Preparation method of small molecule polypeptide of malva sylvestris and amino acid solution and application thereof

Abstract

The application belongs to the technical field of cosmetics, and particularly relates to a preparation method of a small molecule polypeptide of a leaf-eating plant and an amino acid solution and application thereof. The method obtains specific peptide segments by gradually enzymatically digesting proteins contained in the leaf-eating plant and by controlling pH values and enzyme types, so as to realize efficient enzymatic digestion and improve the purity of products, thereby laying a foundation for the application of the products in the field of cosmetics. The method of the application not only has safe and reliable production principles, but also improves the yield of the small molecule polypeptide of the leaf-eating plant, and solves the technical problem that cosmetic protein raw materials are difficult to penetrate the skin.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology, specifically relating to the preparation method and application of a solution of small molecule polypeptides and amino acids from *Eclipta prostrata*. Background Technology

[0002] Aging is a complex process involving multiple factors, mainly divided into aging caused by endogenous and exogenous factors. Exogenous stimuli such as ultraviolet radiation, pollution, and glycation activate NADPH oxidase (NOX) and disrupt mitochondrial energy levels, leading to the proliferation of free radicals (O3). 2- The excessive accumulation of free radicals (ROS) can lead to the accumulation of reactive oxygen species (ROS), which in turn can cause oxidative stress in cells. ROS are both triggering signals for inflammation and secondary byproducts of amplified inflammation. Intracellular ROS accumulation can induce the release of inflammatory factors IL-1β, IL-6, and TNF-α by activating the NF-κB pathway. TNF-α and IL-1β promote the degradation of collagen and elastin by activating matrix metalloproteinases (MMPs); while IL-6 and IL-1 activate the p53-p21 pathway, inducing fibroblasts to enter cell cycle arrest and secrete senescence-associated secretory phenotypes (SASPs), promoting cellular senescence and ultimately leading to typical aging phenotypes in the skin, such as wrinkles, sagging, dryness, pigmentation, and a weakened skin barrier. Currently, plant-derived anti-aging ingredients have become a research hotspot due to their high safety and multi-target effects, but most suffer from low extraction efficiency and unstable activity.

[0003] Rumex patientia L. × Rumex tianschanicus is a perennial plant belonging to the Polygonaceae family and the Rumex genus, bred in 1995 through hybridization of Rumex patientia (female) and Rumex tianschanicus (male). It combines the advantages of both parents: cold hardiness, salt tolerance, rapid growth, and high yield. It can grow in saline-alkali and arid marginal lands in northern and northwestern China, and is now cultivated on a large scale in Hebei, Shandong, Inner Mongolia, Northeast China, Xinjiang, and other regions.

[0004] Edible grass, also known as "amino acid grass" due to its high amino acid content, contains 18 kinds of amino acids, of which essential amino acids account for 45% of the total. Its crude protein content ranges from 34.7% to 48.7%. Furthermore, it contains various vitamins, minerals, and active functional ingredients such as flavonoids and polyphenols. Therefore, edible grass is a "four-high" plant—high in protein, vitamins, minerals, and functional factors—possessing multiple values ​​as food, feed, health care, and ecological restoration, earning it the reputation of a "plant gold mine." However, research on the application of its extracts in the cosmetics field is limited, lacking systematic extraction processes and product development.

[0005] Chinese patent document CN108514526A discloses edible grass plant protein raw materials and their uses in cosmetics and personal care products. The raw materials are pulverized grass powder, ground grass pulp, or extracted grass juice, and are applied to daily necessities such as face masks and toothpaste. However, directly using edible grass powder, pulp, or juice in cosmetics is problematic because the grass powder has a large particle size and contains insoluble fiber residue, which easily leads to flocculation and sedimentation in transparent aqueous solutions and sprays, resulting in poor product stability; the grass juice initially has a total bacterial count of approximately 10. 5 -10 6 CFU / g requires additional irradiation or high-pressure treatment, increasing costs; the high concentration of chlorophyll and carotenoids in straw pulp can easily conflict with fragrances and pigments, limiting its application in various cosmetics.

[0006] Chinese patent document CN117296977A discloses a method for preparing leafy grass protein, including the following steps: Step 1: After appropriately pulverizing dried leafy grass material, mix it with water and inactivate it at 90℃ for 15 minutes; Step 2: After cooling, add compound enzyme A to the leafy grass and water mixture under a water bath at 40-45℃, react for 4-5 hours, then raise the temperature to 50-55℃, react for 2-3 hours, and then inactivate it at high temperature before cooling to room temperature; Step 3: Perform centrifugal filtration, wash the solid with water 3 times, and then centrifuge and filter again, finally taking only the solid. However, key technological shortcomings remain: First, the products are mostly large-molecule proteins or peptides with disordered molecular weight distribution, which are not only difficult to absorb through the skin, but also have a very low proportion of target active peptides such as ACE inhibitors and antioxidants, failing to meet the anti-aging efficacy requirements of cosmetics; Second, the hydrolysis efficiency is low; Third, the purity and safety of the products are insufficient: The solids obtained by the method contain many impurities, including not only undegraded fibers, pigments and other non-target components, but also potentially residual undeactivated enzymes and small-molecule metabolic waste; the presence of these impurities leads to low product purity and dull color, which can easily cause skin irritation when applied to cosmetics, and also reduces the efficacy density of the target active ingredients, making it difficult to meet the core requirements of cosmetics for the purity, safety and efficacy of raw materials.

[0007] Therefore, developing a green, environmentally friendly, healthy, and efficient preparation process for edible leaf grass extract that meets cosmetic production standards is of significant research importance. Summary of the Invention

[0008] To address the issues of large molecular weight and difficulty in transdermal absorption of physalis protein in existing technologies, this invention provides a method for preparing a solution of small molecule peptides and amino acids from physalis, and verifies its anti-aging effects at the cellular level. This method is then applied to cosmetics to achieve antioxidant, cell proliferation-promoting, and skin-improving effects, thus overcoming the absorption bottleneck of physalis protein in cosmetic applications.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A method for preparing a solution of small molecule peptides and amino acids from leafy green plants includes the following steps:

[0011] S1. Prepare a suspension of edible leaf grass powder by mixing it with water, and then sterilize it at high temperature.

[0012] S2. Sonicate the suspension of leafy grass powder, then lower the temperature to below 30°C, add cellulase and pectinase and react for 1-2 hours, then raise the temperature to 50-60°C and soak for 2-3 hours. After high-temperature inactivation, lower the temperature to room temperature.

[0013] S3. Add plant aspartic protease to the reaction system obtained in step S2, inactivate it at high temperature after the reaction is completed, and then cool it to room temperature.

[0014] S4. Adjust the pH of the reaction system obtained in step S3 to 6.0-7.0, add bromelain, inactivate it at high temperature after the reaction is completed, and then cool it to room temperature;

[0015] S5. Adjust the pH of the reaction system obtained in step S4 to 8.0-9.0, add alkaline protease, inactivate at high temperature after the reaction is completed, and then cool to room temperature;

[0016] S6. Add exonuclease-aminopeptidase to the reaction system obtained in step S5, react at 50-70℃ for 2-4 hours, inactivate at high temperature after the reaction is completed, and then cool to room temperature.

[0017] S7. Decolorization and fine filtration: Centrifuge the system obtained in step S6 to obtain the supernatant, adjust the pH of the supernatant to 4.0-5.0, and then finely filter it after treatment with activated carbon to obtain a clear supernatant.

[0018] S8. Concentration and separation: The supernatant obtained in step S7 is concentrated by rotary evaporation and ultrafiltration to obtain a solution of small molecule peptides and amino acids.

[0019] Preferably, in step S1, fresh edible grass that has grown to 50-60cm is taken, dried with hot air at 50-60℃ until the moisture content is ≤8%, and then crushed and passed through a 20-60 mesh sieve to obtain edible grass powder.

[0020] Preferably, in step S1, the leafy grass powder suspension is prepared by mixing leafy grass powder and water at a solid-liquid ratio of 1:10-20 g / mL; the high-temperature sterilization is performed at 80°C for 15 min.

