Dendrobium officinale enzymatic hydrolysate composition, preparation method thereof, and application thereof in anti-aging cosmetics

Abstract

The present invention discloses a Dendrobium officinale protein hydrolysate composition, a preparation method thereof, and an application thereof in anti-aging cosmetics. The Dendrobium officinale protein hydrolysate composition consists of Dendrobium officinale protein hydrolysates TDM-1 and TDM-5, with a mass ratio of 1:(0.5-2); TDM-1 and TDM-5 are peptide components with a molecular weight ≤10 kDa and a peptide component with a molecular weight of 10 kDa < ≤50 kDa, which are obtained by enzymatic hydrolysis of Dendrobium officinale crude protein using a neutral protease and papain. TDM-1 and TDM-5 components have synergistic effects of antioxidant, anti-inflammatory, and barrier repair by inhibiting the activation of the MAPK / NF-κB signaling pathway and promoting ECM repair mediated by the TGF-β pathway, thereby achieving multi-target anti-aging and having low irritation. They can be used in dosage forms such as essences and creams, providing core raw materials for the development of natural anti-aging cosmetics.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and in particular relates to a Dendrobium officinale enzymatic hydrolysate composition, a preparation method thereof, and application thereof in anti-aging cosmetics. Background Art

[0002] While current mainstream anti-aging ingredients like vitamin C, retinol, and synthetic peptides offer significant efficacy, they face significant drawbacks: Vitamin C is easily inactivated by light and heat, and high concentrations can easily cause allergies; retinol is highly irritating, causing peeling and redness in approximately 30% of users; and synthetic peptides have low transdermal penetration and are expensive. A deeper issue lies in the fact that these chemically synthesized ingredients often rely on single mechanisms (such as antioxidant protection or stimulation of collagen synthesis), overlooking the complex multi-pathway regulation of aging, including inflammation (NF-κB pathway) and cellular stress (MAPK pathway). This results in limited efficacy and difficulty maintaining long-term results.

[0003] Dendrobium officinale ( Dendrobium officinale Kimura Dendrobium officinale (Dendrobium officinale) is a traditional and precious Chinese medicinal herb used both as a medicine and a food, boasting benefits such as nourishing yin and clearing heat, promoting fluid production and moistening dryness, and prolonging life. Modern pharmacological research reveals that its stems are rich in active ingredients such as polysaccharides, alkaloids, and proteins. Polysaccharides have become a research focus due to their significant immunomodulatory and antioxidant properties. However, relatively little research has been conducted on proteins and enzymatic peptides derived from Dendrobium officinale. Chinese invention patent application publication number CN113337566A discloses a method for preparing Dendrobium officinale polypeptides. This method involves first obtaining Dendrobium officinale proteins through a non-protease extraction method, then enzymatically hydrolyzing them with neutral protease and papain to obtain a hydrolyzate. The hydrolyzate is then separated into fractions with molecular weights of 1000 to 3000 Da, resulting in a Dendrobium officinale polypeptide extract. This extract retains only the smaller molecular weight peptides, without the full molecular weight functional peptides. At present, there are no reports on obtaining polypeptide components from Dendrobium officinale that can inhibit the activation of the MAPK / NF-κB signaling pathway, promote ECM repair mediated by the TGF-β pathway, have synergistic antioxidant, anti-inflammatory, and barrier repair effects, and achieve multi-target anti-aging. Summary of the Invention

[0004] The purpose of the present invention is to overcome the defects and shortcomings of the existing natural active peptides in the anti-aging field, which only act on a single pathway such as ROS scavenging or RAR receptor activation, and to provide a Dendrobium officinale enzymatic hydrolysate composition with multi-target synergistic anti-aging (antioxidant, anti-inflammatory, and barrier repair) effects.

[0005] The second object of the present invention is to provide a method for preparing the Dendrobium officinale enzymatic hydrolysate composition.

[0006] The third object of the present invention is to provide the application of the Dendrobium officinale enzymatic hydrolysate composition in anti-aging cosmetics.

[0007] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0008] A Dendrobium officinale protein hydrolysate composition is composed of Dendrobium officinale protein hydrolysate products TDM-1 and TDM-5 in a mass ratio of 1:(0.5-2); TDM-1 and TDM-5 are obtained by enzymatically hydrolyzing Dendrobium officinale protein using a neutral protease and papain, and then separating the peptide component with a molecular weight of ≤10 kDa (i.e., TDM-1) and the peptide component with a molecular weight of 10 kDa < ≤50 kDa (i.e., TDM-5).

[0009] This invention utilizes a composite enzyme-directed enzymatic hydrolysis and gradient separation method to fully retain the full molecular weight functional peptides (≤50 kDa) of the Dendrobium officinale protein hydrolysate, avoiding the drawbacks of existing technologies due to single-molecule retention (e.g., retaining only fractions <1 kDa). Furthermore, molecular weight fractionation allows for functional complementarity between TDM-1 and TDM-5. The core active components, TDM-1 (≤10 kDa) and TDM-5 (10 kDa < ≤50 kDa), exhibit significantly superior free radical scavenging (DPPH) activity (2-3 times higher) and anti-photoaging activity compared to the original undigested protein fraction. The ≤10 kDa fraction (TDM-1) specifically regulates the MAPK / NF-κB inflammatory pathway (inhibition rate >60%) and TGF-β-mediated ECM repair (1.8-fold higher collagen synthesis). The 10 kDa < ≤50 kDa fraction (TDM-5) significantly enhances skin barrier function (upregulating FLG and LOR gene expression by 3-fold), creating a complementary and synergistic effect. This fractionation strategy breaks through the limitations of traditional processes that only pursue small molecule peptides, maximizing the utilization of active ingredients. Further compounding of TDM-1 and TDM-5 revealed that TDM-1 and TDM-5 synergistically achieved anti-aging efficacy through dual pathways within a mass ratio of 1:0.5 to 2: Experimental data showed that the compound system increased the survival rate of HaCaT cells to 92.7% (an increase of 18.4% to 34.2% compared to the individual components), reduced the expression of inflammatory factors IL-1β and MMP-1 by 48.8% to 64.6% (1.08±0.1 and 0.89±0.1, respectively, in the 1:1 compound group), and increased the expression of barrier repair genes FLG and HAS-2 by 34.2% to 40.2%. The synergistic mechanism manifests itself in TDM-1 blocking the inflammatory cascade by inhibiting the MAPK / NF-κB pathway (reducing p-p38 / p-JNK phosphorylation levels by 58.3%), while TDM-5 promoting ECM repair by activating the TGF-β / Smad pathway (increasing Smad4 expression by 2.1-fold). Together, they form a closed-loop regulatory network of "inflammation suppression and barrier repair." This system transcends the limitations of a single component target, simultaneously integrating antioxidant, anti-inflammatory, and barrier repair benefits.

