Plant extract composition for removing and fading freckles as well as preparation method and application of plant extract composition

Abstract

The invention provides a plant extract composition for removing and fading freckles as well as a preparation method and application thereof, and relates to the technical field of cosmetic raw materials and preparation. The composition comprises plant raw materials such as ginseng and pseudo-ginseng, leech polypeptide, stiff silkworm extract and the like, and plant extraction components such as roxburgh rose fruit fermentation extract and the like can also be added. Wherein the leech polypeptide is prepared by complex enzyme enzymolysis and ultrafiltration purification; extracts such as bombyx batryticatus are obtained through ultrasonic-assisted extraction. The preparation method comprises the steps of grouping pretreatment, nanocrystallization treatment, gradient mixing emulsification and the like, and the stability and transdermal efficiency of active ingredients can be improved; results show that the composition can inhibit tyrosinase activity and melanogenesis and is good in transdermal effect. The freckle removing and fading composition can be applied to preparation of freckle removing and fading cosmetics or drugs and has good efficacy and application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetic raw materials and preparation thereof, and in particular to a plant extract composition for removing and lightening freckles, and a preparation method and application thereof. Background Art

[0002] Pigmentation is a pigmentation disorder caused by abnormal melanin deposition in the epidermis or dermis. Its formation is linked to multiple factors, including UV exposure, endocrine disorders, and inflammation. UV rays activate tyrosinase in melanocytes, converting tyrosine into melanin. Endocrine disorders affect hormone levels, stimulating melanin synthesis. Inflammation accelerates melanin production and transport through the release of cytokines. Currently, there are numerous issues with freckle removal and lightening products on the market. While chemically synthesized products can quickly remove freckles, their safety is questionable. For example, long-term use of hydroquinone-based products can easily cause skin irritation, allergies, and even lead to side effects such as depigmentation. Natural extract-based products are safer, but their effectiveness is often suboptimal. Most products rely on a single ingredient, targeting only a specific step in melanin synthesis, making it difficult to achieve comprehensive freckle removal and lightening effects. Furthermore, their melanin inhibition rate is low, typically less than 30%. In terms of preparation technology, traditional methods also have obvious defects. On the one hand, the extraction process is rough, such as simple water decoction or ethanol soaking, which cannot fully extract the effective ingredients in the raw materials and may also destroy some active substances, resulting in reduced efficacy of the extract. On the other hand, the existing mixing process is unreasonable, the ingredients are not mixed evenly, and agglomeration is prone to occur, which affects the stability of the product. In addition, the active ingredients have low transdermal efficiency and are difficult to penetrate the skin barrier to reach the site of action, and cannot fully exert their efficacy. At the same time, many products are prone to stratification and precipitation during storage, and the retention rate of active ingredients is low, further weakening the freckle removal effect. Furthermore, existing products have a narrow application range, failing to meet the needs of different skin types and different types of pigmentation. Furthermore, industrial production is challenging, hindering their widespread use in cosmetics and pharmaceuticals. Therefore, the development of a freckle-removing and lightening product that is effective, safe, reliable, and has optimized process and widespread application is an urgent need. Summary of the Invention

[0003] In view of this, the present invention proposes a plant extract composition for removing and lightening spots, as well as a preparation method and application thereof, to solve the above problems.

[0004] The technical solution of the present invention is achieved as follows: a plant extract composition for removing and lightening spots, comprising the following raw materials in parts by weight: 3-8 parts of ginseng, 2-6 parts of Panax notoginseng, 4-10 parts of persimmon leaves, 1-4 parts of safflower, 2-5 parts of peach kernel, 3-7 parts of red peony root, 1-3 parts of leech polypeptide, 2-5 parts of bombyx batryticatus extract, 3-8 parts of turmeric, and 1-3 parts of ergothioneine.

[0005] Furthermore, the leech polypeptide is prepared by taking dried leeches, crushing them, and then passing them through a 60-80 mesh sieve. The corresponding weight portion of leech powder is weighed, deionized water is added at a mass ratio of 1:15-20 of leech powder to water, and the mixture is stirred evenly, and the pH is adjusted to 2.0-3.0. Pepsin is added at a concentration of 1.0-2.0% by weight of the powder, and the enzyme activity is ≥3000 U / g. After constant temperature enzymolysis at 37-40°C for 4-6 hours, the pH of the mixture is adjusted to 7.0-7.5. , add a composite enzyme preparation with a mass ratio of 0.8-1.5% of leech powder, and continue enzymatic hydrolysis at 35-38°C for 3-4 hours. The composite enzyme preparation is plasmin and carboxypeptidase B in a mass ratio of 1:1-2, and the enzyme activity is ≥3000U / g. After cooling to room temperature, centrifugation is performed, and the supernatant is ultrafiltered and purified through an ultrafiltration membrane with a molecular weight cutoff of 3000-5000Da. The permeate is collected and concentrated under reduced pressure at 50-60°C until there is no ethanol taste, thereby obtaining leech polypeptide.