[0021] Preferably, in step S2, the ultrasonic time is 1-2 hours and the ultrasonic power is 100-400W.

[0022] Preferably, in step S2, the cellulase activity is 1:6000-1:8000 U / g, and the mass ratio of cellulase to leafy grass powder is 0.1%-2%:1; the pectinase activity is >30U / mg, and the mass ratio of pectinase to leafy grass powder is 0.1%-2%:1; the mass ratio of cellulase to pectinase is 1-2:1.

[0023] Preferably, the high temperature mentioned in step S2 is 80-90℃.

[0024] Preferably, in step S3, the reaction temperature is 40-50℃ and the reaction time is 2-3 hours.

[0025] Preferably, in step S3, the plant aspartic protease has an enzyme activity >2000 U / g, and its mass ratio with the leafy grass powder is 2%-5%:1.

[0026] Preferably, in step S4, the reaction temperature is 40-55℃, the reaction time is 2-4h, and the pH is 6.9-7.0.

[0027] Preferably, in step S4, the bromelain enzyme with an activity >200 U / mg has a mass ratio of 0.1%-0.5% to the leafy green powder:1.

[0028] Preferably, in step S5, the reaction temperature is 50-60℃ and the reaction time is 2-4h.

[0029] Preferably, in step S5, the alkaline protease activity is >150000 U / g, and its mass ratio with the leafy grass powder is 0.5%-1%:1.

[0030] Preferably, in steps S4 and S5, the pH is adjusted using a 25% (m / m) ammonia solution.

[0031] Preferably, in step S6, the exonuclease-aminopeptidase activity is >15 U / mg, and its mass ratio with the leafy grass powder is 0.1%-0.15%:1.

[0032] The optimal pH range for the exonuclease-aminopeptidase activity is 8.0-9.0. In the method of this invention, the pH of the reaction system after alkaline protease treatment is in the range of 8.0-9.0. This range is within the optimal pH range for the exonuclease-aminopeptidase activity. Therefore, there is no need to adjust the pH of the system, and subsequent enzymatic hydrolysis reactions can be carried out directly.

[0033] Preferably, the centrifugation in step S7 is carried out at a speed of 8000-12000 rpm for 20-30 min.

[0034] Preferably, in step S7, the pH is adjusted using a 20% (m / m) aqueous solution of citric acid.

[0035] Preferably, the step S7, which involves fine filtration after activated carbon treatment to obtain a clear supernatant, specifically involves adding activated carbon to the pH-adjusted supernatant for decolorization, with a mass-to-volume ratio of activated carbon to supernatant of 0.2%–1%: 1 g / mL. The mixture is stirred at 45–60°C for 1–2 hours, centrifuged at 12000 rpm for 20–30 minutes, and the supernatant is collected. The obtained supernatant is then filtered and then vacuum filtered to obtain a clear supernatant.

[0036] Preferably, the rotary evaporation in step S8 is carried out at a temperature of 45-65°C.

[0037] The present invention also provides the application of the small molecule polypeptide and amino acid solution of edible leaf grass prepared according to the above method in the preparation of anti-aging cosmetics, including but not limited to serums, creams, and masks.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] (1) This invention utilizes a stepwise enzymatic hydrolysis method to obtain specific peptides from the proteins contained in edible leaves by controlling the pH value and the type of enzyme. This achieves efficient enzymatic hydrolysis and improves the purity of the product, laying the foundation for its application in the cosmetics field. The method of this invention, through enzymatic hydrolysis at different stages, not only adheres to safe and reliable production principles but also improves the yield of small molecule peptides from edible leaves, solving the technical problem of the difficulty in transdermal absorption of protein raw materials in cosmetics.

[0040] (2) The present invention can continuously enzymatically hydrolyze at different pH levels, which can not only maximize enzyme activity and increase the degree of hydrolysis, thereby reducing the amount of enzyme and cost, but also improve the protein yield and "precisely cut" the protein into smaller pieces, resulting in peptides with smaller target molecular weight (<10KDa).

[0041] (3) The method of the present invention first uses endonuclease to “cut the large protein into 100 kDa medium peptides in the weak acid-neutral-weak base segment, which reduces the viscosity of the solution and exposes more C / N ends. Then, under weak alkaline conditions, sufficient “cut” and enzymatic hydrolysis conditions are provided for subsequent exonuclease. The overall degree of hydrolysis is increased by 15-25% compared with single-segment enzymatic hydrolysis.

[0042] (4) The method of the present invention adds an exonuclease to the weak base segment and cuts off 2-3 amino acids one by one from the exposed end. The product is concentrated in the small peptide range of <10KDa, and the proportion of target active peptides (ACE inhibitors, antioxidants, antibacterial agents, etc.) is significantly increased.

[0043] (5) The method of the present invention allows the endopeptidase and exopeptidase to be in their optimal activity range by stepwise pH control, which can shorten the total reaction time by 20-30% and reduce the amount of enzyme used by 10-15%. The subsequent enzyme inactivation, decolorization and filtration processes are smoother and the overall cost is the lowest.

[0044] (6) This invention obtains the extract of *Hedyotis diffusa* using a green, environmentally friendly, healthy, and safe extraction method. The *Hedyotis diffusa* extract provided by this invention has multiple anti-aging effects, such as scavenging free radicals, anti-oxidation, and promoting collagen regeneration. It can be used as an anti-aging active ingredient and widely applied in various cosmetics such as serums. Attached Figure Description

[0045] Figure 1 This presents the results of the biochemical scavenging rates of different concentrations of *Gnaphalium affine* small molecule polypeptide and amino acid solutions on free radicals in Experiment 2 of this invention. (a) shows the scavenging rate of ·OH, (b) shows the scavenging rate of DPPH, and (c) shows the scavenging rate of O2. 2- The clearance rate results;

[0046] Figure 2 The transdermal absorption rate of small molecule peptides and amino acids from *Gnaphalium affine* and their effect on cell viability are shown in Figure 3 of this invention. (a) is a graph showing the transdermal absorption rate of small molecule peptides and amino acids from *Gnaphalium affine* detected using a TT-6TT-8 transdermal absorption meter, and (b) is a graph showing the effect of different concentrations of small molecule peptides and amino acids from *Gnaphalium affine* on cell viability using a CCK-8 assay.

[0047] Figure 3 This is a diagram showing the results of ROS generation in H2O2-induced oxidation model cells in Experiment Example 4 of this invention, where the bright field shows the morphology and density of cells in each group, ROS shows the fluorescence signal intensity of reactive oxygen species in the cells, CTR is the control group, H2O2 is the oxidation-induced model group, and H2O2+0.5mg / mL indicates the group that was intervened with 0.5mg / mL of foliage small molecule peptide and amino acid solution after H2O2 induction.

[0048] Figure 4 The results of the effects of small molecule peptides and amino acids of edible grass on type I and type III collagen in Experiment Example 5 of the present invention are shown. Among them, (a) is a graph showing the effect of small molecule peptides and amino acids of edible grass on type I collagen, and (b) is a graph showing the effect of small molecule peptides and amino acids of edible grass on type III collagen.

[0049] Figure 5 This is a graph showing the results of the hyaluronidase inhibition experiment in Experiment Example 6 of the present invention;

[0050] Figure 6The following is a diagram showing the cell migration ability results in Experiment Example 6 of the present invention, wherein (a) is a microscopic image of the cell scratch test, and (b) is the statistical result of the relative migration rate of cells in each group;

[0051] Figure 7 In Experiment 7, a model of chicken embryo allantoic membrane was used to demonstrate the safety and non-irritation of small molecule peptides and amino acids from the leafy green plant. Detailed Implementation

[0052] The technical solutions and anti-aging efficacy verification of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All embodiments obtained by those skilled in the art based on the present invention are without creative effort.

[0053] The "water" mentioned below refers to "deionized water".

[0054] The cellulase used in these embodiments of the invention was purchased from Beijing Coollab Technology Co., Ltd., product number CC3281, with an enzyme activity of 1:7000 U / g and a purity >99%.

[0055] Pectinase was purchased from Beijing Coollab Technology Co., Ltd., product number CP8181, with enzyme activity >40U / mg and purity >99%.