[0010] Furthermore, the mass ratio of TDM-1 to TDM-5 in the Dendrobium officinale enzymatic hydrolysate composition is 1:(0.5-1).

[0011] Preferably, the mass ratio of TDM-1 to TDM-5 in the Dendrobium officinale protein hydrolysate composition is 1:1.

[0012] Furthermore, the Dendrobium officinale protein is extracted by using an alkali extraction and acid precipitation method.

[0013] Furthermore, the extraction temperature of the alkali extraction and acid precipitation is 55-65° C., the extraction time is 1.0-2.0 h, the alkali solution concentration is 0.08-0.12 mol / L, the solid-liquid ratio is 1.45-1:65 (g / mL), and the acid precipitation pH is 3.0-4.5.

[0014] Furthermore, the extraction temperature of the alkali extraction and acid precipitation is 60-65° C., the extraction time is 1.5-2.0 h, the alkali solution concentration is 0.08-0.10 mol / L, the material-liquid ratio is 1:55-1:65 (g / mL), and the acid precipitation pH is 3.0-4.0.

[0015] Preferably, the alkali extraction and acid precipitation process is performed at a temperature of 60°C, an extraction time of 1.5 hours, an alkali concentration of 0.10 mol / L, a solid-liquid ratio of 1:55 (g / mL), and an acid precipitation pH of 4.0. Under these conditions, the protein extraction rate reaches 40.95%, and the crude protein purity is 74.81%.

[0016] In a preferred embodiment, the preparation method of Dendrobium officinale protein comprises grinding dried Dendrobium officinale stems into a 60-mesh powder for later use. The powder is then mixed with a 0.1 mol / L NaOH solution at a solid-liquid ratio of 1:55 (g / mL). The mixture is stirred and extracted in a 60°C water bath for 1.5 hours, with continuous stirring to fully dissolve the protein. The extract is then filtered to obtain a solution. The extract is adjusted to pH 4.0 with 1 mol / L HCl, allowed to stand for 30 minutes to allow protein precipitation, and then centrifuged at 3500 rpm for 20 minutes to collect the precipitated protein. The precipitate is washed twice with pure water and placed into a 3 kDa molecular weight cutoff dialysis bag for dialysis to remove impurities (i.e., proteins with a molecular weight greater than 3 kDa are retained in the bag). The solution is dialyzed until the conductivity stabilizes. The resulting solution is filtered through a 0.45 μm membrane filter and freeze-dried to obtain crude Dendrobium officinale protein (TD) with a purity of ≥74.81% and an extraction yield of 40.95%.

[0017] Furthermore, during the combined enzymatic hydrolysis, the mass ratio of neutral protease to papain is 1:1, the enzymatic hydrolysis time is 2 to 5 h, the enzymatic hydrolysis pH is 6.0 to 8.0, the enzymatic hydrolysis temperature is 55 to 65° C., and the total enzyme addition amount is 6000 to 8000 U / g of Dendrobium officinale protein.

[0018] Furthermore, the mass ratio of neutral protease to papain is 1:1, the enzymatic hydrolysis time is 2 to 3 h, the enzymatic hydrolysis pH is 7.0 to 8.0, the enzymatic hydrolysis temperature is 60 to 65°C, and the total enzyme addition amount is 7000 to 8000 U / g of Dendrobium officinale protein.

[0019] Preferably, the mass ratio of neutral protease to papain is 1:1, the enzymatic hydrolysis time is 3 hours, the enzymatic hydrolysis pH is 7.10, the enzymatic hydrolysis temperature is 60°C, and the total enzyme amount is 7100 U / g of Dendrobium officinale protein. Under these conditions, the hydrolysis degree of Dendrobium officinale protein reaches 41.66%.

[0020] As a preferred embodiment, the combined enzymatic hydrolysis method comprises dissolving TD protein powder at a concentration of 0.3% (w / v) in deionized water and adjusting the solution pH to 7.10. Neutral protease and papain (mass ratio 1:1) are added, with a total enzyme dosage of 7100 U / g protein. Enzymatic hydrolysis is carried out in a 60°C water bath for 3 hours. Following enzymatic hydrolysis, the enzyme is inactivated in a 90°C water bath for 15 minutes. The supernatant is collected by centrifugation (4000 rpm, 15 minutes) to obtain the Dendrobium officinale enzymatic hydrolysate (TDM). The degree of hydrolysis of the Dendrobium officinale protein reaches 41.66%.

[0021] Furthermore, the separation adopts gradient ultrafiltration and gradient ultrafiltration fractionation method: 50 kDa and 10 kDa ultrafiltration membranes are used for separation in sequence to obtain TDM-1 (≤10 kDa) and TDM-5 (10 kDa < molecular weight ≤50 kDa) active components, thereby retaining functional peptides in the entire molecular weight range (≤50 kDa).

[0022] Preferably, three-stage gradient ultrafiltration separation is used. Specifically:

[0023] Primary separation: The TDM enzymatic hydrolysate was pre-filtered through a 0.45 μm filter membrane to remove undigested macromolecular impurities and obtain the TDM fraction.

[0024] First-stage ultrafiltration: The TDM component passes through a 50 kDa ultrafiltration membrane, retaining components with a molecular weight greater than 50 kDa (discarded), and collecting the filtrate (molecular weight ≤ 50 kDa).

[0025] Second-stage ultrafiltration: The filtrate is passed through a 10 kDa ultrafiltration membrane to obtain TDM-1 (a small peptide with a molecular weight of ≤10 kDa) and TDM-5 (a peptide fraction with a molecular weight between 10 kDa and ≤50 kDa). This process retains functional peptides across the entire molecular weight range (≤50 kDa), avoiding resource waste associated with single-molecule retention. The TDM-5 and TDM-1 fractions are lyophilized for storage.

