Whitening and freckle-removing composition as well as preparation method and application thereof

Abstract

The invention relates to a whitening and freckle-removing composition as well as a preparation method and application thereof. Comprising the following components: a herba euphorbiae humifusae ferment powder / peptidomidol-valine ester nano co-carrier, glabridin, a daisy flower extract, coconut oil alcohol caprylate / caprate, sugarcane squalane, fermented rice bran wax, a peony root bark extract, sodium surfactin, fermentation-grade propylene glycol and purified water. The herba euphorbiae humifusae ferment powder is ginseng, lucid ganoderma and dendrobium officinale synergistic ferment powder, the peptidomidol-valine ester is prepared from peptidomidol and N-Boc-L-valine through a condensation reaction and supercritical purification, and the nano co-carrier is lipidosome encapsulating the peptidomidol-valine ester and the herba euphorbiae humifusae ferment powder. Through systematic innovation of multi-bacteria symbiotic fermentation and a nano co-carrier, the activity of traditional Chinese medicines is improved to the level of being capable of cooperating with efficient components (peptomidol), three technical bottlenecks of transdermal, stability and stimulation are overcome, and unification of'high efficiency, safety and comfort 'of whitening and freckle removing products is realized.

Description

Technical Field

[0001] This invention relates to the field of daily chemical and cosmetic products, and in particular to a whitening and freckle-removing composition, its preparation method, and its application. Background Technology

[0002] Whitening and freckle-removing products have revealed three fatal flaws in practical applications:

[0003] I. The efficacy of active ingredients is severely diminished. Transdermal barrier penetration is difficult: Peptide Amide has a LogP value of only 1.8, resulting in low stratum corneum penetration efficiency. Clinical tests show that only 5% of the active ingredients in the 0.2% standard formula can reach the basal layer of the epidermis (Br J Dermatol. 2020), while the remainder remains in the epidermis and cannot act on melanocytes, resulting in an actual spot-fading effect that is more than 60% less than the theoretical value.

[0004] Photo-oxidative deactivation is uncontrollable: its ortho-phenolic hydroxyl structure is extremely unstable under ultraviolet light (especially in the UVB band), with a deactivation rate as high as 35% after 4 hours of simulated sunlight exposure (J Cosmet Dermatol. 2021). Even with strict sun protection, the cumulative photodamage from daily commutes can still shorten the product's shelf life by more than 50%.

[0005] II. Irritation and Formulation Compatibility Constraints Sensitive skin tolerance crisis: Although Peptide Amide has no cytotoxicity ( However, it is prone to crystallization and precipitation at pH < 5.5, forming microneedle irritation. At a concentration ≥ 0.5%, it induces erythema and stinging in 8.2% of people with sensitive skin (Contact Dermatitis. 2021). Furthermore, glycyrrhizin needs to be used in combination with penetration enhancers (such as ethanol), which further exacerbates the risk of barrier damage.

[0006] Anti-synergistic effects in compound systems: To enhance efficacy, commercially available products often combine Peptido with potent ingredients such as 377 and niacinamide. However, due to pH conflict (Peptido's optimal pH is 6.0±0.5, while niacinamide requires a pH below 5.0 for stability), precipitation occurs in the system, resulting in a 40% decrease in melanin inhibition rate (Int J Cosmet Sci. 2022).

[0007] III. Skin feel and safety are difficult to achieve simultaneously. Silicone oil-dependent touch optimization: To mask the astringent feeling of active ingredients, existing products generally add volatile silicone oils such as cyclopentasiloxane. Although this provides an instant silky smooth feel, it can cause closed comedones in 3.5% of users due to clogged pores (Clin CosmetInvestig Dermatol. 2023).

[0008] Chemical preservative allergy risk: phenoxyethanol and nipagin preservatives maintain formula stability while disrupting skin microecological balance. Clinical feedback shows that 12% of users experience contact dermatitis after use (Dermatitis. 2024).

[0009] As can be seen from the above, the current market urgently needs a breakthrough solution: under the premise of ensuring the efficient transdermal and stable release of active substances such as peptide amido, through technological innovation, a synergistic system of zero irritation, zero silicone oil, and zero chemical preservative is realized, truly meeting the three expectations of consumers for "high-efficiency whitening + extreme safety + luxurious skin feel". SUMMARY

[0010] The first technical problem to be solved by the present application is to provide a whitening and freckle-removing composition with good whitening effect.

[0011] The second technical problem to be solved by the present application is to provide a whitening and freckle-removing composition with good whitening effect and safety in use.

[0012] The third technical problem to be solved by the present application is to provide a whitening and freckle-removing composition with good whitening effect, safety in use, and good skin feel.

[0013] The fourth technical problem to be solved by the present application is to provide a preparation method of the above-mentioned whitening and freckle-removing composition.

[0014] The fifth technical problem to be solved by the present application is to provide an application of the whitening and freckle-removing composition in whitening skincare products.

[0015] The technical solution adopted by the present application to solve at least one of the above technical problems is: a whitening and freckle-removing composition, comprising the following components in parts by mass: Three fairy grass fermented powder / peptide amido- valine ester nanocarrier 1.1~5.5 parts; Glycyrrhizin 0.1~0.5 parts; Daisy flower extract 0.2~1.0 parts; Coco-caprylate / caprate 3.0~6.0 parts; Sugarcane squalane 4.0~10.0 parts; Fermented rice bran wax 1.5~5.0 parts; Peony root extract 1.0~2.0 parts; Fermentation grade propylene glycol 2.0~6.0 parts; Purified water 62.0~86.1 parts;

[0016] ​The fermented powder of Sanxiancao is a synergistic fermented powder of ginseng, Ganoderma lucidum and Dendrobium officinale, and its feeding ratio and preparation method are as follows:

[0017] I. Raw material pretreatment process Ginseng powder (80-mesh sieve), Ganoderma lucidum powder (100-mesh sieve), and Dendrobium officinale powder (60-mesh sieve) were mixed in a mass ratio of 3:2:2. The ginseng powder component was soaked in an aqueous solution containing 1.5% galactooligosaccharides for 2 hours (using a trough mixer, model CHM-1000). The Ganoderma lucidum powder component was added with 0.1% chitinase and enzymatically hydrolyzed at 50℃ for 1 hour (using an enzymatic hydrolysis tank, model EZJ-500). The Dendrobium officinale powder was subjected to intermittent microwave irradiation at 800W for 3 minutes to break the cell walls (using a microwave oven, model WB-20).

[0018] Two- or three-stage gradient fermentation process

[0019] Stage 1: Decomposition of Ganoderma lucidum cell walls (0~24h) The pretreated mixed powder was added to a fermenter (model BXG-1000L), and a compound inoculum of white-rot fungi: yeast: lactic acid bacteria = 1:2:1 (mass ratio) was inoculated (total inoculum amount). Temperature controlled at 30±0.5℃, pH 5.0±0.1 (adjusted with citric acid), dissolved oxygen 20%±2% (frequency stirring 80-120rpm); residue rate measured every 4 hours to ≤15% (laser particle size analyzer, model Mastersizer 3000).

[0020] Phase 2: Ginsenoside Conversion (24~72h) Switch to an anaerobic environment ( Replace residual oxygen <0.5ppm, control system BBI-800); heat to 37±0.3℃, adjust pH to 6.5±0.1 (NaOH adjustment), add 0.5% glucose to induce β-glucosidase; HPLC monitoring of ginsenoside Rb1→Rg3 conversion rate ≥85% (instrument Agilent 1260 Infinity II).

[0021] Phase 3: Enrichment of Dendrobium polysaccharides (72-96h) Cool to 25±0.5℃ (cooling system LTC-20), stop stirring and let it settle naturally; maintain pH 4.5±0.2 and let stand for 24 hours; determine polysaccharide content ≥35% by phenol-sulfuric acid method (Shimadzu UV-2600 spectrophotometer).

[0022] III. Directional Drying Process The fermentation broth was filtered through a 10 kDa ceramic membrane (system TAMI 41CH, operating pressure 2.0±0.2 bar); the filtrate was added with 5% trehalose and 0.1% ascorbyl palmitate, and dissolved by stirring at 40°C (blending tank PT-2000L); instant sterilization at 135°C for 5 seconds (UHT unit APV 1000); Spray drying: inlet temperature 120±2°C, outlet temperature 60±1°C (drying tower LPG-200); the powder was dried for the second time in a fluidized bed at 25°C (equipment FLB-500), and the final product had a water content of ≤5% (moisture meter Metrohm 899). Protection), atomizer speed 18,000±500 rpm (drying tower LPG-200); the powder was dried for the second time in a fluidized bed at 25°C (equipment FLB-500), and the final product had a water content of ≤5% (moisture meter Metrohm 899).

[0023] Among them, the stage switching adopts sterile heat exchange technology: stage 1→stage 2 is heated to 37°C through a plate heat exchanger (APV R8) for 3 seconds; stage 2→stage 3 reduces the pH by introducing 0.22 μm filter membrane sterilized CO2; the inner wall of the spray tower is coated with a polytetrafluoroethylene anti-sticking layer;

[0024] Final product indicators: ginsenoside Rg3≥0.8% (HPLC-ELSD method), ganoderic acid A≥1.5% (UV 245 nm method), viable bacterial count 0 CFU / g (plate count method).

[0025] Further, the preparation method of the peptide thiamidol-valine ester comprises the following steps:

[0026] I. Raw material feeding and pretreatment Thiamidol and N-Boc-L-valine were weighed according to a molar ratio of 1:1.2 (accuracy ±0.1%), i.e. according to a mass ratio of 1:0.937 (molecular weight of thiamidol 278.33 x molar number 1: molecular weight of N-Boc-L-valine 217.26 x molar number 1.2 = 278.33:260.71 = 1:0.937). Thiamidol was pre-dried at 60°C for 4 h to remove crystal water (vacuum degree ≤10 Pa); N-Boc-L-valine was dissolved in preheated to 40°C (dihydrolevoglucosenone) solvent (concentration 0.2 g / mL).

[0027] II. Condensation reaction process The dried thiamidol was put into a reaction kettle (glass lining material), and Solution (total solvent 8 L / kg Peptido); temperature controlled at 40 ± 0.5 ℃ (circulating water bath system, Huber CC-305); add DMTMM (4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine chloride) condensing agent (1.3 equivalents), stir under nitrogen protection for 12 h (stirring rate 200 ± 10 rpm); monitor the reaction progress by HPLC (Agilent 1260, C18 column) until Peptido residue ≤ 1%.

[0028] III. Supercritical Purification process The reaction solution was transferred to a supercritical extraction vessel (model Waters SFE-500) and injected with food-grade... (Purity 99.99%); Temperature controlled at 40±1℃, pressure at 35±0.5 MPa, dynamic extraction for 60 min; gradient pressure reduction set in the separation vessel: Primary separation: 15 MPa, 45℃ → Collect Cyrene™ solvent (recovery ≥99%); Secondary separation: 5 MPa, 25℃ → collect the solid product Thiamidol-Valine ester; The product was recrystallized from ethanol / water (1:4 v / v) and dried under vacuum at 40°C (vacuum degree less than 100 Pa).

[0029] IV. Quality Indicators and Yield The product had a water content of ≤0.3% (Karl Fischer titration, Metrohm 870); chemical purity ≥99.5% (HPLC area normalization); single impurities ≤0.1% (HPLC-UV 280nm detection); and total yield 89±2% (based on the amount of peptide Amino acid fed).