[0056] The plant aspartic protease was purchased from Xi'an Xinlu Biotechnology Co., Ltd., production batch number: XL20250715, enzyme activity 3000U / g, content >99%;

[0057] Bromelain was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., product number B832358, with an enzyme activity of 300 U / mg and a purity of >99%.

[0058] The alkaline protease was purchased from Beijing Coollab Technology Co., Ltd., product number CA1521, with enzyme activity >200,000 U / g and purity >99%.

[0059] The exonuclease-aminopeptidase (Aminopeptidase M) was purchased from Kekulé (Guangzhou) Biopharmaceutical Co., Ltd. (KKL), product number KE8888, with an enzyme activity of 21 U / mg and a purity of >99%.

[0060] The amino acid (AA) content test kit was purchased from Beijing Solarbio Technology Co., Ltd., item number BC1575;

[0061] The BeyoBCA peptide concentration assay kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd., product number P0397M.

[0062] Example 1

[0063] A method for preparing a solution of small molecule peptides and amino acids from leafy green plants includes the following steps:

[0064] S1. Raw material pretreatment: Take fresh edible grass that has grown to 50-60 cm, dry it with hot air at 60℃ to a moisture content of 7.3%, then crush it and pass it through a 20-60 mesh sieve to obtain edible grass powder. Take 100g of edible grass powder and mix it with water at a solid-liquid ratio of 1:15g / mL to prepare an edible grass powder suspension. Inactivate it at 80℃ for 15min.

[0065] S2. Ultrasonic disruption: The leafy grass powder suspension obtained in step S1 was naturally cooled to 40°C and ultrasonically disrupted for 2 hours at a power of 400W. After ultrasonication, it was naturally cooled to 25°C. 2g of cellulase and 1g of pectinase were added to the leafy grass powder suspension and stirred for 1 hour. Then the temperature was raised to 55°C and the mixture was soaked for another 3 hours to release intracellular proteins, flavonoids, polyphenols and oxalic acid. After the reaction was completed, the mixture was inactivated by high temperature at 90°C and then cooled to room temperature. At this time, the pH was 4.89.

[0066] S3, Acidic enzymatic hydrolysis of proteins: At this point, 3.5g of plant aspartic protease is added to the system and reacted at 45℃ for 3 hours to hydrolyze the large molecular proteins released from the cells into peptides less than 150kDa. After the reaction is completed, the proteins are inactivated by high temperature at 60℃ and then cooled to room temperature.

[0067] S4. Neutral enzymatic hydrolysis of proteins: The pH value was finely adjusted to 6.98 using a 25% (m / m) ammonia solution. 0.25g of bromelain was added to the reaction system and reacted at 50℃ for 2h to further hydrolyze the large molecular proteins released from the cells into peptides less than 100kDa. After the reaction was completed, the proteins were inactivated by high temperature at 90℃ and then cooled to room temperature.

[0068] S5. Alkaline enzymatic hydrolysis of proteins: The pH value was finely adjusted to 8.56 using a 25% (m / m) ammonia solution. 0.75g of alkaline protease was added to the reaction system and reacted at 55℃ for 2h to further hydrolyze the large molecular proteins and peptides released from the cells into peptides smaller than 50kDa. After the reaction was completed, the proteins were inactivated by high temperature at 90℃ and then cooled to room temperature.

[0069] S6. Obtaining small molecule peptides: At this point, the reaction system is generally alkaline. Add 0.1g of exonuclease-aminopeptidase to the reaction system and react at 65℃ for 2h. This will generate small molecule peptides (<30KDa, or even <10KDa) from the peptides digested in steps S3, S4, and S5 above. After the reaction is complete, inactivate the peptides at 95℃ and allow them to stand to return to room temperature.

[0070] S7. Decolorization and Fine Filtration: After centrifuging the above reaction system at 12000 rpm for 30 min at room temperature, take the supernatant. Adjust the pH to 4.52 with a 20% (m / m) citric acid aqueous solution. Add 1-10 nm activated carbon and stir at 50 °C for 2 h. The mass-volume ratio of activated carbon to supernatant is 0.2%:1 g / mL. Adsorb oxalic acid and chlorophyll in the system. After centrifuging at 12000 rpm for 30 min, take the supernatant. Filter the supernatant through a nylon membrane with a pore size of 1-10 µm to obtain a clear supernatant. Then filter it through a 0.22 µm filter membrane to obtain an even clearer and more transparent supernatant.

[0071] S8. Concentration and separation: The supernatant obtained in S7 is concentrated to 1 / 5 of its volume by rotary evaporation at 65°C; the concentrated solution is then ultrafiltered using an ultrafiltration centrifuge tube with a strength of <30 kDa to obtain a solution of small molecule peptides and amino acids with a strength of <30 kDa.

[0072] The ultrafiltration specifically refers to:

[0073] (1) Add 3.5 mL of concentrated sample to the Amicon® Ultra ultrafiltration centrifuge tube.

[0074] (2) Place the capped ultrafiltration centrifuge tube into the centrifuge rotor and balance it with a similar ultrafiltration tube.

[0075] (3) Use a swing drum rotor to centrifuge at a maximum of 4,000 xg for 40 minutes.

[0076] (4) When recovering the concentrated product, insert the pipette (tip) into the inner tube of the ultrafiltration centrifuge tube and move it left and right to draw up the sample to ensure complete recovery. The filtrate can be stored in the outer tube of the centrifuge.

[0077] Example 2

[0078] A method for preparing a solution of small molecule peptides and amino acids from leafy green plants includes the following steps:

[0079] S1. Raw material pretreatment: Take fresh leafy grass that has grown to 50-60 cm, dry it with hot air at 60℃ to a moisture content of 7.5%, then crush it and pass it through a 20-60 mesh sieve to obtain leafy grass powder. Take 100g of leafy grass powder and mix it with water at a solid-liquid ratio of 1:15g / mL to prepare a leafy grass powder suspension. Inactivate it at 80℃ for 15min.

[0080] S2. Ultrasonic disruption: The leafy grass powder suspension obtained in step S1 was naturally cooled to 40°C and ultrasonically disrupted for 1 hour at a power of 300W. After ultrasonication, it was naturally cooled to 25°C. 2g of cellulase and 1g of pectinase were added to the leafy grass powder suspension and stirred for 2 hours. Then, the temperature was raised to 55°C and the mixture was soaked for another 2 hours to release intracellular proteins, flavonoids, polyphenols, and oxalic acid. After the reaction was completed, the mixture was inactivated by high-temperature inactivation at 90°C and then cooled to room temperature. At this point, the pH was 4.85.

[0081] S3, Acidic enzymatic hydrolysis of proteins: At this point, 4g of plant aspartic protease is added to the system and reacted at 45℃ for 3 hours to hydrolyze the large molecular proteins released from the cells into peptides less than 150kDa. After the reaction is completed, the proteins are inactivated by high temperature at 60℃ and then cooled to room temperature.

[0082] S4. Neutral enzymatic hydrolysis of proteins: The pH value was finely adjusted to 6.92 using a 25% (m / m) ammonia solution. 0.20g of bromelain was added to the reaction system and reacted at 45℃ for 4h to further hydrolyze the large molecular proteins released from the cells into peptides less than 100kDa. After the reaction was completed, the proteins were inactivated by high temperature at 90℃ and then cooled to room temperature.

[0083] S5. Alkaline enzymatic hydrolysis of proteins: The pH value was finely adjusted to 8.44 using a 25% (m / m) ammonia solution. 0.5g of alkaline protease was added to the reaction system and reacted at 55℃ for 4h to further hydrolyze the large molecular proteins and peptides released from the cells into peptides smaller than 50kDa. After the reaction was completed, the proteins were inactivated by high temperature at 90℃ and then cooled to room temperature.

[0084] S6. Obtaining small molecule peptides: At this point, the reaction system is generally alkaline. Add 0.1g of exonuclease-aminopeptidase to the reaction system and react at 65℃ for 4h. This will generate small molecule peptides (<30KDa, or even <10KDa) from the peptides digested in steps S3, S4, and S5 above. After the reaction is complete, inactivate the peptides at 95℃ and allow them to stand to return to room temperature.