[0026] The present invention's Dendrobium officinale proteolytic hydrolysates, TDM-1 and TDM-5, achieve functional complementarity through molecular weight classification: TDM-1 (≤10 kDa) focuses on transdermal absorption and inflammation regulation, inhibiting UVB-induced activation of the MAPK / NF-κB pathway; TDM-5 (10 kDa < ≤50 kDa) enhances epidermal barrier function by promoting TGF-β signaling (e.g., upregulating FLG and HAS-2 expression), thereby synergistically promoting multi-target anti-aging effects (antioxidant, anti-inflammatory, and barrier repair). TDM-1 and TDM-5 exhibit significant synergistic anti-aging effects within a mass ratio of 1:0.5 to 2, with the highest synergistic effect achieved at a 1:1 ratio. The two synergistically repair photoaging. Furthermore, neither TDM-1 nor TDM-5 exhibited significant cytotoxicity within a concentration range of 0 to 800 μg / mL. Within a concentration range of 100 to 400 μg / mL, both TDM-1 and TDM-5 significantly improved cell viability, approaching that of normal cells, demonstrating a certain degree of protection against photoaging damage. Therefore, these two ingredients can replace traditional irritating ingredients (such as retinol) and adapt to the development of anti-aging products under the trend of "pure beauty". They have the advantages of natural safety and multi-effect synergy. The active components (TDM-1 and TDM-5) can be directly used as natural anti-aging ingredients in cosmetic dosage forms such as essences, creams, and masks, and have broad market application prospects.

[0027] Based on this, the present invention also provides the use of any of the above-mentioned Dendrobium officinale enzymatic hydrolysate compositions in anti-aging.

[0028] The present invention also provides use of any of the above-mentioned Dendrobium officinale enzymatic hydrolysate compositions in the preparation of anti-aging cosmetics.

[0029] The present invention also provides a cosmetic with anti-aging efficacy, wherein the cosmetic contains any of the above-mentioned Dendrobium officinale enzymatic hydrolysate compositions.

[0030] Preferably, the dosage form of the cosmetic includes but is not limited to essence, cream or mask.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention provides a Dendrobium officinale enzymatic hydrolysate composition, comprising Dendrobium officinale enzymatic hydrolysate products TDM-1 and TDM-5 in a mass ratio of 1:(0.5-2). TDM-1 and TDM-5 are peptide fractions with a molecular weight of ≤10 kDa and peptide fractions with a molecular weight between 10 kDa and ≤50 kDa, obtained by enzymatic hydrolysis of Dendrobium officinale crude protein using a neutral protease and papain. The present invention achieves complete retention of the full molecular weight functional peptides (≤50 kDa) of the Dendrobium officinale enzymatic hydrolysate, avoiding the drawbacks of existing technologies caused by single-molecule retention (e.g., retaining only fractions <1 kDa). Furthermore, TDM-1 and TDM-5 achieve functional complementarity through molecular weight fractionation. By inhibiting MAPK / NF-κB signaling and promoting TGF-β-mediated ECM repair, the TDM-1 and TDM-5 components exhibit synergistic antioxidant, anti-inflammatory, and barrier-repair effects, achieving multi-target anti-aging. TDM-1 and TDM-5 exhibit significant synergistic anti-aging effects within a mass ratio of 1:0.5 to 2, surpassing the single mechanism of action of traditional ingredients such as retinol. Furthermore, the Dendrobium officinale enzymatic hydrolysate composition exhibits low irritation (cell viability >95% at 400 μg / mL). It can be used in formulations such as serums and creams, providing a core raw material for the development of natural anti-aging cosmetics with significant economic and ecological value. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the distribution diagram of Dendrobium officinale protein and enzymatic ultrafiltration products.

[0034] Figure 2 It is the DPPH clearance rate of Dendrobium officinale protein and enzymatic ultrafiltration product.

[0035] Figure 3 The toxic effects of TD, TDM, TDM-5, and TDM-1 on HaCaT cells. Figure 3 A is the TD component; B is the TDM component; C is the TDM-1 component; D is the TDM-5 component; **P<0.01.

[0036] Figure 4 The effects of TD, TDM, TDM-5, and TDM-1 on HaCaT cell viability after UVB radiation. Figure 4 A is the TD component; B is the TDM component; C is the TDM-5 component; D is the TDM-1 component; the vitamin C concentration is 5 μg·mL -1 ; ###P<0.001, model group vs control group; ***P<0.001, **P<0.01, *P<0.05, experimental group vs model group.

[0037] Figure 5 The proliferation of HaCaT cells after UVB irradiation was affected by TD, TDM, TDM-5, and TDM-1. The magnification was 100×, and the concentrations of TD, TDM, TDM-1, and TDM-5 were 200 μg·mL. -1 , vitamin C concentration was 5 μg·mL -1 ; ###P<0.001, model group vs control group; ***P<0.001, **P<0.01, *P<0.05, experimental group vs model group.

[0038] Figure 6 Effects of TD, TDM, TDM-5, and TDM-1 on ROS in HaCaT cells after UVB irradiation. Vitamin C was 5 μg·mL -1 ; ###P<0.001, model group vs control group; ***P<0.001, **P<0.01, *P<0.05, experimental group vs model group.

[0039] Figure 7 Figure 3. Effects of TD, TDM, TDM-5, and TDM-1 on the expression of filaggrin (FLG) mRNA in HaCaT cells after UVB irradiation. Vitamin C was 5 μg·mL-1; ###P < 0.001, model group vs. control group; ***P < 0.001, **P < 0.01, *P < 0.05, experimental group vs. model group.

[0040] Figure 8 Effects of TD, TDM, TDM-5, and TDM-1 on IL-1β mRNA expression in HaCaT cells after UVB irradiation. Vitamin C was 5 μg·mL -1 ; ###P<0.001, model group vs control group; ***P<0.001, **P<0.01, *P<0.05, experimental group vs model group.