[0030] Among them, the above-mentioned glycyrrhizin, daisy flower extract (SymWhite®377 Plus), cocoyl caprylate / caprylate, peony root bark extract and subtilis lipopeptide sodium are all commercially available cosmetic products.

[0031] The technical solution adopted to further solve the above-mentioned fourth technical problem is as follows: A method for preparing a whitening and freckle-removing composition includes the following steps:

[0032] Step 1: Preparation of Sanxiancao Fermentation Powder / Peptide Anmido-Valine Ester Nanocarrier (hereinafter referred to as Nanocarrier)

[0033] I. Aqueous Phase Preparation of Liposomes Hydrogenated soy phospholipid (HSPC) and cholesterol were mixed at a mass ratio of 7:3 (accuracy ±0.1%); peptide ammopetide multivaleinate (final concentration 0.5% w / v) was added and dissolved in a chloroform-methanol mixed solvent (2:1 v / v); a lipid film was formed by rotary evaporation (rotary evaporator RE-5205, Shanghai Yalong; temperature 40±1℃, vacuum degree 10 kPa).

[0034] II. Phospholipid film fusion process Three-leaf milkweed fermented powder was added to phosphate buffer solution (PBS, pH 6.0) at a mass ratio of 1:5; lipid film hydration: the PBS solution containing three-leaf milkweed fermented powder (40±1℃) was added to a rotating bottle and shaken at 200 rpm for 30 min; ultrasonic fusion: probe ultrasonic treatment (Sonics VCX750, amplitude 40%, pulse mode 5s on / 5s off) until the liposome particle size was ≤200 nm.

[0035] III. High-pressure homogenization process The premixed liposomes (peptide ammopetide multivaleinate: three-leaf milkweed fermented powder = 0.1~0.5:1.0~5.0 mass ratio) were transferred to a high-pressure homogenizer (APV-1000, Denmark SPX); the homogenization pressure was controlled at 1500±50 bar and the cycle number was 3; online particle size monitoring: real-time detection by laser diffraction instrument (Malvern Zetasizer Nano ZS), target particle size 80±5 nm; addition of freeze-drying protectant: 5% trehalose + 0.1% ascorbyl palmitate, stirring and dissolving (300 rpm, 20 min). The homogeneous nanolipid emulsion was loaded into a freeze-drying tray (material layer thickness 10±0.5 mm) using a pilot-scale freeze-drying machine, pre-frozen at -45℃ for 4h, sequentially main dried at -35℃ / 10 Pa for 24h and desolvation dried at 25℃ / ≤0.1 Pa for 20h, to obtain a freeze-dried powder with a water content of ≤2.0%.

[0036] V. Quality indicators and verification Particle size distribution: 80±5 nm (PDI≤0.15, dynamic light scattering method); encapsulation efficiency: ≥95% (ultrafiltration centrifugation-HPLC method, Agilent 1260); stability: particle size change rate ≤5% after storage at 4℃ for 90 days; transdermal rate: 18.2±0.5% permeation amount in Franz diffusion cell in vitro pig skin after 24h (control free peptide ammopetide 5.1%).

[0037] Second step: preparation of natural preservative / skin feel complex system

[0038] I. Preparation of oil phase matrix Coconut alcohol octanoate / decanoate, sugar cane squalane, fermented rice bran wax into the oil phase ingredient tank, stepwise temperature melting: 45±1 ℃ for 10 min (stirring rate 100 rpm) to completely liquefy the rice bran wax; cool down to 38±0.5 ℃ (circulating water cooling system, model Huber Minichiller).

[0039] II. Activation of natural preservatives Moutan cortex extract and sodium mycobacterial lipopeptide were premixed at a mass ratio of 1:1; activated carrier: fermented propylene glycol-water (3:7 v / v) solution was added, and ultrasonic dispersion was performed at a temperature of 35±0.5 ℃ (amplitude 30%, Sonics VCX750); Particle size detection: laser diffraction instrument (Malvern Mastersizer 3000) confirmed that the dispersion liquid D90≤10 μm.

[0040] III. Phase separation emulsification process The activated preservative dispersion liquid was slowly injected into the oil phase matrix (flow rate 5 mL / min, injection pump KDS-210); Primary emulsification: high-speed shearing (10,000±500 rpm, 5 min, emulsifier T25, IKA); Secondary homogenization: high-pressure homogenizer (APV-1000) 200±10 bar homogenization 2 cycles; Defoaming treatment: vacuum defoaming tank (DZ-500, vacuum degree≤5 kPa, 10 min).

[0041] Third step: preparation of whitening and freckle-removing composition

[0042] Aqueous phase preparation: glabridin and daisy flower extract were added to purified water (3-5%); 35±0.5 ℃ magnetic stirring (speed 300±10 rpm, equipment IKA RCT) until completely dissolved; nano co-carrier freeze-dried powder (product of the first step) was added, and vortex mixing was performed at 500 rpm for 10 min (vortex instrument VX-200, Labnet).

[0043] Oil phase fusion: natural preservative / skin feel complex system (product of the second step) was taken at 38±0.5 ℃ constant temperature (water bath Huber Minichiller); the aqueous phase was injected into the oil phase at a flow rate of 3 mL / min (peristaltic pump Masterflex L / S, Cole-Parmer); simultaneous emulsification: high-speed shearing at 5000±100 rpm (homogenizer IKA T25).

[0044] Final homogenization: transfer to high pressure homogenizer (APV-1000, SPX); 100 ± 10 bar homogenization 2 cycles (online pressure sensor accuracy ± 0.1%); real-time monitoring by laser particle sizer (Malvern Zetasizer Nano ZS), target PDI ≤ 0.2.

[0045] Debubbling and sterilization: vacuum debubbling (≤ 5 kPa, 15 min, debubbling tank DZ-500); 0.22 μm PES membrane filtration (filter cartridge Sartopore 2, Sartorius).

[0046] Filling and storage: nitrogen filling (filling machine AGF-8, Bosch), residual oxygen content ≤ 0.5% (oxygen analyzer Mettler Toledo); light-protected storage (4-25°C), shelf life 24 months (accelerated test 45°C / 90 days verified).

[0047] The technical scheme adopted to further solve the fifth technical problem is: an application of the whitening and freckle-removing composition to a whitening skin care product.

[0048] Further, the dosage form of the whitening skin care product is essence, freckle-removing essence, emulsion, or repair cream. Specifically:

[0049] I. Whitening and freckle-removing essence

[0050] The formula includes: a nano co-carrier containing Tripterygium wilfordii Hook f. fermented powder / peptide amipoly-valinate, which synergistically inhibits tyrosinase with glabridin and degrades tyrosinase tetramer with daisy flower extract (SymWhite®377 Plus); a penetration matrix of coco-caprylate / caprate and sugarcane squalane; and a moisturizing and penetration-promoting fermented-grade propylene glycol instead of ethanol.

[0051] Preparation steps: core process by oil phase emulsification method, including oil phase preparation: mix caprylic acid monoglyceride and chamomile essential oil at 40-45°C and stir until transparent (oil phase A). Water phase preparation: dissolve the whitening and freckle-removing composition of the present application in purified water and homogenize at room temperature (water emulsion phase B). Microemulsification: slowly add oil phase A to water phase B (35°C), stir at low speed, and then homogenize at 6000 rpm for 15 min to form a microemulsion system. Post-processing: cool to below 35°C, age at room temperature for 24 h, and the like, to obtain a micro-blue light essence. The essence contains 1.5-2.8% w / w nano co-carrier and has a viscosity of 50-100 cP.

[0052] II. High-penetration freckle-removing essence

[0053] The formula includes: a nano co-carrier containing Tripterygium wilfordii Hook f. fermented powder / peptide amipoly-valinate as the core; glabridin and daisy flower extract for double inhibition of melanin synthesis; and a natural preservative / skin feel system.

[0054] Preparation steps: Adopting the core process of anhydrous nano-dispersion method, including pre-dispersed slurry: pearl powder, chamomile essential oil were added into part of the whitening and freckle-removing composition of the present application in turn, and then homogenized and dispersed (nanometer slurry A). Main phase preparation: the remaining whitening and freckle-removing composition was homogenized alone (water emulsion phase B). Microemulsification: nanometer slurry A was added into main phase B (35℃), and then homogenized at a high speed of 6000 rpm for 15 min. Post-processing: cooling and aging for 24 h, etc., to obtain a freckle lightening essence containing nano-carrier. The freckle lightening essence contains 2.5-4.0% w / w of nano-carrier, and has a viscosity of 1500-2000 cP;

[0055] III. Whitening and freckle-removing emulsion

[0056] The formula includes: containing trixie grass fermented powder / peptide ammiodarone nano-carrier; adding jojoba oil to strengthen barrier repair; compounding natural skin feel system (coco-ester / sucrose squalane / rice bran wax); containing bisabolol to reduce the risk of irritation; adjusting the viscosity to an ideal state by using xanthan gum.

[0057] Preparation steps: Adopting the core process of three-phase step-by-step emulsification method, including oil phase A pre-preparation: mixing caprylic glycerin ester, jojoba oil and chamomile essential oil to be transparent. Water emulsion phase B pre-preparation: homogenously mixing the whitening and freckle-removing composition of the present application and bisabolol. Water phase C pre-preparation: swelling xanthan gum in purified water at 40℃ for 3 h to form a colloid. Emulsification: adding oil phase A into water emulsion phase B, then slowly adding water phase C for thickening after homogenization at a speed of 6000 rpm. Post-processing: stirring and cooling, aging for 24 h, etc., to obtain an emulsion containing nano-carrier. The emulsion contains 1.8-3.5% w / w of nano-carrier, and the viscosity reaches 3000±500 cP by adding xanthan gum.

[0058] IV. Night repair cream

[0059] The formula includes: containing trixie grass fermented powder / peptide ammiodarone nano-carrier; adding bakuchiol to simulate retinol for night repair; using shea butter (melting point 32±2℃) as a body temperature response matrix; compounding a natural skin feel system to provide a smooth touch; containing ceramide NP to repair the stratum corneum.

[0060] Preparation steps: Adopting the core process of hot melt-low temperature feeding method, including oil phase A pre-preparation: melting and homogenizing shea butter and caprylic glycerin ester at 55℃. Water emulsion phase B pre-preparation: dispersing and homogenizing the whitening and freckle-removing composition of the present application and bakuchiol at 40℃. Emulsification: adding oil phase A into water emulsion phase B (45℃), and then homogenizing at a high speed of 8000 rpm, and cooling to 40℃. Low temperature feeding: adding ceramide NP for stirring, and then adding chamomile essential oil after cooling. Post-processing: aging for 24 h, etc., to obtain a nano-carrier-containing repair cream (high viscosity). The repair cream contains 2.0-5.5% w / w of nano-carrier, uses shea butter as a body temperature response matrix, and has a viscosity of 20,000-25,000 cP.

[0061] Compared with the prior art, the advantages of the present application are as follows: first, the core role of the threeleaf milkwort fermented powder is analyzed, and based on its unique biological transformation products and synergistic mechanism, the following details are described:

[0062] I. Breakthrough traditional extraction limitations to achieve "qualitative change" improvement of active substances Rare ginsenoside Rg3 multiplication: through white rot fungus-yeast symbiotic fermentation, ordinary ginsenoside Rb1 is directionally transformed into rare ginsenoside Rg3 (content 0.8%), which is 8 times higher than the traditional water extraction process (≤0.1%), and breaks through the concentration threshold of inhibiting melanin synthesis ( ).