[0085] S7. Decolorization and Fine Filtration: After centrifuging the above reaction system at 12000 rpm for 30 min at room temperature, take the supernatant. Adjust the pH to 4.78 with a 20% (m / m) citric acid aqueous solution, add 1-10 nm activated carbon, and stir at 55℃ for 2 h. The mass-volume ratio of activated carbon to supernatant is 0.7%:1 g / mL. Adsorb oxalic acid and chlorophyll present in the system. After centrifuging at 12000 rpm for 30 min, take the supernatant. Filter the supernatant through a nylon membrane with a pore size of 1-10 µm to obtain a clear supernatant. Then filter it through a 0.22 µm filter membrane to obtain an even clearer and more transparent supernatant.

[0086] S8. Concentration: The supernatant obtained in S7 is concentrated by rotary evaporation at 65°C to 1 / 5 of the volume of the supernatant; the concentrated solution is ultrafiltered using an ultrafiltration centrifuge tube with a strength of <10 kDa to obtain a solution of small molecule peptides and amino acids with a strength of <10 kDa; the ultrafiltration process is the same as in Example 1.

[0087] Example 3

[0088] A method for preparing a solution of small molecule peptides and amino acids from leafy green plants includes the following steps:

[0089] S1. Raw material pretreatment: Take fresh edible grass that has grown to 50-60 cm, dry it with hot air at 60℃ to a moisture content of 6.3%, then crush it and pass it through a 20-60 mesh sieve to obtain edible grass powder. Take 100g of edible grass powder and mix it with water at a solid-liquid ratio of 1:15g / mL to prepare an edible grass powder suspension. Inactivate it at 80℃ for 15min.

[0090] S2. Ultrasonic disruption: The leafy grass powder suspension obtained in step S1 was naturally cooled to 40℃ and ultrasonically disrupted for 1.5 hours at a power of 300W. After ultrasonication, it was naturally cooled to 25℃. 2g of cellulase and 1g of pectinase were added to the leafy grass powder suspension and reacted for 1 hour. Then, the temperature was raised to 55℃ and the mixture was soaked for another 3 hours to release intracellular proteins, flavonoids, polyphenols, and oxalic acid. After the reaction was completed, the mixture was inactivated by high-temperature inactivation at 90℃ and then cooled to room temperature. At this point, the pH was 4.95.

[0091] S3, Acidic enzymatic hydrolysis of proteins: At this point, 5g of plant aspartic protease is added to the system and reacted at 50℃ for 3 hours to hydrolyze the large molecular proteins released from the cells into peptides less than 150kDa. After the reaction is completed, the proteins are inactivated by high temperature at 60℃ and then cooled to room temperature.

[0092] S4, Neutral enzymatic hydrolysis of proteins: The pH value was finely adjusted to 6.94 using a 25% (m / m) ammonia solution. 0.5g of bromelain was added to the reaction system and the reaction was carried out at 45℃ for 4h to further hydrolyze the large molecular proteins released from the cells into peptides less than 100kDa. After the reaction was completed, the mixture was passed through 90℃ and then cooled to room temperature.

[0093] S5. Alkaline enzymatic hydrolysis of proteins: The pH value is finely adjusted to 8.05 using a 25% (m / m) ammonia solution. 1g of alkaline protease is added to the reaction system and the reaction is carried out at 55℃ for 2h to further hydrolyze the large molecular proteins and peptides released from the cells into peptides with less than 50kDa. After the reaction is completed, the mixture is passed through 90℃ and then cooled to room temperature.

[0094] S6. Obtaining small molecule peptides: At this point, the reaction system is generally alkaline (pH 8.12). Add 0.15g of exonuclease-aminopeptidase to the reaction system and react at 65℃ for 4h. This will generate small molecule peptides (<30kDa, or even <10kDa) from the peptides digested in steps S3, S4, and S5 above. After the reaction is complete, inactivate the peptides at 95℃ and allow them to stand to return to room temperature.

[0095] S7. Decolorization and Fine Filtration: After centrifuging the above reaction system at 12000 rpm for 30 min at room temperature, collect the supernatant. Adjust the pH to 4.81 with a 20% (m / m) citric acid aqueous solution, add 1-10 nm activated carbon, and stir at 60℃ for 2 h. The mass-volume ratio of activated carbon to supernatant is 1%:1 g / mL. Adsorb oxalic acid and chlorophyll present in the system. After centrifuging at 12000 rpm for 30 min, collect the supernatant. Filter the supernatant through a nylon membrane with a pore size of 1-10 µm to obtain a clear supernatant. Then filter it through a 0.22 µm filter membrane to obtain an even clearer and more transparent supernatant.

[0096] S8. Concentration: The supernatant obtained in S7 is concentrated by rotary evaporation at 65°C to 1 / 5 of the volume of the supernatant; the concentrated solution is ultrafiltered using an ultrafiltration centrifuge tube with a strength of <10 kDa to obtain a solution of small molecule peptides and amino acids with a strength of <10 kDa; the ultrafiltration process is the same as in Example 1.

[0097] Comparative Example 1

[0098] Everything else is the same as in Example 1, except that:

[0099] In step S3, add 2g of plant aspartic protease;

[0100] In step S4, the pH value is finely adjusted to 6.95 using a 25% (m / m) ammonia solution, and 0.1g of bromelain is added to the reaction system.

[0101] In step S5, the pH value is finely adjusted to 8.51 using a 25% (m / m) ammonia solution, and 0.5g of alkaline protease is added to the reaction system.

[0102] Without adding exonuclease-aminopeptidase (step S6 omitted), proceed directly to step S7 for decolorization and fine filtration. During the decolorization and fine filtration process, adjust the pH to 4.75 using a 20% (m / m) citric acid aqueous solution.

[0103] In step S8, the concentrate is not subjected to ultrafiltration.

[0104] Comparative Example 2

[0105] Everything else is the same as in Example 1, except that:

[0106] In step S3, add 1g of plant aspartic protease;

[0107] In step S4, the pH value was finely adjusted to 6.94 using a 25% (m / m) ammonia solution, and 0.5g of bromelain was added to the reaction system.

[0108] In step S5, the pH value is finely adjusted to 8.45 using a 25% (m / m) ammonia solution, and 1g of alkaline protease is added to the reaction system.

[0109] In step S6, 0.15g of exonuclease-aminopeptidase is added to the reaction system;

[0110] In step S7, the pH is adjusted to 4.48 using a 20% (m / m) aqueous solution of citric acid.

[0111] Comparative Example 3

[0112] Everything else is the same as in Example 2, except that:

[0113] In step S3, add 3g of plant aspartic protease;

[0114] In step S4, the pH value was finely adjusted to 6.95 using a 25% (m / m) ammonia solution, and 0.05g of bromelain was added to the reaction system.

[0115] In step S5, the pH value is finely adjusted to 8.42 using a 25% (m / m) ammonia solution, and 1g of alkaline protease is added to the reaction system.

[0116] In step S6, 0.1 g of exonuclease-aminopeptidase is added to the reaction system;

[0117] In step S7, the pH is adjusted to 4.35 using a 20% (m / m) aqueous solution of citric acid.

[0118] Comparative Example 4

[0119] Everything else is the same as in Example 1, except that:

[0120] In step S3, add 5g of plant aspartic protease;

[0121] In step S4, the pH value was finely adjusted to 6.92 using a 25% (m / m) ammonia solution, and 0.5g of bromelain was added to the reaction system.

[0122] In step S5, the pH value is finely adjusted to 8.66 using a 25% (m / m) ammonia solution, and 0.3g of alkaline protease is added to the reaction system.

[0123] In step S6, 0.1 g of exonuclease-aminopeptidase is added to the reaction system;

[0124] In step S7, the pH is adjusted to 4.57 using a 20% (m / m) citric acid aqueous solution;

[0125] Step S8: The obtained concentrate is ultrafiltered using ultrafiltration centrifuge tubes with molecular weights of <100KDa, <50KDa, <30KDa and <10KDa respectively to obtain small molecule peptide and amino acid solutions with different molecular weights.