[0041] Figure 9 Effects of TD, TDM, TDM-5, and TDM-1 on MMP-1 mRNA expression in HaCaT cells after UVB irradiation. Vitamin C was 5 μg·mL -1 ; ###P<0.001, model group vs control group; ***P<0.001, **P<0.01, *P<0.05, experimental group vs model group.

[0042] Figure 10 The effect of TDM-5 on the mRNA expression of keratin barrier-related genes in cells after UVB radiation. Vitamin C was 5 μg·mL -1; #P<0.05, model group vs control group; ***P<0.001, **P<0.01, *P<0.05, experimental group vs model group.

[0043] Figure 11 The effect of TDM-5 on the mRNA expression of moisturizing-related genes in cells after UVB radiation. Vitamin C was 5 μg·mL -1 ; ###P<0.001, model group vs control group; ***P<0.001, **P<0.01, *P<0.05, experimental group vs model group.

[0044] Figure 12 The effect of TDM-1 on the mRNA expression of inflammatory levels in HaCaT cells after UVB irradiation. The concentration of vitamin C was 5 μg·mL -1 ; ###P<0.001, model group vs control group; ***P<0.001, **P<0.01, *P<0.05, experimental group vs model group.

[0045] Figure 13 The effect of TDM-1 on the expression of matrix metalloproteinase mRNA in cells after UVB irradiation. Vitamin C was 5 μg·mL -1 ; ###P<0.001, model group vs control group; ***P<0.001, **P<0.01, *P<0.05, experimental group vs model group.

[0046] Figure 14 Figure 3. Effects of TDM-5 and TDM-1 on the expression of proteins related to the MAPK / AP-1 signaling pathway in photoaged HaCaT cells. Vitamin C concentration was 5 μg·mL-1; ### P < 0.001, model group vs. control group; *** P < 0.001, ** P < 0.01, * P < 0.05, experimental group vs. model group.

[0047] Figure 15 Figure 3. Effects of TDM-5 and TDM-1 on the expression of proteins related to the NF-κB signaling pathway in photoaged HaCaT cells. Vitamin C concentration was 5 μg mL-1; ### P < 0.001, model group vs. control group; *** P < 0.001, ** P < 0.01, * P < 0.05, experimental group vs. model group.

[0048] Figure 16Figure 3. Effects of TDM-5 and TDM-1 on the expression of proteins related to the TGF-β signaling pathway in photoaged HaCaT cells. Vitamin C concentration was 5 μg mL-1; ### P < 0.001, model group vs. control group; *** P < 0.001, ** P < 0.01, * P < 0.05, experimental group vs. model group. DETAILED DESCRIPTION

[0049] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0050] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0051] Example 1 Preparation of Dendrobium officinale enzymatic hydrolysates TDM-5 and TDM-1

[0052] 1. Methods

[0053] 1. Dendrobium officinale protein extraction

[0054] Using powdered Dendrobium officinale as raw material, we systematically investigated the extraction conditions of protein from the herb using an alkaline extraction and acid precipitation method. Through single-factor and response surface experiments, we determined the optimal conditions for protein extraction: extraction temperature of 60°C, extraction time of 1.5 h, extraction concentration of 0.1 mol / L, solid-liquid ratio of 1:55, and acid precipitation pH of 4.0. Under these conditions, a protein extraction rate of 40.95% and a crude protein purity of 74.81% were achieved.

[0055] Specifically, dried Dendrobium officinale stems were ground into a 60-mesh powder and set aside. The powder was then mixed with a 0.1 mol / L NaOH solution at a solid-liquid ratio of 1:55. The mixture was stirred in a 60°C water bath for 1.5 hours, with continuous stirring to fully dissolve the protein. The extract was then filtered to obtain the extract. The extract was adjusted to pH 4.0 with 1 mol / L HCl and allowed to stand for 30 minutes to allow protein precipitation. After standing, the protein precipitate was collected by centrifugation at 3500 rpm for 20 minutes. The precipitate was washed twice with pure water and dialyzed through a 3 kDa molecular weight cutoff dialysis bag to remove impurities. The dialyzation continued until the conductivity stabilized. The resulting solution was filtered through a 0.45 μm membrane filter and freeze-dried to obtain crude Dendrobium officinale protein (TD) with a purity of ≥74.81% and an extraction yield of 40.95%.

[0056] 2. Directed enzymatic hydrolysis of Dendrobium officinale protein using complex enzymes

[0057] Using the freeze-dried protein powder from step 1 as raw material, an enzymatic hydrolysis method (using a combination of neutral protease and papain) was systematically investigated to determine the optimal hydrolysis conditions for the protein. Using single-factor and response surface design, the optimal hydrolysis conditions for the protein were determined to be: substrate concentration of 0.3%, reaction time of 3 hours, enzyme dosage of 7100 U, hydrolysis pH of 7.10, and hydrolysis temperature of 55°C. Under these conditions, the degree of hydrolysis of the protein reached 41.65%.

[0058] Specifically, TD protein powder was dissolved in deionized water at a concentration of 0.3% (w / v), and the pH of the solution was adjusted to 7.10. Neutral protease and papain (mass ratio 1:1) were added, with a total enzyme dosage of 7100 U / g protein. Enzymatic hydrolysis was carried out in a 60°C water bath for 3 hours. After completion of the enzymatic hydrolysis, the enzyme was inactivated in a 90°C water bath for 15 minutes. The supernatant was collected by centrifugation (4000 rpm, 15 minutes) to obtain the Dendrobium officinale enzymatic hydrolysate (TDM). The degree of protein hydrolysis reached 41.65%.

[0059] 3. Preparation of Dendrobium officinale enzymatic hydrolysis ultrafiltration products TDM, TDM-5, and TDM-1

[0060] The Dendrobium officinale enzymatic hydrolysis product in step 2 was separated by three-stage ultrafiltration:

[0061] Primary separation: The Dendrobium officinale enzymatic hydrolysate was pre-filtered through a 0.45 μm filter membrane to remove undigested macromolecular impurities and obtain the TDM fraction.

[0062] First-stage ultrafiltration: The TDM component passes through a 50 kDa ultrafiltration membrane, retaining components with a molecular weight greater than 50 kDa (discarded), and collecting the filtrate (molecular weight ≤ 50 kDa).