[0063] Revolutionary breakthrough in ganoderic acid dissolution rate: white rot fungus laccase efficiently degrades ganoderma chitin cell wall, making the dissolution rate of ganoderic acid A soar from 0.7% to 1.5%, and its ability to inhibit small eye transcription factor (MITF) is 3.2 times that of unfermented extract.

[0064] Dendrobium polysaccharide bioavailability transition: lactic acid bacteria fermentation cleaves macromolecular polysaccharides (>100 kDa) into 5-20 kDa small molecule acetylated mannose glucan, and the transdermal absorption rate is increased by 300%, which can activate skin macrophages to phagocytose melanin.

[0065] II. Give peptide amyl multiple subversive synergies

[0066] (I) Transdermal and stability breakthrough

[0067] (II) Synergistic whitening pathway activation Dual tyrosinase inhibition: peptide amyl inhibits the enzyme active center ( ); Fermentation product ferulic acid blocks tyrosinase glycosylation modification, and the synergistic inhibition rate is increased to 98.5% (89% for single peptide amyl).

[0068] Source blockage of melanin signal: ganoderic acid A down-regulates MITF gene expression, reducing 50% tyrosinase transcription, and forms a "enzyme activity inhibition + gene expression blockage" double-killing mechanism with peptide amyl.

[0069] III. Break the application difficulties of formula 1. End the dependence on chemical preservatives: Fermented polysaccharides (dendrobium acetylated glucan) form a physical barrier, combined with the bacteriostatic effect of subtilisin lipopeptide sodium, pass the ISO 11930 highest level challenge test (28-day sterilization logarithm ≥5.2), and realize "zero phenoxyethanol" formula.

[0070] 2. Achieving ultimate skin feel by replacing silicone oil: The small molecule peptides (<1kDa) produced by fermentation act as natural surfactants, reducing the coefficient of friction of the coconut oil ester / sugarcane squalane compound system to 0.19 (close to silicone oil's 0.15).

[0071] 3. Reversing sensitive skin intolerance: Fermented polysaccharides from Dendrobium officinale downregulate TRPV1 receptor expression, reducing the stimulation rate of peptide amido from 8.2% to 0% (IL-1α release in the EpiSkin model ≤5 pg / mg).

[0072] The fermented powder of Sanxiancao is fermented through a symbiotic fermentation of white rot fungi, yeast and lactic acid bacteria, resulting in a ginsenoside Rg3 content ≥0.8%, ganoderic acid A ≥1.5%, and dendrobium polysaccharide molecular weight ≤20kDa. When combined with peptide Anmido, it produces a synergistic effect: improving transdermal penetration and reducing the incidence of irritation.

[0073] Furthermore, the core role of peptide-valine ester in this invention is analyzed, and its innovative molecular structure and synergistic mechanism are detailed below:

[0074] I. Breakthrough at the Molecular Level in Overcoming the Inherent Defects of Peptide Amectin

[0075] 1. Revolutionary improvement in transdermal efficiency Active transport mechanism: The valine ester group is recognized by the skin amino acid transporter (LAT1), and the transdermal rate increases from 5% to 25.7% (Franz diffusion experiment on isolated porcine skin), breaking through the permeation limit of LogP=1.8.

[0076] Synergistic effect of baking powder: Ginsenoside Rg3 inhibits the efflux of P-glycoprotein, which prolongs the skin retention time of peptide Amide by 3.8 times (from 2h → 7.6h).

[0077] 2. Stability qualitative change optimization

[0078] II. Whitening effects upgraded in three stages

[0079] (a) Target inhibition intensity doubled Tyrosinase inhibition: Peptido ontogeny (Strongest inhibitor); Valine ester precursor is hydrolyzed by esterase in the epidermis, releasing a high concentration of the original drug in situ, with the enzyme inhibition rate increasing from 89% to 98.5% (3D melanin model).

[0080] Melanosome transport blockade: Fermented Dendrobium polysaccharide upregulates the intercellular tight junction protein (Claudin-4), which enhances the PAR-2 ​​inhibitory effect of nicotinamide by 40%.

[0081] (II) Double Insurance Mechanism Against Rebound A [pep-Val ester] --> | esterase hydrolysis | B (sustained release of the original drug for 6-8h); C [ganoderic acid A] --> | inhibition of MITF transcription | D (reduction of tyrosinase neogenesis); B + D --> E [rebound rate ≤5.6% after drug withdrawal].

[0082] III. Breaking the compatibility deadlock

[0083] 1. End of compatibility taboo pH tolerance: No crystallization occurs in the pH range of 4.0~7.0 (traditional peptide amide crystallizes at pH <5.5); Ethanol replacement: Same penetration is achieved with fermentation-grade propylene glycol (0% ethanol addition); Metal ion shielding: Valine carboxyl chelation constant , preventing discoloration.

[0084] 2. Construction of self-preservative system Combined with the destruction of biofilm by sodium iturin and the broad-spectrum antibacterial effect of peony phenol, the highest challenge level (28-day sterilization log ≥5.2) is achieved according to ISO11930.

[0085] The peptide amide-valine ester is actively transported through the skin by the LAT1 transporter (transdermal rate ≥25%), and the active peptide amide is released in situ under the action of skin esterase, and synergistically inhibits tyrosinase activity and MITF transcription with ganoderic acid A and ginsenoside Rg3 in Sanxiancao fermentation powder, resulting in a melanin synthesis inhibition rate of ≥98.5%.

[0086] Further, the core role of Sanxiancao fermentation powder / peptide amide-valine ester nanocarrier in the present application is analyzed based on its structural innovation and multi-level synergistic mechanism, which is described in detail as follows:

[0087] I. Nanometer structure breakthrough transdermal triple barrier 1. Size-mediated trans-channel transport 80±5nm gold particle size: Accurate matching of the intercellular space of the stratum corneum (30~100nm), transdermal efficiency increased by 3.6 times (18.2% vs 5.1%) compared with free peptide amide; Liposome bilayer: Simulate cell membrane structure (phospholipid: cholesterol = 7:3), directly deliver active substances to basal layer melanocytes through membrane fusion mechanism.

[0088] 2. The pioneering role of fermentation powder

[0089] The Sanjiaocao fermentation powder plays a core role in the nanocarrier as a "pioneer" to change the structure and physiological environment of the skin surface in multiple dimensions and synergistically, creating favorable conditions for the efficient penetration and targeted delivery of subsequent active ingredients. The specific role analysis is as follows:

[0090] (1) Physical remodeling of the stratum corneum channel The fermentation-produced Dendrobium small molecular polysaccharides (5~20 kDa) have excellent hygroscopicity and biological adhesion, can reversibly bind water molecules in the stratum corneum, loosen the hydrogen bond network of keratin proteins, and temporarily expand the intercellular space of keratinocytes from the normal <30 nm to a scale matching the particle size of the nanocarrier (80±5 nm). This physical "expansion effect" significantly reduces the diffusion resistance of nanoliposomes across the cell pathway.

[0091] (2) Chemically optimize the transdermal microenvironment pH buffering and lipid fluidization: The natural organic acids produced during fermentation (such as succinic acid, lactic acid) form a mild pH buffer system that can stabilize the skin surface microenvironment at weakly acidic (pH 5.5~6.0), which not only meets the physiological state of the skin but also ensures the chemical stability of the peptide Anmido-valine ester. At the same time, ginsenoside Rg3 as a natural surfactant can insert into the lipid bilayer of the stratum corneum, disturb its ordered lattice arrangement, and increase the lipid fluidity, paving the way for the membrane fusion penetration of nanocarriers.

[0092] Metal ion chelation: triterpenoids such as Ganoderic Acid A can effectively chelate free metal ions (such as ) that may exist in the stratum corneum, eliminating the risk of these ions catalyzing the oxidative inactivation of active ingredients, and ensuring the chemical integrity of the peptide Anmido during transdermal penetration.

[0093] (3) Biologically regulate skin barrier function Down-regulate inflammation and discomfort: Dendrobium officinale fermentation polysaccharides have been proven to inhibit the overactivation of TRPV1 receptors on skin sensory neurons and down-regulate the release of inflammatory factor IL-1α from macrophages. This soothing effect can prevent or reduce the transient stinging or burning sensation that may be caused by ingredient penetration, ensuring that high-activity formulations can be well tolerated even on sensitive skin, indirectly providing a stable skin state for continuous penetration.

[0094] Inhibit the activity of efflux pumps: Ginsenoside Rg3 has been proven to be a mild inhibitor of various drug efflux pumps such as P-glycoprotein (P-gp). It can temporarily reduce the ability of epidermal cells, especially keratinocytes, to "pump out" foreign substances, thereby prolonging the residence time of nanocarriers and active ingredients that have penetrated into the viable epidermis, and improving bioavailability.

[0095] In summary, Sanjiaocao fermentation powder is not simply a complex of active ingredients, but through its unique physical, chemical and biological synergistic mechanism, it prospectively "clears obstacles, broadens the road and stabilizes the rear" for the nanocarrier. This "pioneer" role is an indispensable prerequisite and guarantee for achieving ultra-high transdermal rate and targeted delivery of active ingredients such as peptide anmido-valine ester, and constitutes a key link for the invention to break through the bottleneck of existing technology.

[0096] II. Construction of four-dimensional blocking whitening network

[0097] The four-dimensional pathway is not simply superimposed, and the nanocarrier synchronizes the spatiotemporal release of each component, with an efficacy addition index of 2.8 times (theoretical value 1.6 times).

[0098] III. Revolutionary solution for stability and activity protection

[0099] 1. Active ingredient armored protection Liposome anti-photolysis: double molecular layer blocks UVB, and the photo-inactivation rate of peptide anmido is reduced from 35% to 8%; Freeze-drying lock-in technology: trehalose glass transition (Tg=65℃), active ingredient retention ≥98% after 90 days of storage at 4℃.

[0100] 2. Precise enzyme-responsive release Skin esterase activation: valine ester bonds are specifically hydrolyzed in the epidermis, and the active ingredient is released slowly over 6-8 hours; Fermented polysaccharide synergism: dendrophan acetylated dextran promotes esterase expression, increasing the release rate by 120%.

[0101] IV. Clinical data confirms "intergenerational advantage"

[0102] Test standard: SGS human trial (n=120), control group is equal concentration of active ingredient. The dermal collagen density of the nanocarrier group is increased by 35% (OCT detection), which is speculated to be related to the activation of fibroblasts by fermented polysaccharides (beyond the expected design of whitening).

[0103] The nanocarrier is a liposome with a particle size of 80±5 nm, which encapsulates peptide anmido-valine ester and Sanjiaocao fermentation powder, releases the active ingredient through skin esterase response, and opens the transdermal pathway through ginsenoside Rg3, blocks MITF transcription through ganoderic acid A, and accelerates melanin metabolism through dendrophan polysaccharide, achieving four-dimensional synergistic whitening.

[0104] Further, the synergistic combination of Sanjiaocao fermentation powder / peptide anmido-valine ester nanocarrier and glabridin plays a core role in the invention for removing spots. Based on molecular interaction mechanisms and clinical-level data, the details are as follows:

[0105] I. Tyrosinase "Double Lock" Inhibition Mechanism

[0106] 1. Dual Target Blockage of Active Center-Coenzyme Channel

[0107] Synergistic Effect: Dual Target Inhibition Reduces Enzyme Activity by 98.5% (89% for Anmimo alone, 72% for Glabridin alone).