[0126] Experimental Example 1: Detection of Small Molecule Peptides and Amino Acids in Leafy Grass Solution

[0127] A. Detection of amino acid content:

[0128] (1) Prepare a 1.25 μmol / mL glutamic acid standard using water as the solvent, mix thoroughly, and prepare immediately before use;

[0129] (2) The amino acid (AA) content test kit was tested according to its instructions:

[0130] Mark the blank tube, standard tube, and test tube on the EP tube, and set up 3 parallel samples for each group.

[0131] Add a certain amount of distilled water to the blank tube;

[0132] Add an equal volume of 1.25 μmol / mL glutamic acid standard solution to the standard tube;

[0133] Add an equal volume of the sample solution to the test tube;

[0134] The sample test solution is a small molecule polypeptide and amino acid solution prepared in Examples 1-3 and Comparative Examples 1-4.

[0135] (3) Vortex the blank tube, standard tube and test tube above, place them in a boiling water bath for 15 min, cool them and invert them repeatedly, centrifuge them and take the supernatant into a 96-well plate and use an enzyme-linked immunosorbent assay reader to measure the absorbance value A at 570 nm.

[0136] (4) Calculate the amino acid content:

[0137] Amino acid content (μmol / mL) = 2.5 * ΔA 测定 / ΔA 标准 *F(1),

[0138] ΔA 测定 =A 测定管 -A 空白管 (2),

[0139] ΔA 标准 =A 标准管 -A 空白管 (3);

[0140] Where F is the dilution factor, A 测定管 To measure the absorbance of the tube, A 空白管 A represents the absorbance value of the blank tube. 标准管 This is the absorbance value of the standard tube.

[0141] B. Detection of polypeptide content:

[0142] 1) Preparation of Standards: Prepare peptide standards at concentrations of 0, 15.625, 31.25, 62.5, 125, 250, 500, and 1000 μg / ml according to the table below. Ensure thorough mixing before each dilution.

[0143] Table 1. Preparation of peptide standards

[0144]

[0145] (2) Preparation of BevoBCA working solution: Add 900 μL of Reagent A, 864 μL of Reagent B and 36 μL of Reagent C to a 10 ml test tube and mix thoroughly to prepare BevoBCA working solution. Each reaction requires 180 μL of BevoBCA working solution.

[0146] (3) Take 20 μL of each of the different concentrations of polypeptide standards prepared in (1) and add them to the standard wells of the 96-well plate.

[0147] (4) Add 20 μL of sample to the sample wells of the 96-well plate;

[0148] The samples were small molecule polypeptide and amino acid solutions prepared in Examples 1-3 and Comparative Examples 1-4.

[0149] (5) Add 180 μL of BevoBCA working solution to the standard well and the sample well respectively, mix with a microplate shaker for 1 minute, and place at 37°C for 15 minutes.

[0150] (6) Use an ELISA reader to measure the absorbance at a wavelength of 480 nm. The absorbance measurements of both the standard and the sample should be reduced by the absorbance measurement of the blank standard of 0 μg / ml. The absorbance of the standard and the sample after blank correction is calculated and used to plot the standard curve and calculate the sample concentration.

[0151] (7) Calculate the peptide concentration in the sample wells based on the standard curve;

[0152] Table 2. Results of determination of small molecule peptides and amino acid content

[0153]

[0154] Quantitative analysis confirmed that the small molecule peptides and amino acid solutions prepared in Examples 1-3 and Comparative Examples 1-4 contained both small molecule peptides and amino acids, with small molecule peptides being the main component and amino acid content being relatively low.

[0155] The results of Comparative Example 1 showed that after continuous enzymatic hydrolysis with endonucleases at different pH values, the protein in the leafwort could be fully hydrolyzed into polypeptides with multiple ends, yielding more than 80% of the total polypeptides, but the amino acid content was relatively low at this point. The results of Examples 1 and 2 showed that, based on Comparative Example 1, the addition of an exonuclease-aminopeptidase that met the pH requirements of the final enzymatic hydrolysis could promote further enzymatic hydrolysis of the polypeptides, yielding more small-molecule peptides and amino acids. Further ultrafiltration separated peptides of <30 kDa and <10 kDa and more amino acids, finding that most of the small-molecule polypeptides were concentrated in <10 kDa, with a yield of over 62%. The results of Examples 2 and 3 showed that, from the perspective of being more economical and environmentally friendly, there was no significant difference between the results of Examples 2 and 3. Therefore, the sample obtained in Example 2 was selected for subsequent research on efficacy and application.

[0156] Compared with Comparative Example 2, the results of Example 1 show that the amount of plant aspartic protease added affects whether the hydrolysis of leafy grass protein can proceed smoothly, which is the key to starting the hydrolysis of leafy grass protein.

[0157] Compared with Comparative Example 3, the results of Example 2 show that bromelain plays a bridging role, achieving broad-spectrum cleavage of leafy green protein under mild conditions, and affecting the hydrolysis efficiency of leafy green protein to some extent.

[0158] Compared to Examples 1 and 2 and Comparative Example 4, the results showed that the addition of alkaline protease not only affected the degree of hydrolysis and molecular weight distribution of small protein molecules in leafy greens, but also provided more cleavage sites for the addition of exonucleases during hydrolysis, thus affecting the efficiency of the hydrolysis process. In conclusion, each enzyme plays an important role in the hydrolysis of leafy greens; however, from the perspective of economic efficiency and obtaining smaller molecular weight peptides and amino acids, Example 2 is the best choice and can be used for subsequent research.

[0159] The small molecule peptides and amino acid solutions prepared in Example 2 were freeze-dried to prepare freeze-dried powders of small molecule peptides and amino acids. The freeze-dried powders were used for the analysis of Examples 2-7. The freeze-drying process was as follows: pre-cooling at -45℃ for 3 hours; sublimation at 0℃ for 18 hours.

[0160] In Experiments 2-7, water was used as the solvent and freeze-dried small molecule peptides and amino acids as the solutes to prepare solutions of leafy grass small molecule peptides and amino acids.

[0161] Experimental Example 2: Determination of the antioxidant efficacy (DPPH, ·OH and O) of the small molecule polypeptides and amino acid solutions of *Gnaphalium affine* prepared in Example 2 at the biochemical level. 2- )

[0162] Exogenous skin aging is primarily due to photoaging, which leads to excessive accumulation of free radicals within cells, inducing oxidative stress and ultimately causing skin cell aging. Therefore, this study investigates whether the inhibition of small molecule peptides and amino acids from *Gynostemma pentaphyllum* can inhibit 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH). 2- The generation of ·OH free radicals is used to assess its antioxidant capacity.

[0163] A. Scavenging rate of DPPH by leafy green plant small molecule peptides and amino acid solution

[0164] (1) Using water as a solvent, prepare 0.1 mM DPPH solution and 0.3, 0.6, and 1 mg / ml solutions of small molecule polypeptides and amino acids from the leafy green plant;

[0165] (2) Sample group, control group and blank group were set up respectively; the sample group was prepared with 180µL+90µL DMSO+90µL LPPH of different concentrations of small molecule peptides and amino acids of edible grass, the control group was prepared with 180µL DMSO+90µL LPPH, and the blank group was prepared with 180µL+90µL anhydrous ethanol of different concentrations of small molecule peptides and amino acids of edible grass.

[0166] (3) Shake in the dark for 10 minutes, and zero the container with anhydrous ethanol.

[0167] (4) Test absorbance at 520 nm.

[0168] (5) Record and analyze the data.

[0169] DPPH clearance rate % = (A 对照组 -(A 样品组 -A 空白组 )) / A 对照组 *100% (4)

[0170] Among them, A 对照组 A represents the absorbance of the control group. 样品组 A represents the absorbance of the sample group. 空白组 The absorbance of the blank group is shown.

[0171] B. Scavenging rate of ·OH by leafy green plant small molecule polypeptide and amino acid solution:

[0172] (1) Using water as a solvent, prepare 9 mmol / L ethanol-salicylic acid aqueous solution, 9 mmol / L ferrous sulfate solution, 8.8 mmol / L H2O2 and different concentrations of 0.3, 0.6 and 1 mg / ml of small molecule polypeptides and amino acids from the leafwort plant;

[0173] (2) The reaction system was carried out in a 1.5 mL colorimetric tube, and the order of sample addition is shown in Table 3 below;

[0174] (3) Shake well, incubate in a 37℃ water bath for 15 min, and measure its absorbance; the absorbance of the sample is measured as A. X The absorbance of the blank control is A0, and the absorbance of the sample background is A. X0 ;

[0175] (4) Record and analyze the data. ·OH removal rate % = [A0 - (A X -A X0 )] / A0*100%(5).