[0063] Second-stage ultrafiltration: The filtrate is passed through a 10 kDa ultrafiltration membrane to obtain TDM-1 (a small peptide with a molecular weight of ≤10 kDa) and TDM-5 (a peptide fraction with a molecular weight between 10 kDa and ≤50 kDa). This process retains functional peptides across the entire molecular weight range (≤50 kDa), avoiding resource waste associated with single-molecule retention. The TDM-5 and TDM-1 fractions are lyophilized for storage.

[0064] 2. Results

[0065] The molecular weight distribution of the above-mentioned Dendrobium officinale protein and enzymatic ultrafiltration product was analyzed by silver staining. The results are as follows: Figure 1As shown, the proteins in the TD sample were primarily distributed around 130 kDa, 100 kDa, 35 kDa, and 15 kDa, while those in the TDM sample were concentrated around 100 kDa, 55 kDa, 35 kDa, 15 kDa, and 10 kDa. A comparison of the two revealed that after treatment with neutral protease and papain, the molecular weight of the proteins was significantly reduced, and their distribution became more concentrated and uniform. After filtration and interception with 50 kDa and 10 kDa ultrafiltration membranes, the TDM-5 sample retained proteins only in the 35-40 kDa range. However, after ultrafiltration with 10 kDa, the proteins in the TDM-1 sample were primarily concentrated around 10 kDa. This indicates that, compared to the undigested protein sample, the enzymatically digested Dendrobium officinale protein distribution is more uniform and concentrated, significantly reducing the molecular weight of the proteins. The distribution range of the sample after ultrafiltration is more uniform than that before ultrafiltration, indicating that the sample is purer after ultrafiltration enrichment. Ultrafiltration membrane treatment can significantly improve the purity of the target protein and help enrich proteins in a specific molecular weight range.

[0066] Example 2 Analysis of the in vitro antioxidant activity of the ultrafiltration product of Dendrobium officinale enzymatic hydrolysis

[0067] Reactive oxygen species (ROS) are continuously produced during normal human metabolism. While moderate amounts of ROS can act as intracellular signaling molecules to regulate metabolism, excessive accumulation can trigger severe oxidative stress, damaging cell membranes, proteins, DNA, and other biomacromolecules, leading to a variety of chronic diseases and skin aging. Ultraviolet (UV)-induced ROS accumulation is a key mechanism of photoaging. Therefore, the development of highly effective natural antioxidant ingredients that scavenge free radicals and block oxidative chain reactions has become a core focus of functional skincare product research and development. This study evaluated the antioxidant properties of Dendrobium officinale protein and its enzymatically hydrolyzed ultrafiltration fractions (TD, TDM, TDM-5, and TDM-1) using in vitro antioxidant assays and the DPPH test.

[0068] 1. Method

[0069] DPPH scavenging activity assay

[0070] Weigh 12 mg of DPPH powder and dissolve it in 100 ml of anhydrous ethanol. Ultrasonicate for 30 minutes to prepare a DPPH free radical working solution. DPPH free radicals are sensitive to light and easily affected by environmental oxidation, so the DPPH working solution must be prepared immediately before use. Take 100 μL of the sample solution into a 96-well plate, add 100 μL of DPPH working solution, react at room temperature for 30 minutes, and measure the absorbance A at 517 nm using a microplate reader. XMeasure the absorbance (A0) of a solution containing 100 μL of the solution mixed with 100 μL of anhydrous ethanol. Measure the absorbance (A1) of a solution containing 100 μL of deionized water and 100 μL of the DPPH working solution. Each experiment was performed in triplicate. Calculate the DPPH scavenging rate using the following formula (1).

[0071] DPPH clearance rate%=[1-(A X -A0) / A1]×100%(1)

[0072] Where:

[0073] A X : absorbance of 100 μL sample + 100 μL DPPH;

[0074] A0: absorbance of 100 μL sample + 100 μL anhydrous ethanol;

[0075] A1: Absorbance of 100 μL deionized water + 100 μL DPPH.

[0076] The test in this example was repeated at least three times and the data were analyzed and graphed using SPSS and Design-Expert 13 software. The experimental results were expressed as mean ± standard deviation and variance analysis was performed.

[0077] 2. Results

[0078] Determination of DPPH free radical scavenging activity of Dendrobium officinale protein and its enzymatic ultrafiltration product. Figure 2 It can be seen that when the concentration is increased from 0.47 mg·mL -1 Increased to 60 mg mL -1 When the concentration of TD, TDM, TDM-5 and TDM-1 increased, the scavenging ability of DPPH free radicals gradually increased, showing a significant concentration dependence. According to the half inhibition rate data, the half inhibition rate of TD was 10.08 mg·mL -1 , TDM was 2.772 mg·mL -1 , TDM-5: 9.476 mg·mL -1 , TDM-1: 5.294 mg·mL -1 The results indicate that the DPPH free radical scavenging rate of the enzymatic hydrolysis product is better than that of the unhydrolyzed product, and the antioxidant activity of the small-molecule enzymatic hydrolysis ultrafiltration product TDM-1 is better than that of the larger molecular weight TDM-5. This indicates that enzymatic hydrolysis can effectively enhance the antioxidant activity of Dendrobium officinale protein, and ultrafiltration further enriches the small-molecule components, making their antioxidant activity even better.

[0079] Example 3 Verification of the Photoaging Damage of HACAT Cells by the Ultrafiltration Product of Dendrobium Officinale Hydrolysis

[0080] Exposure to UVB radiation in the epidermis and dermis causes hyperpigmentation, thickening of the epidermis, and the development of loose wrinkles. UVB radiation causes excessive production of reactive oxygen species (ROS) in the skin, which can lead to DNA breakage, impaired mitochondrial function, and cell apoptosis. Furthermore, when the skin is irritated, it induces the secretion of inflammatory factors, triggering an inflammatory response and activating matrix metalloproteinases (MMPs). This leads to the degradation of extracellular collagen, damage to the skin barrier, and accelerated skin cell aging.