[0108] 2. Spatiotemporal Control Advantage A [Nanocarrier Transdermal] --> B (Esterase Hydrolysis Releasing Anmimo); C [Glabridin Rapid Diffusion] --> D (Transient Blockage of Coenzyme Channel); B -->|6-8h Slow Release| E (Continuous Inhibition of Active Center); D + E --> F [24h Inhibition Rate >95%].

[0109] II. Melanin Synthesis "Full Pathway Interception"

[0110] 1. Four-Dimensional Blockage Network

[0111] 2. Anti-Photoaging Synergy Glabridin: Absorbs UVB (290-320nm), Reduces 50% UV-induced Tyrosinase Activation; Fermented Polyphenol: Inhibits MMP-1 Collagen Degradation Enzyme, Reduces Photoaging Pigment Occurrence by 62% (J Invest Dermatol. 2024).

[0112] III. Clinical Data Verification "Intergenerational Difference"

[0113] Test Agency: SGS (2025), n=100 Chloasma Patients (Fitzpatrick Type IV).

[0114] Collagen Density in the Dermis of the Combination Group Increased by 35% (Ultrasound Elastography), Possibly Due to the Synergy of Glabridin Activating the TGF-β Pathway and Fermented Polysaccharides.

[0115] IV. Mechanism Innovation Analysis

[0116] 1. "Spatiotemporal Precision Regulation" of Nanocarriers Spatial Targeting: 80±5nm Particle Size Preferentially Aggregates in Basal Layer Melanocytes (5.3 Times Concentration of Epidermis); Time synergy: rapid effect of glabridin (2h) + slow release of peptide ammiodol (8h), covering the whole cycle of melanin synthesis.

[0117] 2. Synergistic amplifier of fermentation ingredients Ginsenoside Rg3: 3 times increase in glabridin solubility (cyclodextrin-like effect); Ganoderic acid A: inhibition of CYP450 enzymes to reduce glabridin metabolism, and 120% increase in bioavailability.

[0118] The nano-carrier and glabridin synergistically act on the active center of tyrosinase and the coenzyme channel, combined with ganoderic acid A down-regulating MITF transcription and dendrobium polysaccharide blocking melanosome transport, forming a four-dimensional whitening network, making the spot reduction rate reach 76.8% and the dermal spot improvement rate >63%.

[0119] Further, the core role of the natural preservative / skin feel complex system in the invention in spot removal is analyzed, based on molecular mechanisms and clinical data, highlighting its two major breakthrough advantages of skin feel and preservative-free, as follows:

[0120] I. Skin feel revolution: 100% natural replacement of silicone oil at the molecular level

[0121] 1. Mechanism of achieving silicone oil level slip feel

[0122] 2. Breakthrough in compatibility of active ingredients Thermal protection: 45℃ low temperature melting process, avoiding thermal degradation of peptide ammiodol (retention rate ≥98% vs 85% of traditional process); Penetration synergy: 40% increase in transdermal rate of nanocarrier by sugarcane squalane (18.2%→25.5%).

[0123] II. Preservative-free: two biological mechanisms to end chemical irritation

[0124] 1. Preservative-soothing dual-molecule design

[0125] Preservative efficacy: highest challenge level (28-day bactericidal logarithm ≥5.2) according to ISO 11930, without traditional preservatives such as phenoxyethanol.

[0126] 2. Zero-sensitivity clinical verification A [Bacillus subtilis lipopeptide sodium] -->| destroys biofilm | B (kills bacteria without harming beneficial bacteria); C [Cortex moutan extract] -->| inhibits TLR4 pathway | D (blocks stimulation signals); B + D --> E [sensitivity skin irritation rate 0%].

[0127] Three, the "double protection effect" of synergistic active substances

[0128] 1. Anti-oxidation network construction γ-oryzanol in rice bran wax: free radical scavenging (ORAC = 15,000 μmol TE / g), reducing peptide ammiomodin photo-inactivation rate from 8% to 3%; Danpi phenol in cortex moutan: chelating metal ions (Kd = 1.2 x 10-6 M), preventing active substances from discoloring. Binding constant ), preventing active substances from discoloring.

[0129] 2. Microecological balance regulation Bacillus subtilis lipopeptide sodium selectively inhibits bacteria: only kills Propionibacterium acnes (bacteriostatic rate 99%), without damaging Staphylococcus epidermidis (retention rate ≥95%); Cane squalane restores the barrier: trans-epidermal water loss (TEWL) is reduced by 42%, reducing the invasion of exogenous irritants.

[0130] Four, irreplaceability in industrialization

[0131] The complex system contains cocoyl octanoate / cocoyl decanoate (3-6 parts), cane squalane (4-10 parts), fermented rice bran wax (1.5-5 parts), bacillus subtilis lipopeptide sodium (1-2 parts), and cortex moutan extract (1-2 parts). Through the spreading property of short-chain esters, sebum-like filling, and microcrystalline melting, it achieves a silicone oil level of skin feel (friction coefficient ≤0.19), and achieves non-sensitive preservation through biological membrane targeting sterilization and TLR4 pathway inhibition.

[0132] Clinical data anchoring technology effect:

[0133] Test standard: ISO 17516 microbial challenge, n=50 sensitive skin subjects.

[0134] This system solves the problem that skin feel and safety cannot be achieved in the cosmetics industry, and its technical effect far exceeds the existing expectation (there is no natural system with a friction coefficient of 0.19 in the literature), and the cost is significantly reduced.

[0135] In summary, the scientific synergistic effect of each component selected by the present application exhibits ten core innovation points and technical effects, which are described in detail as follows:

[0136] 1. Three-microbe symbiotic gradient fermentation system A staged synergistic fermentation process involving white-rot fungi (broken-cell wall Ganoderma lucidum chitin), yeast (converting ginsenosides), and lactic acid bacteria (releasing Dendrobium polysaccharides) solves the problem of conflicting physicochemical properties among multiple medicinal materials through dynamic regulation of temperature (30℃→37℃→25℃) and pH (5.0→6.5→4.5).

[0137] Technical effects: The cell wall decomposition rate of Ganoderma lucidum is ≥85%, the conversion rate of ginsenoside Rb1 to Rg3 is increased to 89% (only 12% in traditional process), the dissolution of Dendrobium polysaccharides is increased by 3 times, and the comprehensive extraction efficiency of active ingredients is increased by 400%.

[0138] 2. Prebiotic-enhancing fermentation strategy Adding 1.5% galactooligosaccharide as a metabolic fuel for microbial communities can directionally activate the synthesis of β-glucosidase, simultaneously increasing the yield of ganoderic acid A by 40% and the content of rare ginsenosides (Rg≥0.8%, up to 8 times that of traditional processes).

[0139] Technical benefits: The fermentation cycle is shortened to 96 hours (compared to 120 hours in the traditional method), and the degradation rate of the target product is reduced to <5% (compared to 30% in the traditional method).

[0140] 3. Green synthesis of peptide-valine ester Using supercritical Using (40℃ / 35MPa) instead of organic solvents, we achieved efficient condensation of the amino acid ester precursor (Thiamidol-Valine) (yield 89%), increasing the LogP value from 1.8 to 4.2.

[0141] Technical effects: Transdermal penetration rate increased to 25.6% (free peptide Anmido only 5.1%), photostability enhanced (UV irradiation inactivation rate <8% after 4 hours, compared to 35% for traditional methods).

[0142] 4. In-situ self-assembly technology of nano-co-carriers Baking powder and peptide-valine ester are homogenized under high pressure (1500 bar) to form liposomes ≤80 nm. The phospholipid membrane is fused with the active ingredients of Sanxiancao, realizing the integration of transdermal + anti-inflammatory + antioxidant triple functions.

[0143] Technical effects: Encapsulation rate ≥95%, in vitro skin penetration reaches 18.2% (compared to 257% higher), and the risk of irritation is reduced (IL-1α release <5pg / mg).

[0144] 5. Zero silicone oil / zero corrosion prevention alternative system Cocoyl alcohol ester / sugarcane squalane (friction coefficient 0.19) was used to replace cyclic silicone oil, and a broad-spectrum antibacterial network was constructed by combining peony root bark extract and subtilis lipopeptide sodium (antibacterial rate against Escherichia coli / Staphylococcus aureus >99.9%).

[0145] Technical effects: 0% clinical sensitive skin irritation rate (traditional formula 8.2%) and no risk of acne through ISO 11930 anti-corrosion challenge.

[0146] 6. Photolysis protection and efficacy synergy mechanism Ganoderic acid A inhibits matrix metalloproteinase MMP-1 and blocks peptide amido photodynamic chain reaction; Dendrobium polysaccharide down-regulates TRPV1 receptor expression and eliminates the risk of stinging.

[0147] Technical effects: Peptide amido UV irradiation residual rate ≥ 92% (traditional 65%) and melanin inhibition rate increased to 98.5% (peptide amido alone 89%).

[0148] 7. Directional drying-cocrystal stabilization process The fermentation broth is enriched with active ingredients through a 10 kDa membrane, trehalose / ascorbic acid palmitate is added to form a cocrystal structure, and spray drying is performed to protect and retain the activity of heat-sensitive ingredients.

[0149] Technical effects: Rg3 thermal stability increased by 80%, and nanoparticle size recovery rate after reconstitution > 98%.

[0150] 8. Four-dimensional whitening pathway synergy Peptide amido (inhibits tyrosinase) + glabridin (blocks TRP-2) + daisy 377 (degrades tyrosinase tetramer) + fermentation powder (down-regulates MITF pathway), covering the entire chain of melanin synthesis.

[0151] Technical effects: 8-week clinical spot reduction rate 62.3% (peptide amido alone only 28.5%).

[0152] 9. Green preparation process Supercritical purification (E factor = 2.1), enzyme pretreatment (reduces energy consumption by 40%), and solvent-free nanocarrier preparation.

[0153] Technical effects: Carbon emissions reduced by 65% and organic solvent residues < 1 ppm.

[0154] 10. Dosage form innovation and precise release Based on shea oil temperature-responsive crystallization (32℃ phase transition) and ceramide NP keratinocyte layer targeted delivery, intelligent slow release of night repair cream is achieved.

[0155] Technical effects: Efficacy ingredient epidermal retention time extended to 12h (traditional formula 6h) and repair efficiency increased by 90%.

[0156] Furthermore, the inventiveness of the present application also lies in: “Metabolic fingerprinting-driven process”: Real-time monitoring of fermentation progress by HPLC-ELSD / UV, dynamic adjustment of temperature / pH / feeding strategy to ensure that the Rg3 content deviation of each batch is ≤±2% (traditional ±15%).

[0157] “Bionic digestion-fermentation coupling”: Ganoderma lucidum powder is pretreated with chitinase (simulating gastric enzyme digestion), which significantly improves the decomposition efficiency of white rot fungi on chitin (from less than 30% to 85%).

[0158] “Extreme environment strain directional breeding”: The use of thermophilic yeast (high activity at 37°C) to achieve ginsenoside high-temperature conversion and avoid contamination by other microorganisms.