[0176] Table 3. Sample addition sequence for determining hydroxyl radical scavenging ability using the ethanol-salicylic acid method.

[0177]

[0178] C. The effect of leafwort small molecule polypeptides and amino acid solutions on O 2- Sweep rate:

[0179] (1) Prepare 10 mmol / L HCl solution, 5 mmol / L pyrogallol solution, 0.1 mol / L Tris-HCl buffer, and 0.3, 0.6, and 1 mg / ml of leafy green small molecule peptides and amino acids respectively;

[0180] 5 mmol / L pyrogallol solution: Weigh a certain amount of pyrogallol and dissolve it in 10 mmol / L HCl to prepare a 5 mmol / L pyrogallol solution. Prepare and use immediately.

[0181] Preparation of 0.1 mol / L Tris-HCl buffer: Weigh 12.114 g Tris powder, dissolve it in 800 ml of purified water, adjust the pH to 8.2 with concentrated hydrochloric acid, and bring the volume to 1 L with purified water.

[0182] (2) This reaction is carried out in a cuvette, and the order of sample addition is shown in Table 4;

[0183] (3) After rapid mixing and shaking, the absorbance of the solution is measured at 325 nm as the first absorbance value. The absorbance is measured every 1 min for 4 min. The increase of absorbance per minute within the linear range is calculated. △A0 is the auto-oxidation rate of pyrogallol; △A is the auto-oxidation rate of pyrogallol after the sample solution is added.

[0184] (4) Record and analyze the data, O 2- Clearance rate (%) = (△A0-△A) / △A0×100% (6).

[0185] Table 4. Sampling order for superoxide anion scavenging determination by pyrogallol method

[0186]

[0187] Figure 1 The results of this test case on the scavenging rate of free radicals are presented to evaluate its antioxidant efficacy at the biochemical level. (a) shows the scavenging rate of ·OH, (b) shows the scavenging rate of DPPH, and (c) shows the scavenging rate of O2. 2- The clearance rate results showed that small molecule peptides and amino acids from *Gnaphalium affine* inhibited DPPH and O in a dose-dependent manner. 2- The generation of ·OH free radicals, among which, small molecule peptides and amino acids from *Gynostemma pentaphyllum* at 0.6 mg / ml, have an effect on DPPH and O2. 2- The scavenging rates of ·OH free radicals reached 66%, 35%, and 89%, respectively; the scavenging rates of small molecule peptides and amino acids from the leafwort at 1 mg / ml were effective against DPPH and O2. 2- The scavenging rates of ·OH radicals reached 71%, 55%, and 94%, respectively.

[0188] Experiment 3: Determining the transdermal absorption rate of the leafwort small molecule peptides and amino acid solutions prepared in Example 2 and their effect on cell viability (MTT).

[0189] Peptide raw materials have always been popular in the market, but their transdermal absorption is a problem that has attracted much attention from researchers. This study utilized a Franz diffusion cell with porcine ex vivo skin samples. The cell was filled with a buffer solution simulating bodily fluids (such as PBS at pH 7.4) and kept at a constant temperature (typically 32-37°C) with continuous agitation to simulate physiological conditions. Cosmetic raw materials diffused from the supply chamber through various layers of porcine skin (mainly the stratum corneum) to the receiving chamber. Samples were taken periodically from the receiving chamber, and the concentration of permeated raw materials was measured using appropriate methods. The cumulative permeation rate per unit time was calculated to evaluate the transdermal absorption rate of the raw materials.

[0190] From a histological perspective, skin aging is mainly manifested in the decreased activity of fibroblasts in the dermis, reduced collagen synthesis, and accelerated collagen degradation. This disrupts the dense and orderly collagen fiber network structure of the skin, causing it to collapse and ultimately leading to skin laxity and wrinkles. Assessing the impact of raw materials on fibroblast activity can, to some extent, evaluate the anti-aging effect of those materials.

[0191] A. Analysis of the transdermal absorption effect of leafwort small molecule peptides and amino acid solutions:

[0192] This experiment used the Franz diffusion cell-porcine ex vivo skin model to evaluate the efficacy of the penetration enhancer: First, fresh porcine ear skin was taken, subcutaneous fat was removed and the thickness was controlled to 0.8 mm. The skin was then fixed in the diffusion cell (effective area 1 cm²) with the stratum corneum facing upwards. 2 The receiving chamber was filled with a pre-warmed saline solution at 37°C and magnetically stirred at 400 rpm. 2 ml of a 10 mg / ml solution of small molecule peptides and amino acids from the herb *Eclipta prostrata* was added to the supply chamber. Samples were taken from the receiving chamber at 2, 4, 6, 8, and 12 hours. The protein content was detected using a peptide kit. Finally, the cumulative amount of small molecule peptides and amino acids from the herb *Eclipta prostrata* and the amount of transdermal absorption were calculated to evaluate its transdermal absorption rate.

[0193] B. Analysis of the effects of small molecule peptides and amino acid solutions from *Gynostemma pentaphyllum* on fibroblast viability:

[0194] (1) Select Hacat cells that are in good growth condition and in the logarithmic growth phase, digest them with trypsin and centrifuge them, discard the supernatant, and gently pipette the cell pellet with 2 mL of fresh culture medium to obtain a cell suspension.

[0195] (2) Dilute the cell suspension to a concentration of 4×10⁻⁶. 4 After inoculating the cells / mL into a 96-well plate, sterile PBS was used in the edge wells, and three replicates were set up.

[0196] (3) After the cells have completely adhered to the wall and entered the logarithmic growth phase, the control group was replaced with fresh culture medium, and the experimental group was given 200 μL of culture medium containing different concentrations of small molecule peptides and amino acids from the leafy grass, and cultured for another 24 h.

[0197] (4) After the culture is completed, add 10% CCK-8 solution to each well and incubate again for 2-3 hours;

[0198] (5) Select 490 nm as the detection wavelength and use an ELISA reader to measure the absorbance (OD value) of each well.

[0199] (6) Cell survival rate (%) = (OD experimental group - OD zeroing group) / (OD blank control group - OD zeroing group) × 100%.

[0200] Figure 2The results showed the transdermal absorption rate of small molecule peptides and amino acids from *Hedyotis diffusa* and their effect on cell viability. (a) shows the transdermal absorption rate of small molecule peptides and amino acids detected using a TT-6TT-8 transdermal absorption analyzer, and (b) shows the effect of different concentrations of small molecule peptides and amino acids on cell viability using a CCK-8 assay. The transdermal absorption rate results showed that small molecule peptides and amino acids increased the transdermal absorption rate in a time-dependent manner, reaching approximately 70% at 12 h, indicating good transdermal absorption. The cell viability results showed that, compared with the control group (CTR), small molecule peptides and amino acids at 0.1 mg / ml had no significant effect on cell viability. Cell viability decreased between 1-10 mg / ml, with 10 mg / ml reaching the half-inhibitory concentration. However, small molecule peptides and amino acids at 0.5 mg / ml significantly promoted cell proliferation.

[0201] Experimental Example 4: Antioxidant analysis of the small molecule peptides and amino acids of *Gnaphalium affine* prepared in Example 2 at the cellular level.

[0202] ROS (reactive oxygen species) is a collective term for the "reactive oxygen species family," which includes true free radicals (such as O2). 2- ·, ·OH and RO2·), and also some non-free radical but highly reactive oxygen-containing molecules (such as H2O2, O 2- ONOO - ). Continuous skin exposure to ultraviolet radiation (especially UVA / UVB) and pollution can induce the generation of large amounts of ROS / free radicals.