[0081] 1. Method

[0082] In this example, HaCaT cells were used as a model for UVB-induced photoaging damage. A photoaging model was established in HaCaT cells. Different concentrations of Vitamin C, Dendrobium officinale protein, and ultrafiltration fractions were then added for therapeutic intervention. HaCaT cell viability was measured using the CCK-8 assay, and the proliferation of UVB-induced HaCaT cells was determined using the EDU labeling assay. The effects of different dosing groups on cell survival and proliferation in the UVB-induced photoaging model were evaluated. Flow cytometry was used to determine the ROS content in HaCaT cells according to the operation of the reactive oxygen species detection kit. The antioxidant-related indicators in HaCaT cells after UVB irradiation were determined according to the instructions of the MDA, SOD and T-AOC kits. The oxidative stress level was explored by measuring oxidative damage indicators and antioxidant enzyme activities. At the same time, fluorescence quantitative PCR was used to detect the mRNA levels of barrier factor filaggrin (FLG), inflammatory factor (1L-1β), and intracellular matrix metalloproteinase I (MMP-1) in HaCaT cells to evaluate the repair ability of each component on the keratinocyte barrier, the transcription levels of inflammatory factors and MMPs, and to reveal the protective mechanism of Dendrobium officinale protein components in UVB-induced photoaging.

[0083] 2. Results

[0084] (1) Toxic effects of TD, TDM, TDM-5, and TDM-1 on HaCaT cells

[0085] The results are as follows Figure 3 As shown, by comparing the experimental group with the control group, the TD ( Figure 3 A), TDM ( Figure 3 Middle B), TDM-1 ( Figure 3 Middle C), TDM-5 ( Figure 3 D) 0~800 μg·mL -1 No significant cytotoxicity was observed within the concentration range, so subsequent experiments were conducted within the range of 0-400 μg·mL-1.

[0086] (2) Effects of TD, TDM, TDM-5, and TDM-1 on HaCaT cells after UVB irradiation

[0087] The experimental results are as follows Figure 4 , at 40 mJ·cm -2 After UVB irradiation, the cell viability of the model group decreased significantly, about 60% of the cell viability of the control group, which indicated that the photoaging model was successfully established and the cell state was significantly damaged. Then different concentrations of Dendrobium officinale protein and ultrafiltration hydrolyzate were added to HaCaT cells for intervention and cultured for 12 hours. The results showed that TD ( Figure 4 A), TDM ( Figure 4 Middle B), TDM-5 ( Figure 4 Middle C), TDM-1 ( Figure 4 D in the middle) can improve the cell viability after UVB light damage and is positively correlated with the dose. -1 At the concentration of 100 μg·mL, there were signs of cell viability recovery. -1 with 200 μg·mL -1 with 400 μg·mL -1 The ability to restore cell viability was significantly enhanced at 200 μg·mL -1 with 400 μg·mL -1 The above results show that the small molecule protein activity of the ultrafiltration products TDM-5 and TDM-1 is higher than that of TD and TDM before ultrafiltration, and the activity of TDM after protease hydrolysis is higher than that of TD, which indicates that the hydrolysis of Dendrobium officinale protein can improve its therapeutic effect on HaCaT cells damaged by UVB light.

[0088] (3) Effects of TD, TDM, TDM-5, and TDM-1 on HaCaT cell proliferation after UVB irradiation

[0089] The experimental results are as follows Figure 5 As shown in the figure, the proliferation rate of cells in the model group was significantly decreased compared with that in the control group (###P<0.001). Compared with the model group, the vitamin C group could reduce the peroxidation level of cells, reduce the oxidative stress level, and restore the proliferation activity of cells (***P<0.001). Compared with the model group, the products of the TD and TDM groups at 200 μg·mL -1 At the same concentration, both TDM-1 and TDM-5 had a restorative effect on cell proliferation, and TDM had a better restorative effect on cells than TD, indicating that the activity of protein after hydrolysis was higher than that of unhydrolyzed protein. Compared with the model group, the products of TDM-1 and TDM-5 groups at 200 μg·mL -1At the same concentration, it can better reverse the photodamage of keratinocytes caused by UVB (**P<0.01, ***P<0.001). For TDM-1, this indicates that the small molecule peptides after ultrafiltration are more easily absorbed by cells, while for TDM-5, it may participate in the synthesis process of cells to a certain extent, promote cell proliferation, and reduce the photodamage caused by UVB on the cell surface.

[0090] (4) Effects of TD, TDM, TDM-5, and TDM-1 on ROS levels in HaCaT cells after UVB irradiation

[0091] The experimental results are as follows Figure 6 As shown in the figure, compared with the control group, the expression of ROS in the cells of the model group was significantly increased (P<0.001); compared with the model group, the vitamin C group could significantly inhibit the generation of ROS in the cells (P<0.001); compared with the model group, the TD group and the TDM group at 200 μg·mL -1 There were significant differences in the inhibitory effects on ROS between the two groups (P<0.01, P<0.001), which confirmed that the activity of the protein after hydrolysis was better than that before hydrolysis. -1 Compared with the model group, the TDM-5 and TDM-1 groups had a significant inhibitory effect at 100 μg·mL -1 , 200 μg·mL -1 At a dose of 100mg / L, the production of intracellular ROS was significantly inhibited, confirming that the ultrafiltration fraction had a better inhibitory effect on ROS than TDM (P<0.01, P<0.001). This indicates that TD, TDM, TDM-5, and TDM-1 can inhibit the expression level of intracellular ROS and reduce the degree of oxidative stress in cells, and the inhibitory effect of TDM-5 and TDM-1 is better.

[0092] (5) Regulatory effects of TD, TDM, TDM-5, and TDM-1 on antioxidant indicators in HaCaT cells after UVB irradiation

[0093] The experimental results are shown in Table 1. Compared with the normal control group, the expression level of MDA in the model group was significantly increased (P < 0.01), while the expression levels of SOD and T-AOC were significantly decreased (P < 0.001 and P < 0.05, respectively). This indicates that UVB-induced photoaging leads to significant damage to the function of the cellular antioxidant system; the positive group 5 μg·mL -1 Vitamin C can significantly improve these three indicators.