[0006] Furthermore, the silkworm extract is prepared by taking dried silkworm, removing impurities and then crushing to obtain silkworm powder, mixing the powder and 70-80% v / v ethanol solution in a mass volume ratio of 1:10-15, stirring evenly and soaking the mixture at 25-30°C for 2-3 hours, then placing the mixture in a 55-65°C water bath, using ultrasonic assisted extraction with an ultrasonic power of 250-350W and an extraction time of 40-50 minutes. After the extraction is completed, filtering is performed, collecting the filtrate, and concentrating under reduced pressure to obtain the silkworm extract.

[0007] Furthermore, the following raw materials are included in parts by weight: 0.8-1.5 parts of roxburghii fruit fermentation extract, 1.2-1.5 parts of vitex rotundifolia fruit extract, and 0.5-0.7 parts of nasturtium enzymatic hydrolysis extract.

[0008] Furthermore, the roxburghii fruit fermentation extract is prepared by removing impurities from fresh roxburghii fruit, drying it to a moisture content of ≤8%, crushing it through a 40-60 mesh sieve to obtain roxburghii fruit dry powder, adding deionized water at a mass ratio of 1:8-12 between the roxburghii fruit dry powder and water, stirring evenly, adjusting the pH to 4.0-5.0, adding a composite fermentation agent in an amount of 2-5% by mass of the roxburghii fruit dry powder, and standing and fermenting it at 30-35° C. for 48-72 hours. After the fermentation is completed, sterilizing the fermentation liquid at 80-90° C. for 15-20 minutes, cooling it to room temperature, centrifuging it to obtain the supernatant, and concentrating it under reduced pressure to 1 / 5-1 / 3 of the original volume to obtain the roxburghii fruit fermentation extract.

[0009] Furthermore, the composite fermentation agent is a mixture of saccharomyces cerevisiae and Lactobacillus plantarum in a mass ratio of (3.5-5.5):1, and the viable count of Lactobacillus plantarum is greater than 1.0×10 5 cfu / mL.

[0010] Furthermore, the vitex rotundifolia extract is prepared by taking dried vitex rotundifolia, crushing the fruit, and then passing it through a 20-40 mesh sieve to obtain a powder; adding 8-12 times the weight of the vitex rotundifolia powder to a 60-70% v / v ethanol solution; stirring the mixture evenly and then soaking it for 1-3 hours; placing the soaked mixture in a constant temperature water bath at 50-60°C; and extracting it for 30-45 minutes under ultrasonic power of 200-300W and frequency of 30-40kHz; filtering after the extraction is completed, collecting the filtrate, repeatedly extracting the filter residue 1-3 times, combining all the filtrates, and concentrating under reduced pressure to obtain the vitex rotundifolia extract.

[0011] Furthermore, the nasturtium enzymatic hydrolysis extract is prepared by taking fresh nasturtium whole herb, washing it, chopping it, drying it, and grinding it through a 40-60 mesh sieve to obtain nasturtium dry powder. The mass ratio of the dry powder to water is 1:10-15. Tris-HCl buffer is added, and after stirring evenly, the pH is adjusted to 4.5-5.5. The following are added in order according to the mass of the dry powder: Stage 1: Enzymatic hydrolysis with complex enzyme A at 35-45°C for 60-90 minutes, wherein the complex enzyme A comprises 1200±50U / g of cellulase and 800±20U / g of pectinase; Stage 2: Enzyme complex B, 48-52°C for 30-50 minutes, wherein the enzyme complex B comprises 300±10 U / g of myrosinase and 200±5 U / g of β-glucuronidase; Add 1-2 times the volume of limonene to the enzymatic hydrolysate, and simultaneously perform ultrasonic assisted extraction at a power of 300-500W and a frequency of 25-35kHz for 15-25 minutes.

[0012] Furthermore, a method for preparing a plant extract composition for removing and lightening freckles comprises the following steps: S1. Group preprocessing: a. Ginseng, Panax notoginseng, and turmeric were crushed through a 50-70 mesh sieve and extracted twice with 60-70% v / v ethanol under reflux at a mass volume ratio of g / mL of 1:10-14. The mixture was extracted at 70-90°C for 1-3h. The combined extracts were concentrated under reduced pressure to a relative density of 1.15 at 60°C to obtain an alcohol extract A. b. The persimmon leaves, safflower, peach kernel, red peony root were crushed through a 50-70 mesh sieve, and a citric acid solution of 40-80% v / v was added at a mass volume ratio of g / mL of 1:12-17. Ultrasonic extraction was performed at 45-55 ° C for 30-50 minutes at an ultrasonic power of 300-500 W. The supernatant was centrifuged and purified, and the 60% v / v ethanol elution fraction was collected to obtain a refined solution B; S2. Nano-processing: a. Leech polypeptide: Redissolve the lyophilized powder in phosphate buffer (pH 7.0) to a concentration of 4-6%. Microfluidize at a pressure of 1300-1700 bar for 2-4 cycles to obtain a leech polypeptide nanoliquid. b. Bombyx batryticatus extract: Mix the Bombyx batryticatus powder, polysorbate 80, and water in a mass / volume ratio of (0.8-1.2):(0.03-0.07):10. Cold sonicate at 3-5°C for 15-25 minutes at a power of 500-700W to obtain a Bombyx batryticatus extract dispersion. S3, gradient mixing emulsification: a. The alcohol extract A and refined liquid B were concentrated under reduced pressure at 40-50 ℃ to a moisture content of ≤5%, thioneine was added, and the fermented extract of roxburghii fruit was stirred at 300-400 rpm for 10-15 minutes until dissolved; b. Slowly add leech polypeptide nanofluid and silkworm extract dispersion, maintain 35-38 ° C and stir at 700-900 rpm for 12-17 minutes; c. Finally, add Vitex rotundifolia extract and Nasturtium enzymatic extract, continue stirring for 5-10 minutes, degas under -0.09±0.01 MPa vacuum pressure for 8-10 minutes, and then package.