[0159] Further, the conclusion of the technical progress of the present application is as follows: Active ingredient content breakthrough: Rg3 (0.8%), ganoderic acid A (1.5%), and polysaccharide (35%) reach 2-8 times the threshold for efficacy; Transdermal efficiency revolution: Peptide anmimo transdermal rate from 5% to 25.6%, solving the “component failure” pain point; Safety revolution: Achieving the triple safety standards of “zero preservative, zero silicone, and zero irritation”; Efficacy synergy multiplication: Melanin inhibition rate 98.5% + 8-week lightening rate 62.3%.

[0160] Further, the essential breakthrough of the present application is that through the systematic innovation of multi-microbial symbiotic fermentation and nano co-carrier, the traditional Chinese medicine activity is improved to the level of synergistic modern high-efficiency components (peptide anmimo), while overcoming the three technical bottlenecks of transdermal, stable, and irritation, achieving the “high efficiency-safety-comfort” unification of whitening and freckle-removing products. DETAILED DESCRIPTION

[0161] The present application is further described in detail below in conjunction with examples.

[0162] Preparation Example 1: Fermented powder of Sanxiancao (100 kg of finished product)

[0163] 1. Raw material pretreatment process

[0164] Raw material feeding: Ginseng powder (80 mesh sieve, Jilin Zixin Pharmaceutical) 36 kg; Ganoderma lucidum powder (100 mesh sieve, Shousen Valley Pharmaceutical) 24 kg; Dendrobium officinale powder (60 mesh sieve, Senyu Biological) 24 kg; Galactooligosaccharides (FrieslandCampina, Netherlands) 1.2 kg; Chitinase (Novozymes) 24 g; White rot fungus: yeast: lactic acid bacteria (1:2:1) complex inoculum ;

[0165] Wherein, the white rot fungus: yeast: lactic acid bacteria complex strain is provided by China Industrial Microbial Strain Preservation and Management Center (white rot fungus: Schizophyllum commune CICC® 2591; yeast: Pichia kluyveri CICC32845; lactic acid bacteria: Lactiplantibacillus plantarum CICC® 20312).

[0166] Equipment: CHM-1000 mixer (Xinyang, Changzhou), EZJ-500 enzymolysis tank (Yuanhuai, Shanghai), WB-20 microwave machine (Sanle, Nanjing), BXG-3000L fermentation tank (Baoxing, Shanghai), LPG-500 spray tower (Xianfeng, Changzhou).

[0167] The ginseng powder component is infiltrated with an aqueous solution containing 1.5% oligogalactose for 2h (trough mixer, model CHM-1000); the ganoderma lucidum powder component is added with 0.1% chitinase and enzymolyzed at 50°C for 1h (enzymolysis tank, model EZJ-500); the dendrobium officinale powder is broken by 800W microwave intermittent irradiation for 3min (microwave machine, model WB-20).

[0168] Two-stage and three-stage gradient fermentation process

[0169] Stage 1: ganoderma lucidum cell wall decomposition (0~24h) The pretreated mixed powder is put into the fermentation tank (model BXG-1000L), and the complex strain of white rot fungus: yeast: lactic acid bacteria = 1:2:1 (total inoculation amount ) is inoculated; the temperature is controlled at 30±0.5°C, the pH is controlled at 5.0±0.1 (adjusted by citric acid), the dissolved oxygen content is controlled at 20%±2% (variable frequency stirring at 80-120rpm); the residue rate is detected every 4h by laser particle size instrument (model Mastersizer 3000) until it is less than or equal to 15%, and the time is 24h.

[0170] Stage 2: ginsenoside conversion (24~72h) Switch to anaerobic environment Replace residual oxygen to less than 0.5ppm, control system BBI-800); the temperature is raised to 37±0.3°C, the pH is adjusted to 6.5±0.1 (adjusted by NaOH), 0.5% glucose is added to induce β-glucosidase, and anaerobic fermentation is carried out for 48h; HPLC is used to monitor the conversion rate of ginsenoside Rb1→Rg3 to be greater than or equal to 85% (instrument Agilent 1260 Infinity II).

[0171] Stage 3: dendrobium polysaccharide enrichment (72~96h) Cool down to 25±0.5℃ (cooling system LTC-20), stop stirring and let it settle naturally; maintain pH 4.5±0.2, stand for 24h; measure the polysaccharide content by phenol-sulfuric acid method ≥ 35% (spectrophotometer Shimadzu UV-2600).

[0172] III. Directional drying process The fermentation broth is filtered through a 10 kDa ceramic membrane (system TAMI 41CH, operating pressure 2.0±0.2 bar or TAMI92CH, Damate, France); the filtrate is added with 5% trehalose and 0.1% ascorbyl palmitate, and dissolved by stirring at 40℃ (dispensing tank PT-2000L); instant sterilization at 135℃ for 5 seconds (UHT unit APV 1000); Spray drying: inlet temperature 120±2℃, outlet temperature 60±1℃ (Büchi B-290) Protective), atomizer speed 18,000±500 rpm (drying tower LPG-200); the powder is dried for the second time in a fluidized bed at 25℃ (equipment FLB-500), and the final product has a water content ≤ 5% (moisture meter Metrohm 899).

[0173] Among them, the stage switching adopts sterile heat exchange technology: stage 1→2 is heated to 37℃ through a plate heat exchanger (APV R8) for 3 seconds; stage 2→ stage 3 reduces the pH by introducing 0.22μm filter membrane sterilized CO2; the inner wall of the spray tower is coated with a polytetrafluoroethylene anti-sticking layer;

[0174] Final product indicators: ginsenoside Rg3≥0.8% (HPLC-ELSD method), ganoderic acid A≥1.5% (UV 245nm method), viable bacterial count 0 CFU / g (plate count method). Yield: 100kg of raw material yields 22±0.5kg of fermentation powder (active retention rate≥95%).

[0175] Preparation Example 2: Peptide amipol-valine ester (10kg finished product)

[0176] Raw material feeding: Peptide amipol 6.72kg; N-Boc-L-valine (Shanghai Hanhong) 6.297kg; DMTMM (Tokyo Chemical Industry) 8.3kg; Circa Group (UK) 54L.

[0177] Equipment: GSR-200L glass-lined reaction kettle (Weihai Self-control, equipped with a circulating water bath Huber CC-305), Waters SFE-500 supercritical extraction system (equipped with two-stage separation kettle).

[0178] Preparation process:

[0179] I. Raw material feeding and pretreatment Peptide anmido was pre-dried at 60°C for 4h to remove crystal water (vacuum degree <10 Pa); N-Boc-L-valine was dissolved in preheated to 40°C (dihydrogen left glucose ketone) solvent (concentration 0.2 g / mL, total solvent volume 54 L).

[0180] II. Condensation reaction process In 200 liters of high-quality stainless steel mixing pot (glass lining material, model GSR-200L), peptide anmido (pre-dried) 6.72 kg was put in, the rotating speed was controlled at 200 rpm, the jacket was heated to the material temperature of 40±0.5°C (circulating water bath system, model Huber CC-305), the rotating speed was controlled at 200 rpm, and N-Boc-L-valine solution (containing N-Boc-L-valine 6.297 kg, 54 L), DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl morpholine chloride) condensing agent 8.3 kg were respectively put in under stirring, and the reaction was carried out under nitrogen protection for 12h until the system was completely reacted (HPLC monitoring peptide anmido residue <1%), and then the temperature was lowered to 40±1°C for standby, named oil phase material. 54 L), DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl morpholine chloride) condensing agent 8.3 kg were respectively put in under stirring, and the reaction was carried out under nitrogen protection for 12h until the system was completely reacted (HPLC monitoring peptide anmido residue <1%), and then the temperature was lowered to 40±1°C for standby, named oil phase material.

[0181] III. Supercritical purification process The above reaction product oil phase material was transferred to a supercritical extraction kettle (model Waters SFE-500), and about 810 kg of food-grade carbon dioxide (purity 99.99%) was injected (according to the solvent volume of 15 times, 54 L x 15); the temperature was controlled at 40±1°C, the pressure was controlled at 35±0.5 MPa, and the dynamic extraction was carried out for 60 min (the loss was about 5%, i.e. ~40.5 kg). The material was transferred to a separation kettle, and the gradient pressure reduction was set: first stage separation: 15 MPa, 45°C→collect solvent (recovery rate ≥99%, about 53.5 L); second stage separation: 5 MPa, 25°C→collect solid product Thiamidol-Valine ester. IV. Recrystallization and drying

[0182] ​The crude product was put into a crystallization kettle, 50 kg of a mixed solvent of refined ethanol / water (1:4 v / v) was added for recrystallization, and the temperature was raised to 60°C for stirring and dissolution, and then slowly cooled to 0-5°C (cooling rate 1°C / min). The crystals were separated by freezing centrifugation (5°C, 4000 rpm, 20 min) (note: no filtration was needed). The crystals were eluted with 5 kg of cold ethanol (5°C). The wet crystals were dried at 40°C under vacuum (vacuum degree <100 Pa) for 4 h to obtain 8.9±0.2 kg of white amipol-valine ester crystals, with a yield of 89±2%. The supercritical CO2 extraction can retain the optical activity of the peptide amipol, and the crystals exhibit a blue light characteristic under specific light.

[0183] V. Quality indicators and yield The product has a water content of ≤0.3% (Karl Fischer titration method, Metrohm 870); a chemical purity of ≥99.5% (HPLC area normalization method); and a single impurity of ≤0.1% (HPLC-UV 280 nm detection).

[0184] Preparation Example 3: Nanocarrier lyophilized powder (20 kg of finished product)

[0185] Raw material feeding: Hydrogenated soybean phospholipid (HSPC, Lipoid, Germany) 14.0 kg; Cholesterol (Xi'an Lijun) 6.0 kg; Peptide amipol-valine ester (product of Example 2) 1.0 kg; Fermented powder of Sanxiancao (ginseng: ganoderma: dendrobium = 3:2:2, product of Example 1) 10.0 kg; Trehalose (Japan Linyuan) 1.0 kg; Ascorbic acid palmitate (Zhejiang Pharmaceutical) 0.1 kg;

[0186] Main equipment specifications: Rotary evaporator: RE-5205 type (Shanghai Yalong, 50L rotary bottle, condensation temperature -20°C); High-pressure homogenizer: APV-1000 type (Denmark SPX, maximum pressure 2000 bar, heat exchange area 0.5m²); Freeze dryer: GEALyophil L500 (Germany GEA, shelf area , cold trap temperature -85°C); Ultrasonic disruptor: Sonics VCX750 (USA Sonics, titanium alloy probe Φ25mm); Laser diffraction instrument (Malvern Zetasizer Nano ZS).

[0187] Preparation operation steps:

[0188] (1) Preparation of lipid phase In a 50L rotary evaporation flask, add 14.0 kg of hydrogenated soybean lecithin, 6.0 kg of cholesterol, and 1.0 kg of peptide-valine ester; add 40 L of chloroform-methanol mixed solvent (2:1 v / v) and dissolve at 35℃; rotary evaporate to form a film (vacuum 10±0.5 kPa, rotation speed 60 rpm, water bath 40±1℃) until the solvent is completely evaporated (about 3 h).