[0094] Compared with the model group, the experimental group had a -1 TDM-5 and 50 μg·mL -1TDM-1 could significantly reduce the expression of MDA in cells (P<0.001). -1 The effect was similar to that of 5 μg·mL -1 Equivalent to vitamin C. At the same time 200 μg·mL -1 TDM-5 and 50 μg·mL -1 TDM-1 at 200 μg·mL can effectively improve the activity of SOD and T-AOC in cells (P<0.01, P<0.001). -1 The improvement of intracellular SOD and T-AOC was similar to that of 5 μg·mL -1 The other two groups showed no statistically significant differences. These results demonstrate that the ultrafiltration fractions TDM-5 and TDM-1 can mitigate the peroxidative damage caused by UVB photoaging.

[0095] Table 1 Effects of TD, TDM, TDM-5, and TDM-1 on MDA, SOD, and T-AOC in HaCaT cells after UVB irradiation

[0096]

[0097] Note: ###P<0.001, model group vs. control group; ***P<0.001, **P<0.01, *P<0.05, experimental group vs. model group.

[0098] (6) Regulatory effects of TD, TDM, TDM-5, and TDM-1 on FLG gene in HaCaT cells after UVB irradiation

[0099] Depend on Figure 7 It can be seen that the mRNA expression level of FLG in the model group was significantly decreased compared with the control group (P < 0.01), which indicates that UVB radiation damages the skin hydration function of HaCaT cells. Compared with the positive drug group, vitamin C can restore FLG expression in the model group (P < 0.001) and is close to the normal expression level. In the experimental group, TDM-5 at 50, 100 and 200 μg·mL -1 The expression level of FLGmRNA was effectively up-regulated at the concentration of 200 μg·mL (P<0.01, P<0.001). -1 This shows that the TDM-5 component has the potential to repair the barrier.

[0100] TDM-5 and TDM-1 at 100 and 200 μg·mL -1The expression level of FLG (mRNA) can be restored when TDM-5 is added to the TD group. The experimental results show that TDM-5 is better than TDM-1. Compared with the model group, the expression level of FLG (mRNA) can be restored when TDM-5 is added to the TD group at 50-200 μg·mL. -1 There was no statistical difference in the expression level of FLG (mRNA) within the range.

[0101] According to the above experimental results, TDM-1 and TDM-5 can improve HaCaT cell damage caused by UVB radiation and restore the expression level of FLG (mRNA) in cells, thereby reversing skin photoaging damage.

[0102] (7) Effects of TD, TDM, TDM-5, and TDM-1 on IL-1β gene expression in HaCaT cells after UVB irradiation

[0103] The experimental results are as follows Figure 8 As shown in the figure, compared with the normal control group, the model group had a significantly increased IL-1β mRNA expression level in HaCaT cells after UVB irradiation (P<0.001), indicating that the HaCaT cells activated the inflammatory response after being irradiated by ultraviolet light, indicating that the model effect was good. Compared with the model group, the vitamin C-positive drug group was able to restore the expression level to a close normal state. After the intervention treatment with the four components, compared with the model group, 200 μg·mL -1 TDM-1, 100 μg mL -1 TDM had a significant restorative effect on the expression level of IL-1β mRNA in HaCaT cells (P<0.01); TD at 100 and 200 μg·mL -1 The expression level of IL-1β mRNA in HaCaT cells could be restored under these conditions (P<0.05); the TDM-5 group had a -1 There was no statistical effect on IL-1β mRNA expression levels within the range of 4.

[0104] (8) Regulatory effects of TD, TDM, TDM-5, and TDM-1 on MMP-1 in HaCaT cells after UVB irradiation

[0105] The experimental results are as follows Figure 9 As shown in the figure, the mRNA level of MMP-1 in HaCaT cells in the model group after UVB modeling was higher than that in the normal control group (P<0.001), indicating that light significantly induced the expression of this gene. -1 After vitamin C intervention, the expression level of MMP-1 was significantly downregulated (P<0.001), indicating that vitamin C has good anti-photoaging and collagen protection effects.

[0106] The TDM-1 and TD components in the experimental group were 50, 100, and 200 μg·mL -1 The expression of MMP-1 mRNA in HaCaT cells was significantly reduced at the concentrations of TDM-5 (P<0.001, P<0.01). TDM-5 at low concentrations could also slightly reduce the expression of MMP-1 mRNA. -1 The expression of MMP-1 mRNA was most significantly affected under the conditions of .

[0107] The above experimental results show that the enzymatically hydrolyzed small molecule peptide components TDM-1 and TDM-5 of Dendrobium officinale play a role in anti-inflammatory and cell keratin barrier repair, respectively, and the effects are better than TDM before ultrafiltration and non-enzymatically hydrolyzed Dendrobium officinale protein TD. Therefore, subsequent experiments will select TDM-1 and TDM-5 components for research to explore their lowest effective concentration in the photoaging damage model caused by UVB, and explore possible anti-photoaging damage mechanisms.

[0108] Example 5 Evaluation of the anti-skin aging effects and mechanism studies of TDM-5 and TDM-1

[0109] UVB can induce skin cells to generate large amounts of reactive oxygen species (ROS), triggering oxidative stress and activating multiple intracellular signaling pathways, particularly the mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) pathways. Activation of these signaling pathways can trigger a series of physiological and pathological reactions. On the one hand, it promotes the secretion of inflammatory factors (IL-1β, IL-6, and TNF-α), exacerbating skin inflammation. On the other hand, it upregulates the expression of AP-1 and MMPs, causing extracellular matrix (ECM) degradation and disrupting collagen structure and function.

[0110] The present invention found that TDM-5 and TDM-1 components have a better effect on cells damaged by photoaging after UVB radiation. Among them, TDM-5 component showed better barrier repair ability, while TDM-1 component had better anti-inflammatory and anti-wrinkle effects.

[0111] 1. Method

[0112] Based on the above research results, this example first used real-time fluorescence quantitative RT-qPCR to investigate the effects of the TDM-5 component on the expression levels of keratin barrier-related proteins (FLG, LOR, INV) and moisturizing factor-related proteins (AQP-3, Caspase-14, HAS-2), and determined its minimum effective concentration. Furthermore, the inhibitory effect of TDM-1 on the expression levels of inflammatory factors (IL-1β, IL-6, TNF-α) and MMPs (MMP-1, MMP-3) was investigated, and the effective concentration was determined. Finally, Western blotting was used to examine the protein expression levels of proteins involved in the MAPK / AP-1, NF-κB, and TGF signaling pathways (p38, JNK, ERK, p65, IκBα), as well as TGF-β, smad2 / 3, smad4, and smad7, to explore the potential anti-photoaging mechanisms of the TDM-5 and TDM-1 components.