[0203] Excessive accumulation of free radicals promotes cellular aging by inducing oxidative stress, while intracellular antioxidant enzymes can scavenge free radicals generated in the body. For example, superoxide dismutase (SOD) maintains metabolic homeostasis by catalyzing the dismutation of superoxide anion free radicals into hydrogen peroxide and oxygen. By constructing an oxidative damage model, the antioxidant capacity of Hacat small molecule peptides and amino acids was assessed by detecting their ability to scavenge intracellular ROS.

[0204] A. Effects of leafy green plant small molecule peptides and amino acid solutions on ROS scavenging ability:

[0205] (1) Select Hacat cells that are in good growth condition and in the logarithmic growth phase at a concentration of 1*10 4 The cells were seeded at a density of 100% in a six-well plate and processed after they adhered to the plate.

[0206] (2) After the experimental group was treated with 200µM H2O2 for 2-3 hours, it was washed 3 times with PBS. The control group was replaced with fresh culture medium. The experimental group was treated with 0.5mg / ml of leafwort small molecule peptide and amino acid solution for 24 hours, and then the ROS index was detected.

[0207] (3) Dilute the DCFH-DA probe with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10 μM;

[0208] (4) Discard the original culture medium, add an appropriate volume of diluted DCFH-DA probe, and incubate at 37°C for 20 min.

[0209] (5) Wash the cells three times with serum-free cell culture medium to thoroughly remove the DCFH-DA probes that have not entered the cells;

[0210] (6) Observe under a fluorescence microscope.

[0211] Figure 3 This image shows the results of ROS generation in H2O2-induced oxidation model cells in this experiment using small molecule peptides and amino acids from *Gnaphalium affine*. Bright field plots show the morphology and density of cells in each group, ROS plots show the fluorescence intensity of reactive oxygen species in the cells, CTR plots represent the control group, H2O2 plots represent the oxidation-induced model group, and H2O2+0.5mg / mL plots represent the group that was treated with 0.5mg / mL *Gnaphalium affine* small molecule peptide and amino acid solution after H2O2 induction. Figure 3 The results showed that, compared with the control group, H2O2 treatment of Hacat cells significantly promoted the accumulation of ROS, while the addition of small molecule peptides and amino acids from the herbaceous plant significantly inhibited the production of ROS.

[0212] Experimental Example 5: Determination of the effects of small molecule peptides and amino acids from *Eclipta prostrata* prepared in Example 2 on collagen (ELISA)

[0213] Collagen is the main structural protein of the skin. With age, collagen synthesis slows down while degradation accelerates, leading to decreased skin elasticity and firmness, resulting in wrinkles and sagging. To investigate whether small molecule peptides and amino acids from *Hedyotis diffusa* can delay aging by promoting collagen expression, we used ELISA to examine the effects of these peptides and amino acids on the levels of type I and type III collagen.

[0214] A. Effects of leafwort small molecule peptides and amino acid solutions on type I and type III collagen:

[0215] (1) Select HSF cells that are in good growth condition and in the logarithmic growth phase, digest them with trypsin and centrifuge them, discard the supernatant, and gently pipette the cell pellet with 2 mL of fresh culture medium to obtain a cell suspension.

[0216] (2) After thoroughly mixing the cell suspension, take 10 μL and count the cells using a hemocytometer. Dilute the cell suspension to a concentration of 4 × 10⁻⁶. 4 cells / mL;

[0217] (3) Mix the cell suspension thoroughly and then seed it into a six-well plate. Set up three replicates for each concentration gradient. Note that after adding a few wells, you need to resuspend the cells by pipetting.

[0218] (4) The next day, the old culture medium was discarded, the blank control group was replaced with fresh culture medium, and the sample group was given 0.5 mg / ml of small molecule peptides and amino acids of edible grass and placed in a cell culture incubator for 24 h.

[0219] (5) After 24 hours of treatment, discard the original culture medium, wash the cells twice with PBS, add cell lysis buffer and lyse for 30 minutes, sonicate for 5-10 minutes, centrifuge to obtain supernatant for subsequent experiments.

[0220] (6) After incubation, the corresponding type I and type III collagen ELISA kits were used for experimental determination;

[0221] (7) Remove the required strips from the aluminum foil bag after equilibration at room temperature for 20 minutes, and seal the remaining strips in a self-sealing bag and return them to 4℃;

[0222] (8) Set up standard wells and sample wells, and add 50 μL of standard at different concentrations to each standard well;

[0223] (9) Add 50 μL of the sample to be tested to the sample well first; do not add any to the blank well;

[0224] (10) Except for the blank wells, add 100 μL of horseradish peroxidase (HRP) labeled antibody to each of the standard wells and sample wells, seal the reaction wells with sealing film, and incubate in a water bath or incubator at 37°C for 60 min.

[0225] (11) Discard the liquid, pat dry on absorbent paper, fill each well with washing liquid, let stand for 1 minute, shake off the washing liquid, pat dry on absorbent paper, and repeat this washing process 5 times.

[0226] (12) Add 50 μL of substrate A and B to each well and incubate at 37°C in the dark for 15 min;

[0227] (13) Add 50 μL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm within 15 min;

[0228] (14) Standard curve plotting and sample concentration calculation: Establish a standard curve based on the concentration (x, pg / mL) of the standard tube and the absorbance ΔA standard (y, ΔA standard). Based on the standard curve, substitute the ΔA measurement (y, ΔA measurement) into the formula to calculate the sample concentration (x, pg / mL).

[0229] Figure 4 The results of the effects of small molecule peptides and amino acids of edible grass on type I and type III collagen in Experiment Example 5 of the present invention are shown. Among them, (a) is a graph showing the effect of small molecule peptides and amino acids of edible grass on type I collagen, and (b) is a graph showing the effect of small molecule peptides and amino acids of edible grass on type III collagen. Figure 4 The results showed that, compared with the control group, the addition of small molecule peptides and amino acids from edible leaves promoted the synthesis of type I and type III collagen, indicating that small molecule peptides and amino acids from edible leaves have good anti-aging effects.

[0230] Experimental Example 6: Analysis of the soothing and repairing effects of the small molecule peptides and amino acids of *Hydrocotyle vulgaris* prepared in Example 2 (scratch and hyaluronidase).

[0231] Hyaluronidase regulates the extracellular matrix by degrading hyaluronic acid (HA), exhibiting multiple functions in skin repair, including accelerating healing, soothing, and promoting collagen production. Cellular repair capacity is crucial for skin repair, and scratch assays reflect, to some extent, the ability of cells to repair or migrate. This study evaluated the soothing and repairing efficacy of raw materials by detecting the inhibition rates of hyaluronidase and cellular repair capacity of small molecule peptides and amino acids from *Hydrangea macrophylla*.

[0232] A. Effects of small molecule peptides and amino acid solutions from leafy green plants on hyaluronidase

[0233] (1) At room temperature, hyaluronidase, sodium hyaluronate and buffer solution were added to the reaction system, and the pH of the reaction system was controlled within the range of 5.6-6.0. At the same time, a blank control group (without hyaluronidase) and a negative control group (without sample) were set up.

[0234] (2) Add the raw material solution to be tested into the reaction system to ensure that the sample and hyaluronidase are in full contact. Incubate at 37°C for 30 min to allow the hyaluronidase and sodium hyaluronate to react fully.

[0235] (3) Add acetylacetone and p-dimethylaminobenzaldehyde to carry out a color reaction, which is usually carried out under alkaline conditions;

[0236] (4) The absorbance of the reaction system was measured at 530 nm using a UV spectrophotometer;

[0237] (5) Calculate the hyaluronidase inhibition rate: Hyaluronidase inhibition rate (%) = {1 – (B1-B2) / (A1-A2)}*100, where: A1 is the absorbance of the reaction solution without the sample; A2 is the absorbance of the reaction solution without the sample and enzyme; B1 is the absorbance of the reaction solution containing the sample and enzyme; B2 is the absorbance of the reaction solution containing the sample and without the enzyme.

[0238] (6) Data analysis: The soothing effect of the sample was determined by comparing the hyaluronidase inhibition rate of the sample group and the negative control group.