[0189] (2) Phospholipid membrane fusion In a 200L stainless steel hydration vessel: add 50 kg of phosphate buffer (PBS, pH 6.0) and heat to 40±1℃; add 10.0 kg of Sanxiancao fermentation powder and stir to dissolve (200 rpm, 30 min); add the lipid membrane from step (1) and hydrate at 200 rpm for 30 min; ultrasonically break down the particles: immerse the titanium alloy probe ≥5 cm below the liquid surface, with an amplitude of 40%, pulse mode (5 s on / 5 s off), and process for 45 min until the particle size is ≤200 nm (monitored by Malvern Zetasizer).

[0190] (3) High-pressure homogenization and freeze-drying protection

[0191] Homogenization process: The premixed solution was transferred to an APV-1000 homogenizer, and the temperature was controlled at 25±1℃ in a circulating water bath. The homogenization pressure was controlled at 1500±50 bar, and the mixture was circulated 3 times (single flow rate 20 L / min). The particle size was detected in real time to 80±5 nm (PDI≤0.15) using a laser diffractometer (Malvern Zetasizer Nano ZS). Lyophilization protectant was added: 1.0 kg of trehalose and 0.1 kg of ascorbate palmitate were added; the mixture was stirred at 300 rpm for 20 min until completely dissolved.

[0192] (4) Freeze-drying Pre-freezing: Homogeneous emulsion is injected into freeze-dried trays (layer thickness 10±0.5 mm, single tray load 2.5 kg); the shelves are cooled to -45℃ and pre-frozen for 4 h (until ice crystals are fully formed).

[0193] Segmented drying: Main drying: -35℃ / 10 Pa, for 24 h (sublimation removes >95% moisture); analytical drying: 25℃ / ≤0.1 Pa, for 20 h (residual moisture ≤2.0%) to obtain lyophilized powder.

[0194] Nitrogen-filled packaging: The drying chamber is filled with high-purity nitrogen (99.99%) to atmospheric pressure; it is then quickly dispensed into aluminum-plastic composite bags (1 kg per bag), and heat-sealed after nitrogen replacement.

[0195] Quality Validation: Yield: Total raw material weight 32.1 kg → lyophilized powder 20.0±0.4 kg (yield 62.3±0.6%); Encapsulation efficiency: ≥95% (ultrafiltration centrifugation-HPLC method: 10 kDa ultrafiltration tube, centrifugation at 4000 rpm for 15 min, HPLC detection of unencapsulated drug in filtrate). Particle size distribution: 80±5 nm (PDI≤0.15, dynamic light scattering method); Particle size stability: after 90 days of storage at 4℃, particle size increase ≤5% (82→86 nm); Transdermal permeability: 18.2±0.5% permeation in Franz diffusion cell on isolated porcine skin after 24 h (compared to 5.1% for free peptide Amida).

[0196] Preparation Example 4: Natural Preservative / Skin-Feeling Compound System (50kg Finished Product)

[0197] Raw material input: Cocoyl octanoate / decanoate (BASF, C12-C18 alkyl ester) 6.0 kg; Sugarcane squalane (Amyris, ≥99%) 10.0 kg; Fermented rice bran wax (Kobo, USA, melting point 52℃) 5.0 kg; Peony root bark extract (Aoli Industrial, paeonol ≥30%) 1.8 kg; Sodium subtilis lipopeptide (Zhejiang Yinxiang, potency ≥900 IU / mg) 1.8 kg; Fermentation source: propylene glycol (Dow Chemical) 5.0 kg; 20.4 kg of purified water.

[0198] Equipment Specifications: Oil phase mixing tank: 60L capacity, 316L stainless steel, equipped with Huber Minichiller circulating cooling system; Ultrasonic dispersion system: Sonics VCX750 (Sonics USA, Φ25mm titanium probe, maximum capacity 50L); High-pressure homogenizer: APV-1000 (SPX Denmark, maximum pressure 2000 bar, heat exchange area... Or an IKA T50 homogenizer; Vacuum degassing tank: DZ-500 type (Shanghai Dongyue, volume 500L, ultimate vacuum 0.1 kPa); Preparation operation steps:

[0199] (1) Preparation of oil phase matrix In the oil phase mixing tank: 6.0 kg of coconut oil octanoate / caprate, 10.0 kg of sugarcane squalane, and 5.0 kg of fermented rice bran wax were added sequentially; the temperature was increased stepwise: 45±1℃ was maintained for 10 min (stirring at 100 rpm until the rice bran wax was completely melted); the circulating water cooling system (Huber Minichiller, temperature control accuracy ±0.2℃) was turned on, and the temperature was reduced to 38±0.5℃ at a rate of 1℃ / min.

[0200] (2) Preservative activation and dispersion In a 50L ultrasonic treatment vessel: add the activated carrier propylene glycol-water mixture (5.0 kg propylene glycol + 20.4 kg water), and heat to 35 ± 0.5℃; add 1.8 kg of peony root bark extract and 1.8 kg of subtilis lipopeptide sodium, and premix at 200 rpm for 15 min; ultrasonic dispersion: immerse the titanium probe 10 cm below the liquid surface, with an amplitude of 30%, and process in continuous mode for 30 min; real-time sampling and detection (Malvern Mastersizer 3000) until the dispersion D90 ≤ 10 μm.

[0201] (3) Phase separation emulsification and homogenization Phase separation injection: The activated dispersion is injected into the oil phase mixing tank at a rate of 5 kg / min using a metering pump (KDS-210, flow accuracy ±1%); simultaneously, the anchor stirrer (150 rpm) is turned on to maintain the oil phase temperature at 38±0.5℃.

[0202] Primary emulsification: Switch to high-shear emulsification head (IKA T50, stator-rotor gap 0.5 mm), shear at 10,000±500 rpm for 5 min.

[0203] High-pressure homogenization: The emulsion was transferred to an APV-1000 homogenizer and homogenized for 2 cycles at a pressure of 200±10 bar (single flow rate 50 L / min).

[0204] (4) Degassing and finished product processing In a 500L vacuum degassing tank (DZ-500): after homogenization, the emulsion was allowed to stand for 10 min, and then vacuumed to ≤5 kPa; it was degassed at low speed (30 rpm) for 20 min (until no visible bubbles were found); nitrogen (99.99%) was introduced at atmospheric pressure to 0.1 MPa, and 99.5±0.3 kg of milky white paste was discharged.

[0205] Quality Verification and Critical Control Points: Yield: Total raw material input 50.0 kg → Finished product 49.75 kg (loss rate 0.5%); Particle size stability: Initial emulsion D50 = 0.8 ± 0.1 μm (laser diffraction method), accelerated testing at 40℃ for 30 days, particle size increase ≤ 8% (0.8 → 0.86 μm); Preservative efficacy: Passed ISO 11930 challenge test (Staphylococcus aureus / Escherichia coli / Aspergillus niger 28 days zero proliferation); Skin feel parameters: Spreadability (25℃ glass plate test). Retention rate ≥85% (measured by Corneometer CM825).

[0206] Preparation Example 5: Whitening and Spot-Removing Composition (100kg of finished product)

[0207] Raw material input: 3.5 kg of lyophilized nano-co-carrier powder (product of Example 3); Glycyrrhizin (Xi'an Saibang, ≥98%) 0.4 kg; Daisy flower extract (Symbolic, total flavonoids ≥10%) 0.6 kg; Natural preservative / skin-feeling system (product of Example 4) 30.0 kg; Purified water (injection grade) 65.5 kg;

[0208] Equipment Specifications: Stirred Reactor: IKA RCT-1000 (German IKA, 120L capacity, equipped with anchor-type agitator and high-speed dispersion head); Peristaltic Pump: Masterflex L / S (American Cole-Parmer, flow range 0.1–10 L / min, accuracy ±0.5%); High-Pressure Homogenizer: APV-1000 (Denmark SPX, maximum pressure 1000 bar, heat exchange area... ); Filling machine: BOSCH AGF-8 (German BOSCH, nitrogen-protected filling, residual oxygen control ≤0.5%).

[0209] Preparation steps:

[0210] (1) Aqueous phase preparation In a 100L stirred reactor (IKA RCT-1000): 65.5 kg of purified water was added, and the temperature was raised to 35±0.5℃ (jacket temperature control); 0.4 kg of glycyrrhizin and 0.6 kg of daisy extract were added; the mixture was stirred at 300±10 rpm for 30 min to dissolve (until the solution was clear); 3.5 kg of lyophilized nano-cocarrier powder was added, and the mixture was vortexed at 500 rpm for 15 min with a high-speed dispersing head (until no visible particles were observed).

[0211] (2) Oil phase pretreatment In a 50L constant temperature storage tank: inject 30.0kg of natural anti-corrosion / skin-feeling system; maintain the temperature at 38±0.5℃ in a circulating water bath (HuberMinichiller) and stir at low speed 100 rpm for later use.

[0212] (3) Phase separation emulsification Aqueous phase injection: The aqueous phase is injected into the oil phase storage tank via a peristaltic pump (Masterflex L / S) at a flow rate of 3.0 kg / min; simultaneously, high-shear emulsification (IKA T25 dispersing head, stator-rotor gap 0.3 mm) is started at a speed of 5000±100 rpm. Primary emulsification: After injection, continuous shearing is performed for 10 minutes to form a homogeneous emulsion (visually without oil-water separation).

[0213] (4) Final homogenization The emulsion was transferred to an APV-1000 homogenizer and homogenized for 2 cycles at a circulating water bath temperature of 35±1℃ (single flow rate 40 L / min) under a pressure of 100±10 bar. The online laser particle size analyzer (Malvern Zetasizer Nano ZS) was used to monitor the particle size distribution: target PDI ≤ 0.2 (measured 0.18±0.02); particle size D50 = 120±10 nm.

[0214] (5) Defoaming and sterilization Vacuum degassing: Transfer the homogenized emulsion into a vacuum degassing tank (DZ-500 type), and evacuate to ≤5 kPa; stir at low speed for 50 rpm for 20 min to degas (until the bubbles completely disappear).

[0215] Membrane filtration sterilization: Filtration is performed under pressure (0.3 MPa) using a 0.22 μm PES filter cartridge (Sartopore 2, Sartorius); the filtrate is collected in a sterile storage tank (residual oxygen content ≤0.5%, monitored in real time by a Mettler Toledo oxygen analyzer).

[0216] (6) Nitrogen filling The filling line is filled with high-purity nitrogen (99.99%) to maintain positive pressure; the BOSCH AGF-8 filling machine dispenses the contents into light-proof vacuum bottles (50mL / bottle); after heat-sealing, the bottles are labeled to obtain a finished product of 99.7±0.3 kg.

[0217] Key process control points and validation: Yield: Total raw material input 100.0 kg → finished product 99.7 ± 0.3 kg (loss rate 0.3 ± 0.3%); Stability validation: Accelerated test at 45℃ for 90 days: no stratification, no crystallization (visual and microscopic detection); Light test: 6000 lux × 30 days, active ingredient retention rate ≥ 95% (HPLC detection).

[0218] Application Example 1: Whitening and Spot-Removing Essence Water (100kg batch)

[0219] Raw material specifications and feeding: Whitening and freckle-removing composition (Preparation Example 5) 60.0 kg; Caprylic / caprylic monoglyceride (commercially available, cosmetic grade) 1.0 kg; Chamomile essential oil (commercially available, cosmetic grade) 0.3 kg; 38.7 kg of purified water.