[0013] Furthermore, the plant extract composition for removing and lightening spots is used in the preparation of cosmetics or medicines with the effect of removing and lightening spots.

[0014] Compared with the prior art, the present invention has the following beneficial effects: (1) Strong synergistic effects, with significant freckle removal and lightening effects: Leech polypeptide and silkworm extract can inhibit tyrosinase activity, reducing melanin synthesis from the source; plant extracts such as ginseng and Panax notoginseng can improve skin microcirculation and accelerate melanin metabolism; ergothioneine and roxburghii fruit fermented extract can scavenge free radicals and block ultraviolet-induced pigmentation. This synergistic effect overcomes the limitation of traditional single ingredients that only act on a single link in melanin synthesis. In vitro tests show that the melanin inhibition rate is over 45%, which is better than conventional natural extract products.

[0015] (2) Low irritation of raw materials and process optimization: The raw materials are all natural plant or biological extracts. Leech polypeptides are purified by ultrafiltration to remove impurities. Extracts such as silkworm are extracted using a gentle ultrasound-assisted extraction process to retain active ingredients while reducing irritation. Cell viability tests showed that the cell survival rate exceeded 90%. Nano-processing controls the particle size of active ingredients such as leech polypeptides to the nanoscale, and the cumulative transdermal dose exceeds 90 μg / cm in 12 hours. 2, which solves the problem of low transdermal efficiency of traditional extracts; the gradient mixing and emulsification process mixes raw materials in different states in stages, and combines with vacuum degassing to significantly improve the stability of the system, overcoming the defects of component agglomeration and poor stability caused by traditional mixing processes. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 、 2 All of them are finished product compositions of the embodiment groups. DETAILED DESCRIPTION

[0017] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0018] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods.

[0019] Unless otherwise specified, the materials, reagents, etc. used in the examples of the present invention can be obtained from commercial sources.

[0020] Example 1 A plant extract composition for removing and lightening spots comprises the following raw materials in parts by weight: 3 parts of ginseng, 2 parts of Panax notoginseng, 4 parts of persimmon leaves, 1 part of safflower, 2 parts of peach kernel, 3 parts of red peony root, 1 part of leech polypeptide, 2 parts of bombyx batryticatus extract, 3 parts of turmeric, 1 part of ergothioneine, 0.8 parts of roxburghii fruit fermented extract, 1.2 parts of vitex rotundifolia fruit extract, and 0.5 parts of nasturtium enzymatic hydrolysis extract.

[0021] Example 2 A plant extract composition for removing and lightening spots comprises the following raw materials in parts by weight: 8 parts of ginseng, 6 parts of Panax notoginseng, 10 parts of persimmon leaves, 4 parts of safflower, 5 parts of peach kernel, 7 parts of red peony root, 3 parts of leech polypeptide, 5 parts of bombyx batryticatus extract, 8 parts of turmeric, 3 parts of ergothioneine, 1.5 parts of roxburghii fruit fermented extract, 1.5 parts of vitex rotundifolia fruit extract, and 0.7 part of nasturtium enzymatic hydrolysis extract.

[0022] Example 3 A plant extract composition for removing and lightening freckles, comprising the following raw materials in parts by weight: 5 parts of ginseng, 4 parts of Panax notoginseng, 7 parts of persimmon leaves, 3 parts of safflower, 4 parts of peach kernel, 6 parts of red peony root, 2 parts of leech polypeptide, 3 parts of bombyx batryticatus extract, 5 parts of turmeric, 2 parts of ergothioneine, 1.2 parts of roxburghii fruit fermented extract, 1.3 parts of vitex rotundifolia fruit extract, and 0.6 parts of nasturtium enzymatic hydrolysis extract; Preparation methods of the above raw materials: (1) Leech polypeptide: Take dried leeches, crush them and sieve them through a 70-mesh sieve. Weigh the corresponding weight portion of leech powder, add deionized water at a mass ratio of 1:18 between leech powder and water, stir evenly and adjust the pH to 2.5, add pepsin at a mass ratio of 1.5% of the powder mass, with an enzyme activity of ≥3000 U / g, and perform enzymatic hydrolysis at 38°C for 5 hours. Adjust the pH of the mixture to 7.2, add a composite enzyme preparation at a mass ratio of 1.2% of the leech powder mass, and continue enzymatic hydrolysis at 37°C for 3.5 hours. The composite enzyme preparation is plasmin and carboxypeptidase B at a mass ratio of 1:2, with enzyme activities of both ≥3000 U / g, cool to room temperature and centrifuge. Purify the supernatant by ultrafiltration through an ultrafiltration membrane with a molecular weight cutoff of 4000 Da, collect the permeate, and concentrate under reduced pressure at 55°C until there is no ethanol smell, thereby obtaining leech polypeptide.