[0239] B. Effects of small molecule peptides and amino acid solutions from leafy green plants on cell repair capabilities

[0240] (1) Select cells in good growth condition, digest them with trypsin, and count the cells at a rate of 1×10⁻⁶. 5 Seed cells at a density of 100 cells / well in 24-well plates (mark the back of the 24-well plates with a pencil for easy photography), shake to mix, and place in an incubator;

[0241] (2) The next day, when the cell growth density reaches 80%~90%, remove the culture medium, scratch each well with a 200 μL sterile pipette tip, so that the scratch area and area in each well are as consistent as possible, and wash the cells three times with PBS buffer to remove floating cells.

[0242] (3) Randomly select the scratch area of ​​each hole to take a picture, that is, the scratch width at 0 hours, and record the picture position;

[0243] (4) Add cell culture medium containing 1% serum (control group) or cell culture medium containing 1% serum to each well containing small molecular polypeptides and amino acids of edible grass (experimental group), and set up 3 replicates for each group;

[0244] (5) After 20 hours of treatment, take a picture at the original location and calculate the migration rate = (0-hour scratch width - 20-hour scratch width) / 0-hour scratch width × 100%.

[0245] Figure 5 The results of the hyaluronidase inhibition experiment showed that the small molecule peptides and amino acids of *Eclipta prostrata* inhibited the activity of hyaluronidase in a concentration-dependent manner. At 0.6 mg / ml, the inhibition rate of hyaluronidase reached 85%, and at 1 mg / ml, the inhibition rate of hyaluronidase reached 93%, indicating that it has a good soothing effect. Figure 6 The results of cell migration ability in this experiment are shown in the figure. (a) is a microscopic image of the cell scratch test, and (b) is the statistical result of the relative migration rate of cells in each group. Figure 6The results of the scratch test showed that small molecule peptides and amino acids of *H. coli* significantly promoted the repair ability of *H. coli* cells at 0.5 mg / ml, indicating that it has good repair efficacy.

[0246] Experiment 7: Determination of the safety and non-irritation of the small molecule peptides and amino acids of the leafwort prepared in Example 2.

[0247] To further verify its safety, the safety of low (5 mg / mL), medium (10 mg / mL), and high (50 mg / mL) concentrations of leafy green small molecule peptides and amino acids was first verified by taking advantage of the intact, clear, and transparent chorioallantoic membrane vascular system of the hatched chicken embryos.

[0248] (1) CAM preparation: Select 7-day-old chicken embryos, check by candling, place the air cell end of the chicken embryo upwards, and draw the outline of the air cell of the chicken embryo with a pencil.

[0249] (2) Use pointed tweezers to drill a hole directly above the air cell of the chicken embryo. Carefully peel off the eggshell above the air cell along the outline of the air cell with tweezers. Blow away the eggshell that has fallen onto the air cell membrane. Add 1 mL of physiological saline to moisten the eggshell membrane and discard the remaining physiological saline. Carefully tear off the eggshell membrane with sterilized tweezers (do not puncture the allantoic membrane and blood vessels during this process) to expose the allantoic membrane with a diameter of 2-3 cm.

[0250] (3) At this point, observe the structure of the vascular system again and make a judgment on its integrity and suitability for the experiment;

[0251] (4) Endpoint assessment method: Since the sample was a transparent liquid, the time assessment method was selected for detection. That is, 0.3 mL of the above transparent liquid was directly dropped onto the CAM surface, the CAM reaction was observed, and the time of occurrence of each toxic effect within 5 min was recorded;

[0252] (5) The reaction time method is used to conduct the test. The stimulus score (IS) is calculated using the following formula and the result is kept to two decimal places. According to the IS value, the eye irritation of the test substance is classified according to the following table.

[0253] IS=(301- sec H) 5 / 300+(301-sec L) x7 / 300+(301-sec C) x9 / 300

[0254] Note: sec H (bleeding time) ----- The average time for the onset of bleeding observed on the CAM membrane, in seconds (s);

[0255] sec L (vascular dissolution time) – The average time, measured in seconds (s), to the onset of vascular dissolution observed on the CAM membrane.

[0256] sec C (clotting time) ----- The average time for clotting to begin to occur as observed on the CAM membrane, measured in seconds (s).

[0257] Table 5 Evaluation of Reaction Time Method Results

[0258]

[0259] Figure 7 The results showed that the IS scores of the negative control group, positive control group (0.1 mol NaOH), low concentration (5 mg / ml), medium concentration group (10 mg / ml), and high concentration group (50 mg / ml) were 0, 17.1, 0.05, 0.15, and 0.63, respectively. Since the IS score of the positive control group was between 10 and 19, this experiment was reliable. The IS values ​​of the sample groups at different concentrations were all less than 1. Therefore, the small molecule peptides and amino acids of the leafwort were safe and non-irritating at low, medium, and high concentrations.

Claims

1. A method for preparing a solution of small molecule polypeptides and amino acids from leafy green plants, characterized in that, Includes the following steps: S1. Prepare a suspension of edible leaf grass powder by mixing it with water, and then sterilize it at high temperature. S2. Sonicate the suspension of leafy grass powder, then lower the temperature to below 30°C, add cellulase and pectinase and react for 1-2 hours, then raise the temperature to 50-60°C and soak for 2-3 hours. After high-temperature inactivation, lower the temperature to room temperature. S3. Add plant aspartic protease to the reaction system obtained in step S2, inactivate it at high temperature after the reaction is completed, and then cool it to room temperature. S4. Adjust the pH of the reaction system obtained in step S3 to 6.0-7.0, add bromelain, inactivate it at high temperature after the reaction is completed, and then cool it to room temperature; S5. Adjust the pH of the reaction system obtained in step S4 to 8.0-9.0, add alkaline protease, inactivate at high temperature after the reaction is completed, and then cool to room temperature; S6. Add exonuclease-aminopeptidase to the reaction system obtained in step S5, react at 50-70℃ for 2-4 hours, inactivate at high temperature after the reaction is completed, and then cool to room temperature. S7. Decolorization and fine filtration: Centrifuge the system obtained in step S6 to obtain the supernatant, adjust the pH of the supernatant to 4.0-5.0, and then finely filter it after treatment with activated carbon to obtain a clear supernatant. S8. Concentration and separation: The supernatant obtained in step S7 is concentrated by rotary evaporation and ultrafiltration to obtain a solution of small molecule peptides and amino acids. The leafy grass powder suspension in step S1 is prepared by mixing leafy grass powder and water at a solid-liquid ratio of 1:10-20 g / mL. In step S2, the cellulase activity is 1:6000-1:8000, and the mass ratio of cellulase to leafy grass powder is 0.1%-2%:1; the pectinase activity is >30U / mg, and the mass ratio of pectinase to leafy grass powder is 0.1%-2%:1; the mass ratio of cellulase to pectinase is 1-2:

1. In step S3, the plant aspartic protease with an enzyme activity >2000U / g is reacted at a mass ratio of 2%-5% to 1 with the leafy grass powder; the reaction temperature is 40-50℃ and the reaction time is 2-3h. Step S4: Bromelain, enzyme activity >200U / mg, with a mass ratio of bromelain to leafy green powder of 0.1%-0.5%:1; reaction temperature 40-55℃, reaction time 2-4h, pH 6.9-7.0; Step S5: Alkaline protease activity >150000U / g, its mass ratio with leafy grass powder is 0.5%-1%:1, reaction temperature is 50-60℃, reaction time is 2-4h; Step S6: The exonuclease-aminopeptidase activity is >15 U / mg, and its mass ratio with the leafy grass powder is 0.1%-0.15%:1; Step S7 involves fine filtration after activated carbon treatment to obtain a clear supernatant. Specifically, activated carbon is added to the supernatant after pH adjustment for decolorization. The mass-volume ratio of activated carbon to supernatant is 0.2%–1%:1g / mL. The mixture is stirred at 45–60℃ for 1–2 hours, centrifuged at 12000rpm for 20–30 minutes, and the supernatant is collected. The obtained supernatant is then filtered and then vacuum filtered to obtain a clear supernatant. The rotary evaporation described in step S8 is carried out at a temperature of 45-65℃.

2. The application of the *Eclipta prostrata* small molecule polypeptide and amino acid solution prepared by the method according to any one of claims 1 in the preparation of anti-aging cosmetics.

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