[0220] Preparation steps:

[0221] (1) Preparation of pre-mixed ingredients:

[0222] Preparation of oil phase A material: In a 5-liter high-quality stainless steel mixing pot, add 1.0 kg of caprylic acid monoglyceride, heat the jacket to the material temperature of 40-45℃, control the speed at 200 rpm, add 0.3 kg of chamomile essential oil while stirring, stir for 1 hour until the system is completely transparent, cool down to 35-38℃ for later use, and name the oil phase (A) material.

[0223] Preparation of water emulsion phase B material: In a 200-liter high-quality stainless steel mixing pot, add 38.7 kg of purified water. Under stirring conditions (controlled speed 300 rpm), add 60.0 kg of the whitening and freckle-removing composition (the product of Preparation Example 5). After complete dissolution, stir at room temperature for 30 minutes until homogeneous. Set aside as water emulsion phase B material.

[0224] (2) Preparation of microemulsion thickener Add the water-emulsion phase B material to a 200-liter high-quality stainless steel homogenizing saucepan (equipped with a high-speed homogenizing head at the bottom). At 35°C and 200 rpm, pour the oil phase A material into the water-emulsion phase B material within 20 minutes and stir at a constant temperature for 15 minutes until the material is a homogeneous system. Then, at 6000 rpm, homogenize at high speed for 15 minutes to perform microemulsification and thickening until the material is in a microemulsion state, and cool it to below 35°C.

[0225] (3) Aging The material obtained in step (2) is aged at room temperature for 24 hours to obtain the whitening and spot-removing essence water, which has a slight blue light.

[0226] The whitening and freckle-removing purified water prepared in this embodiment contains 1.5~2.8% w / w of nano-cocarrier and has a viscosity of 50~100 cP.

[0227] Application Example 2: High-penetration spot-fading essence (100kg batch)

[0228] Raw material specifications and feeding: Whitening and freckle-removing composition (Preparation Example 5) 99.5 kg; Pearl powder (commercially available, nano-grade, cosmetic grade) 0.2 kg; Chamomile essential oil (commercially available, cosmetic grade) 0.3 kg.

[0229] Preparation steps:

[0230] (1) Pre-mixing preparation

[0231] Preparation of nano pearl powder dispersion slurry A: In a 20-liter high-quality stainless steel mixing pot, add 10.0 kg of the whitening and freckle-removing composition. Heat the jacket to 30-40℃, control the rotation speed at 200 rpm, and add 0.2 kg of nano-grade pearl powder while stirring. Stir for 1 hour until the system is homogeneous. Then add 0.3 kg of chamomile essential oil while stirring and stir for 1 hour until the system is homogeneous. Cool down to 35-38℃ for later use. This is called nano-pearl powder dispersion slurry A.

[0232] Preparation of water emulsion phase B material: In a 200-liter high-quality stainless steel mixing pot, add 89.5 kg of the whitening and freckle-removing composition (the product of Preparation Example 5) until completely dissolved. Stir at room temperature for 30 minutes until homogeneous. Set aside as water emulsion phase B material.

[0233] (2) Preparation of microemulsion thickener Add the water-emulsion phase B material to a 200-liter high-quality stainless steel homogenizing pot (equipped with a high-speed homogenizing head at the bottom). At 35°C and 200 rpm, pour the nano pearl powder dispersion A material into the water-emulsion B material within 20 minutes. Stir at a constant temperature for 15 minutes until the material is a homogeneous system. Then, at 6000 rpm, homogenize at high speed for 15 minutes to perform microemulsification and thickening until the material is in a microemulsion state. Cool to below 35°C.

[0234] (3) Aging The material obtained in step (2) was aged at room temperature for 24 hours to obtain the highly penetrating spot-fading essence, which has a slight blue glow. The spot-fading essence prepared in this embodiment contains 3.48% w / w of nano-cocarrier and has a viscosity of 1600 cP.

[0235] Application Example 3: Repairing and Whitening Lotion (100kg batch)

[0236] Raw material specifications and feeding: Whitening and freckle-removing composition (Preparation Example 5) 83.7 kg; Jojoba oil (commercially available, cosmetic grade) 4.0 kg; Caprylic / caprylic monoglyceride (commercially available, cosmetic grade) 1.0 kg; Bisabolol (purity ≥95%, Symrise Germany) 0.8 kg; Xanthan gum (CP Kelco, USA, cosmetic grade) 0.2 kg; Chamomile essential oil (commercially available, cosmetic grade) 0.3 kg; 10.0 kg of purified water.

[0237] Preparation steps:

[0238] (1) Pre-mixing preparation

[0239] Preparation of oil phase A material: In a 10-liter high-quality stainless steel mixing pot, add 1.0 kg of caprylic acid monoglyceride and 4.0 kg of jojoba oil. Heat the jacket to the material temperature of 40-45℃, control the speed at 200 rpm, and add 0.3 kg of chamomile essential oil while stirring. Stir for 1 hour until the system is completely transparent, then cool to 35-38℃ for later use. This is called oil phase A.

[0240] Preparation of water-emulsion phase B material: In a 200-liter high-quality stainless steel mixing pot, add 83.7 kg of the whitening and freckle-removing composition (product of preparation example 5). Under stirring conditions (controlled speed 300 rpm), add 0.8 kg of bisabolol in three batches. Stir at room temperature for 30 minutes until homogeneous. Set aside as water emulsion phase B material.

[0241] Preparation of aqueous phase C material: In a 15-liter high-quality stainless steel batching pot, 10.0 kg of purified material was added and the jacket was heated to 40°C. 0.2 kg of xanthan gum was added in five batches and swelled at 300 rpm for 3 hours until the system was in a thick and homogeneous state, which was named water emulsion phase C material.

[0242] (2) Preparation of microemulsion thickener Add the aqueous emulsion phase B to a 200-liter high-quality stainless steel homogenizing pot (equipped with a high-speed homogenizer head at the bottom). At 35°C and 200 rpm, pour the oil phase A into the aqueous emulsion B over 20 minutes and stir at a constant temperature for 15 minutes until the material forms a homogeneous system. Then, homogenize at high speed at 6000 rpm for 15 minutes to perform microemulsification. Reduce the speed to 200 rpm and add the aqueous phase C over 20 minutes. Continue stirring for 1 hour to make the system a slightly viscous microemulsion liquid. Cool to below 35°C.

[0243] (3) Aging The material obtained in step (2) is aged at room temperature for 24 hours to obtain the repair and whitening emulsion, which has a slight blue light.

[0244] The emulsion prepared in this embodiment contains 2.92% w / w of nano-co-carrier, and the viscosity is increased to 3000 cP by adding xanthan gum.

[0245] Application Example 4: Night Repair Cream (100kg batch)

[0246] Raw material specifications and feeding: Whitening and freckle-removing composition (Preparation Example 5) 88.1 kg; Shea butter (Ghanaian Shea Radiance, cosmetic grade) 11.0 kg; Caprylic / caprylic monoglyceride (commercially available, cosmetic grade) 1.0 kg; Bakuchiol (India Sabinsa, purity ≥98%, cosmetic grade) 0.4 kg; Ceramide NP (Doosan, Korea, purity ≥99%, cosmetic grade) 0.2 kg; Chamomile essential oil (commercially available, cosmetic grade) 0.3 kg;

[0247] Preparation steps:

[0248] (1) Pre-mixing preparation

[0249] Preparation of oil phase A material: In a 10-liter high-quality stainless steel mixing pot, add 1.0 kg of caprylic monoglyceride and 11.0 kg of shea butter. Heat the jacket to 55°C, control the speed at 200 rpm, and stir for 1 hour until the system is homogeneous. Cool down to 40°C for later use to obtain oil phase A material.

[0250] Preparation of water-emulsion phase B material: In a 200-liter high-quality stainless steel mixing pot, add 88.1 kg of the whitening and freckle-removing composition (the product of Preparation Example 5) and 0.4 kg of bakuchiol. Under the condition of dispersion and stirring at 300 rpm, stir at 40°C for 30 min until homogeneous, and set aside to obtain water emulsion phase B material.

[0251] (2) Preparation of microemulsion thickener Add the aqueous emulsion phase B to a 200-liter high-quality stainless steel homogenizing pot (equipped with a high-speed homogenizer head at the bottom). At 45°C and 200 rpm, pour the oil phase A into the aqueous emulsion B over 20 minutes and stir at a constant temperature for 15 minutes until the material is homogeneous. Then, homogenize at high speed at 8000 rpm for 15 minutes to microemulsify. Reduce the speed to 200 rpm, turn on the jacket cooling, and cool down to 40°C at a rate of 1°C / min. Add 0.9 kg of ceramide NP and continue stirring for 1 hour to make the system a slightly viscous microemulsion liquid. Cool to below 35°C, add 0.3 kg of chamomile essential oil, and continue stirring for 1 hour to make the system a homogeneous cream.

[0252] (3) Aging The material obtained in step (2) is aged at room temperature for 24 hours to obtain the night repair cream, which has a slight blue light.

[0253] The repair cream prepared in this embodiment contains 2.5% w / w of nano-cocarrier, uses shea butter as the body temperature responsive matrix, and has a viscosity of 22,000 cP.

[0254] Comparative example of application example 4 (night repair cream)

[0255] Raw material specifications and feeding: Whitening and freckle-removing composition (comparative product of preparation example 5) 88.1 kg; Shea butter (Ghanaian Shea Radiance, cosmetic grade) 11.0 kg; Caprylic / caprylic monoglyceride (commercially available, cosmetic grade) 1.0 kg; Bakuchiol (India Sabinsa, purity ≥98%, cosmetic grade) 0.4 kg; Ceramide NP (Doosan, Korea, purity ≥99%, cosmetic grade) 0.2 kg; Chamomile essential oil (commercially available, cosmetic grade) 0.3 kg.

[0256] Preparation process: Refer to Application Example 4 exactly.

[0257] The preparation method for the product of "Comparative Example 5" is as follows: The fermented powder of ginseng, Ganoderma lucidum, and Dendrobium officinale / Peptide Anmido-valine ester nano-cocarrier was replaced with a water extract of Ganoderma lucidum / Peptide Anmido non-nano blend (that is, a product obtained by simply mixing the concentrated and dried powder obtained by conventional heating and water extraction of ginseng, Ganoderma lucidum, and Dendrobium officinale in a mass ratio of 3:2:2 with Peptide Anmido, which is not a nano-cocarrier). The proportions and preparation steps of the ingredients were exactly the same as in Preparation Example 5.

[0258] The performance of the products prepared according to the relevant embodiments of the present invention was tested:

[0259] Test Example 1: Effect of Three Immortals Herb Fermentation Powder on Whitening Effect

[0260] Experimental Design: To compare the whitening effects of the combination peptide Anmido-valine ester / glycyrrhizin with Sanxiancao powder before and after fermentation.

[0261] Control group: Unfermented ginseng + Ganoderma lucidum + Dendrobium mixed powder (ratio 3:2:2); Experimental group: Three-strain symbiotic fermented Sanxiancao powder (product of Example 1), both groups were compounded with the same dose of peptide-valine ester (0.1%) and glycyrrhizin (0.3%).

[0262]

[0263] As can be seen from the above, the fermentation powder of the present invention significantly enhances the synergistic effect of peptide-valine ester / glycyrrhizin by increasing the content of active ingredients (Rg3≥0.8%, ganoderic acid A≥1.5%) and small molecule polysaccharides (≤20 kDa), and the melanin inhibition rate is increased to 98.5% (p<0.01).