[0023] (2) Bombyx batryticatus extract: Dry Bombyx batryticatus was taken, impurities removed and then crushed to obtain Bombyx batryticatus powder. The powder was mixed with 75% v / v ethanol solution at a mass volume ratio of 1:12. After stirring evenly, the mixture was soaked at 28°C for 2.5 hours and then placed in a 60°C water bath. Ultrasonic assisted extraction was performed with an ultrasonic power of 300W and an extraction time of 45 minutes. After the extraction was completed, the mixture was filtered, the filtrate was collected, and the mixture was concentrated under reduced pressure to obtain Bombyx batryticatus extract.

[0024] (3) Rosa roxburghii fruit fermentation extract: fresh Rosa roxburghii fruit is removed from impurities and dried to a moisture content of ≤8%, and then crushed through a 50-mesh sieve to obtain Rosa roxburghii fruit powder. Deionized water is added at a mass ratio of 1:10 between the Rosa roxburghii fruit powder and water, and the mixture is stirred evenly, and the pH is adjusted to 45. A composite fermentation agent is added at a mass ratio of 4% of the mass of the Rosa roxburghii fruit powder, and the mixture is allowed to ferment at 32°C for 60 hours. After the fermentation is completed, the fermentation liquid is sterilized at 85°C for 18 minutes, cooled to room temperature, centrifuged, and the supernatant is collected. The supernatant is concentrated under reduced pressure to 1 / 4 of the original volume to obtain the Rosa roxburghii fruit fermentation extract. The composite fermentation agent is a mixture of Saccharomyces cerevisiae and Lactobacillus plantarum at a mass ratio of 4.5:1, and the number of viable bacteria of the Lactobacillus plantarum is >1.0×10 5 cfu / mL.

[0025] (4) Vitex rotundifolia extract: dried Vitex rotundifolia fruit is crushed and passed through a 30-mesh sieve to obtain a powder. A 65% v / v ethanol solution of 10 times its weight is added to the Vitex rotundifolia powder, stirred evenly and then soaked for 2 hours. The soaked mixture is placed in a constant temperature water bath at 55°C and extracted for 40 minutes under ultrasonic power of 250W and frequency of 35kHz. After the extraction is completed, the mixture is filtered and the filtrate is collected. The residue is extracted twice repeatedly. All the filtrates are combined and concentrated under reduced pressure to obtain the Vitex rotundifolia extract.

[0026] (5) The enzymatic hydrolysis extract of Nasturtium is prepared by washing the whole herb of Nasturtium, chopping it, drying it, and then grinding it through a 50-mesh sieve to obtain Nasturtium dry powder. Tris-HCl buffer is added at a mass ratio of powder to water of 1:12, stirring evenly and adjusting the pH to 5.0. The following are added in order according to the mass of the dry powder: Stage 1: Enzymatic hydrolysis with complex enzyme A at 40°C for 80 minutes, wherein the complex enzyme A comprises 1200 U / g of cellulase and 800 U / g of pectinase; Stage 2: Enzymatic hydrolysis with complex enzyme B at 50°C for 40 minutes, wherein the complex enzyme B comprises 300 U / g of myrosinase and 200 U / g of β-glucuronidase; 1.5 times the volume of limonene was added to the enzymatic hydrolysate, and ultrasonic assisted extraction was performed simultaneously at a power of 400 W and a frequency of 30 kHz for 20 minutes.

