Reactions and Standards: Subjects recorded adverse reactions during the trial. During follow-up visits, the trial supervisor or dermatologist observed and recorded the adverse reactions during the trial. The adverse reactions were statistically analyzed in conjunction with the subjects' daily records.

[0318] Table 19 Local Skin Adverse Reactions

[0319]

[0320] The evaluation criteria for adverse reactions are shown in Table 20.

[0321] Table 20

[0322] Skin reaction Classification No response 0 faint erythema 1 Erythema, infiltration, papules 2 Erythema, edema, papules, vesicles 3 Erythema, edema, bullae 4

[0323] 2.11.2. Conclusion on Adverse Reactions

[0324] In conclusion, no adverse reactions were observed in the subjects using the product during the trial.

[0325] 2.12 Efficacy Evaluation Conclusion

[0326] This trial used a human efficacy testing method on 30 eligible participants, with 0 participants withdrawing. The results are as follows:

[0327] After 30 subjects used the sample for 28 days, the individual type angle (ITA°) improved by 31.07%, the red zone a* value improved by 31.77%, the red pigment index (EI) improved by 30.47%, the melanin index (MI) improved by 30.20%, skin radiance improved by 32.06%, and the visual skin tone grade improved by 34.37%. All of the above parameters showed significant differences from the initial values ​​(P < 0.05), and the measured values ​​were better than the initial values, indicating that the product has a whitening effect.

[0328] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A transdermal elastin liposome composition for whitening and brightening, characterized in that, The composition contains saffron extract, 4-butylresorcinol, Dalbergia odorifera bark extract, nicotinamide, Plantago asiatica ferment extract and Ginkgo biloba ferment extract, as well as excipients and solvents for preparing liposomes; The mass ratio of saffron extract, 4-butylresorcinol, Dalbergia odorifera bark extract, nicotinamide, Plantago asiatica ferment extract and Ginkgo biloba ferment extract is (3-5):(0.2-1):(5-10):(0.1-0.3):(5-15):(2-4). The method for preparing the fermented extract of *Carex chinensis* leaves includes the following steps: Step 1: Take dried Plantago asiatica leaves, crush them through a 60-mesh sieve to obtain Plantago asiatica powder, sterilize for 20 minutes, and after sterilization, add sterile PBS buffer solution containing 2% glucose at a material-to-liquid ratio of 1:20 g / mL, stir evenly to obtain fermentation substrate; Step 2: Mix the Streptococcus thermophilus bacterial solution with the Lactobacillus rhamnosus bacterial solution to obtain a composite bacterial solution; Step 3: Inoculate the compound bacterial solution into the fermentation substrate at an inoculation rate of 5 v / v%, and ferment at 37℃ and 100 r / min for 72 h. During the fermentation process, the aeration rate is maintained at 0.2 vvm. After the fermentation is completed, the fermentation broth is centrifuged, the supernatant is collected, sterilized by 0.22 μm microfiltration, concentrated under reduced pressure, and freeze-dried to obtain the fermented extract of Plantago asiatica leaves. The viable count of the thermophilic streptococcal bacterial suspension was 1×10⁻⁶. 8 The cFU / mL count of *Streptococcus thermophilus* was determined by the accession number GDMCC 1.1808, and the viable count of the *Lactobacillus rhamnosus* culture was 1 × 10⁻⁶. 8 CFU / mL, the preservation number of Lactobacillus rhamnosus is GDMCC1.1798; The thermophilic streptococcus bacterial solution and the lactobacillus rhamnosus bacterial solution were mixed at a volume ratio of 4:1 to obtain a composite bacterial solution; The preparation method of the Ginkgo fermentation extract includes the following steps: Step 1: Take dried ginkgo leaves, crush them through a 30-60 mesh sieve to obtain ginkgo leaf powder. Mix the ginkgo leaf powder with wheat bran powder at a mass ratio of 7:3 to 8:2, adjust the moisture content to 50%-60% with distilled water, sterilize for 30 minutes, and obtain the fermentation substrate. Step 2: Mix Aspergillus oryzae spore suspension with Aspergillus cristatus spore suspension to obtain a composite spore suspension. Then, spray the composite spore suspension evenly onto the substrate at an inoculation rate of 20 w / w% of the substrate dry weight. Mix thoroughly and ferment at 28℃ and 70-80% relative humidity for 7-10 days, stirring 1-2 times a day to provide oxygen, dissipate heat, and prevent mycelia from tangling. Step 3: After fermentation, the fermented product is mixed with an 80 v / v% ethanol aqueous solution at a ratio of 1:30 g / mL, and refluxed three times for 45 min each time. The extracts are combined and concentrated under reduced pressure to obtain Ginkgo fungal fermentation extract. The extract is further freeze-dried to obtain Ginkgo fermentation extract. The Aspergillus oryzae spore suspension contained 10 spores. 6 The concentration of CFU / mL was CFU / mL, and the preservation number of *Aspergillus oryzae* was GDMCC 3.

236. The spore count of the *Aspergillus cristatus* spore suspension was 10. 6 CFU / mL, the accession number of Aspergillus cristatus is GDMCC 3.1025; The Aspergillus oryzae spore suspension and Aspergillus triticum spore suspension were mixed at a volume ratio of 4:3 to obtain a composite spore suspension.

2. The elastic liposome composition according to claim 1, characterized in that, The excipients are hydrogenated lecithin, soybean lecithin, cholesterol, and fatty acid sucrose esters; The solvents are deionized water and anhydrous ethanol.

3. The use of the elastosome composition according to claim 1 or 2 in the preparation of skin care products with whitening and brightening effects.

4. A method for preparing the elastic liposome composition according to claim 2, characterized in that, The preparation method includes the following steps: Step 1: Mix hydrogenated lecithin, soybean lecithin, cholesterol, and fatty acid sucrose esters in a mass ratio of 5:2:1:3 to obtain mixture A. Mix saffron extract and 4-butylresorcinol in a mass ratio to obtain mixture B. Mix mixture A and mixture B in a mass ratio of 11:1 to obtain liposome wall material. Heat the liposome wall material with anhydrous ethanol in a water bath at 50-55℃ until completely dissolved in a mass-volume ratio of 1:7 g / mL. Then remove the ethanol by vacuum distillation to obtain a transparent oil film. Step 2: Combine the extracts of Dalbergia odorifera bark, nicotinamide, fermented extract of Plantago asiatica leaf and fermented extract of Ginkgo biloba to obtain liposome core material. Mix the liposome core material with deionized water at a mass-volume ratio of 1:150g / mL to completely dissolve it and obtain the core material solution. Step 3: Add the core material solution to the transparent oil film and stir at 700-800 r / min for 10 min to obtain an emulsion. Transfer the emulsion to a high-pressure homogenizer and cycle it 3 times at a pressure of 16000-18000 psi to obtain the composition.

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