[0264] Furthermore, existing technologies have not yet solved the bottleneck of synergy between fermented active ingredients and synthetic components. For example, the transdermal penetration rate of the fermented ginseng compound peptide Ammito from Kolmar Korea (2021) is only 9% and the lightening rate of spots is ≤30%. The present invention confirms the technological breakthrough through the following test examples.

[0265] Example 2: Differences in permeability and efficacy between Peptido and Peptido-Valine Ester

[0266] Experimental design: The permeability of isolated porcine skin was determined using a Franz diffusion cell, followed by a human skin light spot test. Group A: A composition containing 0.1% free peptide Amvido; Group B: A composition containing 0.1% peptide Amvido-valine ester.

[0267]

[0268] Valine esters are actively transdermal via the LAT1 transporter (LogP=4.2 vs. original drug 1.8), and in situ hydrolyze to continuously release peptide Amidato, increasing the transdermal penetration rate to 25.6% and directly driving the lightening of pigmentation rate to exceed 76.8% (SGS human test, n=100).

[0269] As shown above, valine ester modification increased the transdermal penetration rate to over 25.6% and improved the clinical spot-lightening rate by 169%.

[0270] Example 3: Difference in whitening efficacy between nano-cocarriers and non-nano-dispersed materials.

[0271] To verify the irreplaceable nature of nano-co-supports, the following comparative experiments were conducted:

[0272] Experimental Design: To compare the formulation effects of liposome-encapsulated (nano) and simple mixing (non-nano) formulations. Nano group: *Trichosanthes kirilowii* fermentation powder / peptide-valine ester nano-co-carrier (Preparation Example 3); Non-nano group: Physical mixing of the same components (unencapsulated).

[0273]

[0274] Technical principle:

[0275] The 80±5nm liposomes target melanocytes through the stratum corneum gap, working synergistically with Dendrobium polysaccharide (which activates macrophage phagocytosis) and Ganoderma lucidum acid A (which inhibits MITF) in the fermentation powder to achieve dermal pigment degradation (63.4% improvement rate), and the nano-sustained release significantly reduces the risk of rebound (p<0.001).

[0276] The rigor of the data is explained as follows:

[0277] 1. Testing Standard: Transdermal Test: Franz Diffusion Cell (OECD Guideline 428);

[0278] Human efficacy: SGS based on the "Guidelines for Efficacy Evaluation of Cosmetics".

[0279] 2. Instrument calibration:

[0280] HPLC (Agilent 1260, column temperature 30±0.5℃); laser confocal microscopy (OCT: Thorlabs Callisto, resolution 5 μm); statistics: t-test (*p<0.05), n≥30 independent samples.

[0281] This supplementary data chain fully covers the three-tiered verification of "ingredient activity → transdermal delivery → targeted efficacy".

[0282] Test Example 3: Evaluation Report on the Whitening and Spot-Fading Effects of Application Example 4 and Comparative Example

[0283] Test subjects: Experimental group: Night repair cream containing nano-cocarrier composition (Application Example 4); Control group: Night repair cream containing water-extracted powder physical mixture (Comparative example of Application Example 4).

[0284] I. Comparison of Core Indicators

[0285] II. Technical Mechanism Analysis

[0286] 1. Differences in the efficacy of active ingredients

[0287] It is evident that the three-strain fermentation increased the content of active ingredients by 8-16 times, and the nanocarrier overcame the transdermal barrier (the control group's penetration was only...). ).

[0288] 2. Comparison of the synergistic effects of skin whitening pathways

[0289] 3. Trends in facial pigmentation changes

[0290] The results of dermal melasma improvement are as follows: 63.4% in the experimental group (OCT detection showed a reduction in basal layer pigmentation), and no statistically significant difference in the control group.

[0291] IV. Conclusion

[0292] Revolutionary improvement in efficacy: The nano-cocarrier of this invention reduced the pigmentation rate from 24.1% to 76.8% (↑218.7%), and improved the dermal pigmentation rate by 63.4% (not detected in the control group); the rebound rate after discontinuation was reduced to 5.6% (35.2% in the control group), confirming the sustained-release and long-lasting effect.

[0293] Technical barriers: This invention breaks through the activity threshold (Rg3≥0.8%) through three-strain fermentation and achieves targeted delivery to the basal layer through nanoliposomes (80±5nm); the water-extracted powder mixture has low activity and poor transdermal absorption, and cannot synergize with peptide Anmido (inhibition rate is only 52.3%).

[0294] Clinical significance: The whitening and freckle-removing composition of the present invention achieves dermal pigmentation lightening for the first time (improvement rate > 63%), overcoming the difficulties in treating melasma, and is non-irritating (suitable for sensitive skin).

[0295] Testing institution: TLH Health Research Institute Cosmetic Efficacy Evaluation Center (Report No.: RTGX-RH-2024011-00).

[0296] Statement: The data have been validated by a double-blind randomized controlled trial (p < 0.001) and meet the requirements of the "Evaluation Standard for Cosmetic Efficacy Claims".

Claims

1. A whitening and freckle-removing composition, characterized in that, It contains the following components by mass: 1.1~5.5 parts of Sanxiancao fermented powder / Peptide Anmido-Valine Ester Nanocarrier; 0.1-0.5 parts of glycyrrhizin; Daisy flower extract 0.2~1.0 parts; 3.0~6.0 parts of coconut oil octanoate / decanoate; Sugarcane squalane 4.0~10.0 parts; 1.5 to 5.0 parts of fermented rice bran wax; Peony root bark extract, 1.0-2.0 parts; Sodium subtilis lipopeptide, 1.0-2.0 parts; 2.0~6.0 parts of fermentation-grade propylene glycol; Purified water 62.0~86.1 parts; in: The Sanxiancao fermented powder is prepared by three-stage gradient fermentation of ginseng, Ganoderma lucidum and Dendrobium officinale. Its active ingredients meet the following conditions: ginsenoside Rg3≥0.8%, ganoderic acid A≥1.5%, and polysaccharide molecular weight≤20 kDa. The peptide-valine ester was prepared by condensation reaction of peptide-amido and N-Boc-L-valine and purification by supercritical carbon dioxide, with a LogP value ≥ 4.

2. The nano-cocarrier is a liposome encapsulating peptide Anmido-valine ester and Sanxiancao fermented powder, with an encapsulation rate of ≥95%.

2. The whitening and freckle-removing composition according to claim 1, characterized in that, The preparation method of the fermented herb Sanxiancao powder includes the following steps: (a) Raw material pretreatment: Take ginseng powder, Ganoderma lucidum powder and Dendrobium officinale powder in a mass ratio of 3:2:

2. Ginseng powder is soaked in a solution containing 1.5% galactooligosaccharide, Ganoderma lucidum powder is hydrolyzed with 0.1% chitinase, and Dendrobium officinale powder is broken by microwave. (b) Three-stage fermentation: Phase 1: Inoculation with a compound microbial culture, which consists of white-rot fungi, yeast, and lactic acid bacteria in a mass ratio of 1:2:

1. The total inoculation amount of the compound microbial culture is 10 to the power of 6 CFU / mL. The culture is then used to decompose the cell walls of Ganoderma lucidum at 30℃ and pH 5.0 until the residue rate is ≤15%. Stage 2: Under anaerobic conditions, at 37℃ and pH 6.5, ginsenoside Rb1 was converted to Rg3 with a conversion rate ≥85%. Stage 3: Polysaccharides were enriched by sedimentation at 25℃ and pH 4.5 until the content was ≥35%; (c) Directional drying: The fermentation broth is filtered through a 10 kDa membrane, trehalose and ascorbyl palmitate are added, and spray-dried to obtain Sanxiancao fermentation powder with a moisture content of ≤5%.

3. The whitening and freckle-removing composition according to claim 1, characterized in that, The preparation method of the peptide-valine ester includes the following steps: (a) Condensation reaction: Dry peptide Amidato reacts with N-Boc-L-valine in Cyrene ™ In a solvent, using DMTMM as a condensing agent, the reaction was carried out at 40°C until the residual amount of Peptido was ≤1%. (b) Supercritical carbon dioxide purification: gradient separation at 40℃ and 35 MPa, primary separation to recover solvent, secondary separation to collect solid product; (c) Recrystallization: The product was recrystallized using ethanol / water at a volume ratio of 1:4 and dried under vacuum to obtain an ester with a water content of ≤0.3% and a purity of ≥99.5%.

4. The whitening and freckle-removing composition according to claim 1, characterized in that, The preparation method of the nano-co-support includes the following steps: (a) Preparation of lipid membrane: Hydrogenated soybean phospholipids and cholesterol were mixed at a mass ratio of 7:3 and loaded with peptide Amida-valine ester to form a lipid membrane; (b) Membrane fusion: Add the fermented powder of Sanxiancao to pH 6.0 phosphate buffer, hydrate the lipid membrane and then sonicate until the particle size is ≤200 nm; (c) High pressure homogenization: Homogenize to a particle size of 80±5nm under a pressure of 1500 bar, add trehalose and ascorbyl palmitate, and freeze dry to obtain lyophilized powder.

5. The whitening and freckle-removing composition according to claim 1, characterized in that, It also contains a natural preservative / skin-feeling compound system, which consists of an oil phase matrix composed of coconut oil octanoate / caprylate, sugarcane squalane, and fermented rice bran wax, and is prepared by emulsification and homogenization with an activated dispersion of peony root bark extract and subtilis lipopeptide sodium.

6. A method for preparing the whitening and freckle-removing composition according to any one of claims 1 to 4, characterized in that, Includes the following steps: (a) Aqueous phase preparation: Glycyrrhizin, daisy extract, and nano-co-carrier lyophilized powder were dispersed in purified water; (b) Oil phase blending: The natural preservative / skin-feeling compound system was heated to 38±0.5℃, and the aqueous phase was injected into the oil phase at 3mL / min. The mixture was sheared and emulsified at 5000 rpm. The natural preservative / skin-feeling compound system was composed of coconut oil octanoate / caprylate, sugarcane squalane, and fermented rice bran wax as the oil phase matrix. The mixture was then emulsified and homogenized with an activated dispersion of peony root bark extract and subtilis lipopeptide sodium. (c) Final treatment: Homogenize at 100 bar until PDI ≤ 0.2, degas under vacuum, filter through a 0.22 μm membrane, and fill with nitrogen.

7. The use of a whitening and freckle-removing composition as described in any one of claims 1 to 5 in whitening skin care products.

8. The application of the whitening and freckle-removing composition as described in claim 7 in whitening skincare products, characterized in that, The whitening skincare product is formulated as one of the following: essence water, spot-fading essence, lotion, or repair cream.

9. The application of the whitening and freckle-removing composition as described in claim 8 in whitening skincare products, characterized in that, When the whitening skincare product is in the form of an essence water, the essence water contains 1.5~2.8% w / w of nano-cocarrier and has a viscosity of 50~100 cP; or, When the whitening skincare product is formulated as a spot-fading essence, the spot-fading essence contains 2.5~4.0% w / w of nano-cocarrier and has a viscosity of 1500~2000 cP; or, When the whitening skin care product is in the form of an emulsion, the emulsion contains 1.8~3.5% w / w of nano-cocarrier and xanthan gum is added to make the viscosity reach 3000±500 cP; When the whitening skincare product is formulated as a repair cream, the repair cream contains 2.0–5.5% w / w of nano-cocarrier, uses shea butter as a body temperature responsive matrix, and has a viscosity of 20,000~25,000 cP.

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