[0027] Preparation process of composition: S1. Group preprocessing: a. Ginseng, Panax notoginseng, and turmeric were ground through a 600-mesh sieve and extracted twice with 65% v / v ethanol under reflux at a mass-to-volume ratio of g / mL of 1:12. The extracts were extracted at 80°C for 2 h. The combined extracts were concentrated under reduced pressure to a relative density of 1.15 at 60°C to obtain alcohol-extracted extract A. b. The persimmon leaves, safflower, peach kernel, and red peony root were crushed through a 60-mesh sieve, and a 60% v / v citric acid solution was added at a mass volume ratio of g / mL of 1:15. Ultrasonic extraction was performed at 50 ° C for 40 minutes at an ultrasonic power of 400 W. The supernatant was centrifuged and purified, and the 60% v / v ethanol elution fraction was collected to obtain a refined solution B. S2. Nano-processing: a. Leech polypeptide: The lyophilized powder was reconstituted with phosphate buffer to a mass concentration of 5%. The mixture was homogenized by microfluidization at a pressure of 1500 bar for three cycles to obtain a leech polypeptide nanoliquid. b. Bombyx batryticatus extract: Bombyx batryticatus powder, polysorbate 80, and water were mixed at a mass volume ratio of 1:0.05:10 (g / mL). The mixture was sonicated at 4°C for 20 minutes at an ultrasonic power of 600 W to obtain a Bombyx batryticatus extract dispersion. S3, gradient mixing emulsification: a. The alcohol extract A and refined liquid B were concentrated under reduced pressure at 45°C to a moisture content of ≤5%, thioneine was added, and the fermented extract of roxburghii fruit was stirred at 350rpm for 12 minutes until dissolved; b. Slowly add leech polypeptide nanofluid, silkworm extract dispersion, maintain 36 ° C and stir at 800 rpm for 15 minutes; c. Finally, add Vitex rotundifolia extract and Nasturtium enzymatic extract, continue stirring for 8 minutes, degas under -0.09 MPa vacuum for 10 minutes and then encapsulate.

[0028] Comparative Example 1 The difference between this comparative example and Example 3 is that the composition includes the following raw materials in parts by weight: 1 part of ginseng, 7 parts of Panax notoginseng, 2 parts of persimmon leaves, 5 parts of safflower, 1 part of peach kernel, 8 parts of red peony root, 4 parts of leech polypeptide, 8 parts of bombyx batryticatus extract, 1 part of turmeric, and 5 parts to 1.5 parts of ergothioneine.

[0029] Comparative Example 2 The difference between this comparative example and Example 3 is that the composition does not contain leech polypeptide and silkworm extract, but is replaced by collagen peptide and pearl powder in equal amounts. The remaining raw materials and weight parts are the same as those in Example 3.

[0030] Comparative Example 3 The difference between this comparative example and Example 3 is that the composition does not contain the roxburghii fruit fermentation extract, vitex rotundifolia fruit extract, and nasturtium enzymatic hydrolysis extract, and the remaining raw materials and weight portions are the same as those in Example 3, and the corresponding raw material addition step is omitted in S3 of the preparation method.

[0031] Comparative Example 4 The difference between this comparative example and Example 3 is that the leech polypeptide and silkworm extract were not subjected to nano-processing, and the remaining steps were the same as in Example 3. Comparative Example 5 The difference between this comparative example and Example 3 is that the gradient mixing emulsification is replaced by synchronous mixing, and all components are stirred and emulsified at 36° C. and 800 rpm for 30 minutes.

[0032] Test Example 1: Spot Removal and Lightening Effect Test 1. Test materials: B16 melanoma cells (purchased from a cell bank), the compositions of Examples 1-3 and Comparative Examples 1-5 (diluted to a mass concentration of 0.1% with serum-free culture medium), and a positive control (arbutin solution, concentration 0.2%).

[0033] 2. Test method: B16 cells were seeded in 96-well plates (5 × 10 3 After culturing for 24 hours, the corresponding composition solution and positive control were added, and the blank group was added with an equal amount of serum-free culture medium. Three replicates were set up for each group. After 72 hours of continuous culture, melanin was extracted by NaOH lysis method, and the absorbance (OD value) was measured at 475 nm. Calculate the melanin inhibition rate: melanin inhibition rate = (OD value of blank group - OD value of experimental group) / OD value of blank group × 100%; At the same time, the CCK-8 method was used to detect cell viability (to ensure that the composition was non-cytotoxic and the viability was required to be >80%).

[0034] 3. Test results:

[0035] Conclusion: The test results show that all three examples exhibit excellent performance in terms of melanin inhibition rate and cell viability. Among them, Example 3 has the highest melanin inhibition rate, reaching 47.3%. Examples 2 and 1 also have rates of 46.1% and 45.5%, respectively, all significantly higher than the positive control (38.7%). This demonstrates that the plant extract composition for freckle removal and lightening of the present invention can effectively inhibit melanin production and has excellent freckle removal and lightening potential. Furthermore, the cell viability of all three examples was above 92%, with Example 3 reaching 93.8%, indicating that while the composition exerts a melanin inhibitory effect, it also has extremely low toxicity to cells and good biocompatibility.

[0036] The melanin inhibition rate of Comparative Example 1 was 39.8%, slightly higher than the positive control, but still lower than all other examples. This shows that a reasonable ratio of raw materials is an important basis for ensuring the melanin inhibition effect, and deviation from the reasonable range will lead to a decrease in effect. The melanin inhibition rate of Comparative Example 2 was only 26.9%, the lowest among all groups. This fully confirms that leech polypeptide and silkworm extract, as core effective ingredients, play an irreplaceable role in synergistically inhibiting melanin production. After replacing them, the core freckle removal efficacy of the composition is greatly weakened.

[0037] The melanin inhibition rates of Comparative Examples 3 (30.5%), 4 (32.1%), and 5 (31.3%) were significantly lower than those of the examples. This is because the fermented extract of roxburghii fruit is rich in antioxidants such as polyphenols and vitamin C, which can inhibit tyrosinase activity and block melanin synthesis; the flavonoids contained in the vitex rotundifolia extract can reduce melanin production by regulating related signaling pathways; and the active peptides in the enzymatic hydrolysis extract of nasturtium can interfere with melanocyte metabolism. Nano-sizing significantly increases the specific surface area of ​​the extract, improving its transdermal absorption efficiency; and the gradient mixing and emulsification process optimizes the synergistic effects between the ingredients, allowing the active ingredients of each extract to more precisely act on the key targets of melanin production.

[0038] In addition, the cell viability of each comparative example was 89.7% or above, indicating that all compositions had low overall toxicity to cells, and the examples achieved better melanin inhibition effects while maintaining high cell viability, with more outstanding overall performance.

[0039] Test Example 2: Tyrosinase inhibition rate test 1. Test Materials: Mushroom tyrosinase (activity ≥ 1000 U / mg), L-tyrosine (purity ≥ 99%), the compositions of Example 3 and Comparative Examples 1-5 (diluted to a mass concentration of 0.1% with 0.05 mol / L phosphate buffer (pH 6.8), and a positive control (arbutin solution, concentration 0.2%).

[0040] 2. Test method: Reaction system configuration: 50 μL of sample solution and 50 μL of tyrosinase solution (0.5 mg / mL) were added to a 96-well plate in sequence. After incubation at 37°C for 10 minutes, 50 μL of L-tyrosine solution (0.5 mmol / L) was added. The blank group was replaced with phosphate buffered saline solution. Three replicate wells were set up for each group. After constant temperature reaction at 37°C for 30 minutes, the absorbance (OD value) at 475 nm was measured using a microplate reader; Calculate the tyrosinase inhibition rate: inhibition rate = (OD value of blank group - OD value of experimental group) / OD value of blank group × 100%.

[0041] 3. Test results:

[0042] Conclusion: Judging from the test results, the tyrosinase inhibition rates of the three examples were all outstanding, among which Example 3 had the highest inhibition rate of 49.8%, while Example 1 and Example 2 also reached 48.9% and 48.6%, respectively, and were significantly higher than the positive control (40.3%). This fully demonstrates that the plant extract composition for removing and lightening spots described in the present invention has an excellent effect in inhibiting tyrosinase activity.

[0043] The tyrosinase inhibition rates of all comparative examples were lower than those of the examples. While comparative example 1 (38.5%) was close to the positive control, it was still lower than all the examples, indicating that a reasonable raw material ratio is essential for ensuring tyrosinase inhibition, and deviations from this ratio can affect the effect. The lower inhibition rate of comparative example 2 (27.9%) suggests that leech polypeptide and Bombyx batryticatus extract, as core ingredients, play a key role in synergistically enhancing tyrosinase inhibition, with the effect significantly reduced after substitution. The inhibition rates of comparative examples 3 (32.4%), 4 (34.2%), and 5 (33.1%) were also lower than those of the examples. This is because antioxidants such as superoxide dismutase (SOD) and vitamin C in the roxburghii fruit fermentation extract can scavenge reactive oxygen species and inhibit the oxidative activation of tyrosinase; nano-processing increases the specific surface area of ​​the raw materials, making them more accessible to the enzyme; and the gradient mixing and emulsification process optimizes the dispersion of the ingredients, ensuring their uniform distribution and full effectiveness. The absence or alteration of these three factors significantly weakens the composition's ability to inhibit tyrosinase activity.

[0044] Test Example 3: Determination of transdermal efficiency 1. Test materials Samples: the compositions of Example 3, Comparative Example 4, and Comparative Example 5 (all prepared as essences containing 5% active ingredients, with physiological saline as the solvent); Transdermal model: Franz diffusion cell (donor chamber volume 2 mL, receiving chamber volume 8 mL), pig ear skin (subcutaneous fat removed, thickness 0.3-0.5 mm, sterilized for use); Detection instrument: High performance liquid chromatography (HPLC), used to determine the content of leech polypeptide (marker).

[0045] 2. Test methods Model construction: Pig ear skin was fixed between the donor and receiver chambers of a Franz diffusion cell, with the skin stratum corneum facing the donor chamber. Physiological saline (32°C ± 0.5°C, magnetic stirring rate 300 rpm) was added to the receiver chamber and allowed to equilibrate for 30 minutes after removing bubbles. Sample loading: Add 1 mL of the sample of Example 3, Comparative Example 4, and Comparative Example 5 to the donor chamber, seal the chamber, and start timing. Take 1 mL of sample from the receiving chamber at 2 h, 4 h, 6 h, 8 h, and 12 h, respectively (simultaneously add an equal amount of 32°C normal saline). Content determination: HPLC was used to detect the concentration of leech polypeptide in the sample (chromatographic conditions: C18 column, mobile phase of methanol-water = 30:70, flow rate 1 mL / min, detection wavelength 220 nm); Calculation indicators: Cumulative transdermal dose (μg / cm 2 ) = total content of active ingredients in the receiving compartment / effective diffusion area of ​​the skin; Transdermal rate (μg / cm 2 ・h) = linear fitting slope of cumulative transdermal penetration from 6 to 12 hours.

[0046] 3. Test results

[0047] Conclusion: The cumulative transdermal dose of Example 3 reached 92.4 μg / cm in 2 hours. 2 , transdermal rate 7.8μg / cm 2 h, significantly higher than those in Comparative Examples 4 and 5. This is mainly due to the nano-processing that reduces the particle size of the active ingredient to enhance skin penetration. The leech polypeptide nanofluid has a small particle size and easily penetrates the skin barrier. The gradient mixing and emulsification process forms a stable dispersion system, reduces the agglomeration of the active ingredient, further promotes transdermal penetration, and improves the skin penetration efficiency of the active ingredient. Comparative Example 4 had the lowest cumulative transdermal dose and transdermal rate (only 45.9 μg / cm in 12 h). 2 ), because they have not been nano-processed, the particles of leech polypeptides and silkworm extracts are large (>1μm), which makes it difficult for them to pass through the gaps in the skin's stratum corneum, limiting their transdermal efficiency; The transdermal effect of Comparative Example 5 is better than that of Comparative Example 4 but weaker than that of Example 3. The synchronous mixing process causes some active ingredients to be unevenly dispersed and easily form local agglomerations. Although it does not completely hinder transdermal penetration, it reduces the effective penetration rate.

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A plant extract composition for removing and lightening freckles, characterized in that: The invention comprises the following raw materials in parts by weight: 3-8 parts of ginseng, 2-6 parts of Panax notoginseng, 4-10 parts of persimmon leaves, 1-4 parts of safflower, 2-5 parts of peach kernel, 3-7 parts of red peony root, 1-3 parts of leech polypeptide, 2-5 parts of bombyx batryticatus extract, 3-8 parts of turmeric and 1-3 parts of ergothioneine.

2. The plant extract composition for removing and lightening freckles according to claim 1, wherein: The leech polypeptide is prepared by taking dried leeches, crushing them, and then passing them through a 60-80 mesh sieve. The corresponding weight portion of leech powder is weighed, deionized water is added at a mass ratio of leech powder to water of 1:15-20, the mixture is stirred evenly, the pH is adjusted to 2.0-3.0, 1.0-2.0% of the weight of the powder is added with pepsin, the enzyme activity is ≥3000 U / g, and the mixture is enzymatically hydrolyzed at a constant temperature of 37-40° C. for 4-6 hours. The pH of the mixture is adjusted to 7.0-7.5, and the mixture is added. A composite enzyme preparation containing 0.8-1.5% by weight of leech powder is added, and enzymatic hydrolysis is continued at 35-38°C for 3-4 hours. The composite enzyme preparation comprises plasmin and carboxypeptidase B in a mass ratio of 1:1-2, and the enzyme activity of both is ≥3000U / g. The mixture is cooled to room temperature and centrifuged. The supernatant is purified by ultrafiltration through an ultrafiltration membrane with a molecular weight cutoff of 3000-5000Da. The permeate is collected and concentrated under reduced pressure at 50-60°C until there is no ethanol smell, thereby obtaining leech polypeptides.

3. The plant extract composition for removing and lightening freckles according to claim 1, wherein: The silkworm extract is prepared by removing impurities from dried silkworms and crushing them to obtain silkworm powder. The powder is mixed with a 70-80% v / v ethanol solution at a mass volume ratio of 1:10-15, stirred evenly, and then soaked at 25-30° C. for 2-3 hours. The mixture is then placed in a 55-65° C. water bath, and ultrasonic-assisted extraction is performed with an ultrasonic power of 250-350 W and an extraction time of 40-50 minutes. After the extraction is completed, the mixture is filtered, the filtrate is collected, and the mixture is concentrated under reduced pressure to obtain the silkworm extract.

4. The plant extract composition for removing and lightening freckles according to claim 1, wherein: The invention also includes the following raw materials in parts by weight: 0.8-1.5 parts of roxburghii fruit fermentation extract, 1.2-1.5 parts of vitex rotundifolia fruit extract, and 0.5-0.7 parts of nasturtium enzymatic hydrolysis extract.

5. The plant extract composition for removing and lightening freckles according to claim 3, wherein: The roxburghii fruit fermentation extract is prepared by removing impurities from fresh roxburghii fruit, drying the fruit to a moisture content of ≤8%, crushing the fruit to pass through a 40-60 mesh sieve to obtain roxburghii fruit dry powder, adding deionized water at a mass ratio of 1:8-12 between the roxburghii fruit dry powder and water, stirring evenly, adjusting the pH to 4.0-5.0, adding a composite fermentation agent in an amount of 2-5% by mass of the roxburghii fruit dry powder, and standing and fermenting the mixture at 30-35° C. for 48-72 hours. After the fermentation is completed, sterilizing the fermentation liquid at 80-90° C. for 15-20 minutes, cooling the mixture to room temperature, centrifuging the supernatant, and concentrating the mixture under reduced pressure to 1 / 5-1 / 3 of the original volume to obtain the roxburghii fruit fermentation extract.

6. The plant extract composition for removing and lightening freckles according to claim 5, characterized in that: The composite fermentation agent is a mixture of saccharomyces cerevisiae and Lactobacillus plantarum in a mass ratio of (3.5-5.5):1, and the number of viable bacteria of the Lactobacillus plantarum is greater than 1.0×10 5 cfu / mL.

7. The plant extract composition for removing and lightening freckles according to claim 3, wherein: The vitex chinensis fruit extract comprises the following steps: taking dried vitex chinensis fruit, crushing the fruit, and then passing the powder through a 20-40 mesh sieve to obtain a powder; adding 8-12 times the weight of the vitex chinensis fruit powder with a 60-70% v / v ethanol solution; stirring the mixture evenly; and soaking the mixture for 1-3 hours. The mixture is placed in a constant temperature water bath at 50-60° C. and extracted for 30-45 minutes under ultrasonic conditions with an ultrasonic power of 200-300 W and a frequency of 30-40 kHz. After the extraction is completed, the mixture is filtered to collect the filtrate, and the extraction of the filter residue is repeated 1-3 times. All the filtrates are combined and concentrated under reduced pressure to obtain the vitex chinensis fruit extract.

8. The plant extract composition for removing and lightening freckles according to claim 3, wherein: The nasturtium enzymatic hydrolysis extract is prepared by taking fresh nasturtium whole herb, washing it, chopping it, drying it, and grinding it through a 40-60 mesh sieve to obtain nasturtium dry powder. Tris-HCl buffer is added at a powder to water mass ratio of 1:10-15, stirring it evenly, and then adjusting the pH to 4.5-5.

5. The following are added in order according to the mass of the dry powder: Stage 1: Enzymatic hydrolysis with complex enzyme A at 35-45°C for 60-90 minutes, wherein the complex enzyme A comprises 1200±50U / g of cellulase and 800±20U / g of pectinase; Stage 2: Enzyme complex B, 48-52°C for 30-50 minutes, wherein the enzyme complex B comprises 300±10 U / g of myrosinase and 200±5 U / g of β-glucuronidase; Add 1-2 times the volume of limonene to the enzymatic hydrolysate, and simultaneously perform ultrasonic assisted extraction at a power of 300-500W and a frequency of 25-35kHz for 15-25 minutes.

9. The method for preparing a plant extract composition for removing and lightening freckles according to any one of claims 1 to 8, wherein: The following steps are involved: S1. Group preprocessing: a. Ginseng, Panax notoginseng, and turmeric were crushed through a 50-70 mesh sieve and extracted twice with 60-70% v / v ethanol under reflux at a mass volume ratio of g / mL of 1:10-14. The mixture was extracted at 70-90°C for 1-3h. The combined extracts were concentrated under reduced pressure to a relative density of 1.15 at 60°C to obtain an alcohol extract A. b. The persimmon leaves, safflower, peach kernel, red peony root were crushed through a 50-70 mesh sieve, and a citric acid solution of 40-80% v / v was added at a mass volume ratio of g / mL of 1:12-17. Ultrasonic extraction was performed at 45-55 ° C for 30-50 minutes at an ultrasonic power of 300-500 W. The supernatant was centrifuged and purified, and the 60% v / v ethanol elution fraction was collected to obtain a refined solution B; S2. Nano-processing: a. Leech polypeptide: The freeze-dried powder was reconstituted with phosphate buffer to a mass concentration of 4-6%. The mixture was homogenized by microfluidization at a pressure of 1300-1700 bar for 2-4 cycles to obtain a leech polypeptide nanoliquid. b. Bombyx batryticatus extract: Bombyx batryticatus powder, polysorbate 80, and water were mixed at a mass volume ratio of (g / mL) (0.8-1.2):(0.03-0.07):

10. The mixture was sonicated at 3-5°C for 15-25 minutes at a power of 500-700W to obtain a Bombyx batryticatus extract dispersion. S3, gradient mixing emulsification: a. The alcohol extract A and refined liquid B were concentrated under reduced pressure at 40-50 ℃ to a moisture content of ≤5%, thioneine was added, and the fermented extract of roxburghii fruit was stirred at 300-400 rpm for 10-15 minutes until dissolved; b. Slowly add leech polypeptide nanofluid and silkworm extract dispersion, maintain 35-38 ° C and stir at 700-900 rpm for 12-17 minutes; c. Finally, add Vitex rotundifolia extract and Nasturtium enzymatic extract, continue stirring for 5-10 minutes, degas under -0.09±0.01 MPa vacuum pressure for 8-10 minutes, and then package.

10. Use of the plant extract composition for removing and lightening spots according to any one of claims 1 to 8 in the preparation of cosmetics or medicines having the effect of removing and lightening spots.

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