[0113] 2. Results

[0114] (1) Effect of TDM-5 on the mRNA expression of intracellular keratin barrier-related genes after UVB radiation

[0115] The stability of the epidermal barrier depends on the terminal differentiation of keratinocytes. Keratin is the foundation of the skin barrier. Keratin intermediate filament-related proteins play a regulatory role in this process, working together to form a stable defense system in the stratum corneum. Filaggrin (FLG) plays a supporting role in stratum corneum formation. It promotes the aggregation of keratin, forming stable fiber bundles and thereby enhancing the barrier function of the stratum corneum. Furthermore, involucrin (INV) and loricrin (LOR), precursors of the cornified envelope (CE), are abundantly expressed during the late stages of keratinocyte differentiation. Under the catalysis of transglutaminase (TGM), they cross-link with each other to form a stable network structure. This process enables the stratum corneum to construct an insoluble cornified envelope scaffold, ensuring the stability of the stratum corneum barrier. FLG mainly provides a solid framework for the stratum corneum by promoting the aggregation of keratin, while INV and LOR serve as the structural basis and form a dense keratin envelope through cross-linking to ensure the integrity of the barrier. The three work together to enable the stratum corneum to effectively resist external physical, chemical and microbial invasions, and ensure the skin's moisture balance and repair ability. The expression of the above three proteins is the core mechanism of epidermal barrier stability, and also plays a vital role in skin health and damage repair. The expression of the three proteins by epidermal cells after UVB radiation is as follows Figure 10As shown in Figure 2, compared with the control group, the mRNA expression levels of FLG, LOR and INV in the model group were significantly decreased (P<0.001). -1 , 200 μg·mL -1 Compared with the model group, the transcription level of FLG in the TDM-5 group was significantly improved (P<0.001, P<0.01), and at 200 μg·mL -1 The effect was comparable to that of Vitamin C (P<0.001). TDM-5 components were 100, 200, and 400 μg·mL -1 Compared with the model group, the TDM-5 group showed an improvement in the transcription of LOR and INV (P<0.001). The experimental results showed that TDM-5 components can effectively improve the expression of FLG, INV, and LOR in HaCaT cells after UVB radiation.

[0116] (2) Effect of TDM-5 on the transcriptional level of moisturizing-related proteins in cells after UVB radiation

[0117] Aquaporin-3 (AQP-3) is a channel protein located on the skin cell membrane. Its primary function is to transport small molecules such as water and glycerol, promoting cellular water absorption, regulating skin hydration, and, to a certain extent, participating in the repair of damaged skin cells. When skin cells are damaged by light, FLG is degraded by Caspase-14, releasing NMFs. These small molecules are highly hygroscopic and help maintain the stratum corneum's water content, enhancing the skin's moisturizing capacity. Hyaluronic acid (HA) is also involved in skin hydration. HA can promote skin repair and barrier stability by regulating intercellular signaling. Hyaluronan synthase 2 (HAS-2) is a key enzyme in HA synthesis. HAS-2 catalyzes the polymerization of N-acetylglucosamine and glucuronic acid to form long-chain HA molecules. These long-chain HA molecules are components of the extracellular matrix and play a role in maintaining skin moisture retention, regulating osmotic pressure, and maintaining barrier function. When hyaluronic acid (HA) levels decrease, the skin's moisturizing function weakens, leading to roughness, deeper wrinkles, and reduced skin repair capacity. Hyaluronic acid also possesses anti-inflammatory properties. Interleukin-1β (IL-1β), a pro-inflammatory cytokine in the expression of inflammatory factors, also regulates the HA-mediated anti-inflammatory response. Reduced IL-1β expression also downregulates the activity of various matrix metalloproteinases (MMPs), reducing extracellular matrix degradation and thereby increasing HA's stability, enabling it to better exert its moisturizing and anti-inflammatory effects. Therefore, skin hydration depends on AQP-3-mediated water transport, NMFs generated by FLG degradation, and HA synthesis promoted by HAS-2. By maintaining skin hydration and reducing the damage to the skin barrier caused by inflammatory factors, the progression of photoaging damage can be delayed and the integrity of the skin barrier maintained.

[0118] The results are as follows Figure 11 As shown in the results, the mRNA levels of AQP-3, Caspase-14 and HAS-2 in the control group were significantly higher than those in the model group (P<0.001). -1 When TDM-5 was added to the stratum corneum, it could improve the transcription of AQP-3, Caspase-14, and HAS-2 (P<0.001, P<0.01). TDM-5 may restore the transcription level of HAS-2 by reducing the transcription levels of IL-1β and MMP-1, and reduce the expression levels of inflammatory factors and MMP-1 through the HA pathway. This shows that TDM-5 can participate in the repair process of the stratum corneum and has a significant effect at 50 μg·mL. -1The effect begins to appear at 100 μg·mL -1 The mRNA levels of AQP-3, Caspase-14 and HAS-2 in skin keratinocytes were significantly improved, confirming its good moisturizing effect.

[0119] (3) Effect of TDM-1 on the transcriptional level of intracellular inflammatory factors after UVB radiation

[0120] When UVB is irradiated on the surface of the skin, it will cause damage to the mitochondria of skin keratinocytes, produce excessive ROS, activate inflammatory receptors, release excessive inflammatory factor signals, and oxidize DNA, lipids, and proteins, further activating the NF-κB and MAPK signaling pathways. At the same time, NF-κB, as a transcription factor, will enter the cell nucleus and bind to the pro-inflammatory gene promoter in the cell nucleus, upregulating the gene transcription of IL-1β, IL-6, IL-8, and TNF-α. In order to explore the regulation of TDM-1 on the transcription level of intracellular inflammatory factors after UVB radiation, PCR (RT-qPCR) technology was used to detect the expression levels of IL-1β, IL-6, IL-8, and TNF-α mRNA in HaCaT cells after UVB radiation. The results are shown in the figure. Figure 12 As shown in the results, compared with the control group, the model group had significantly increased the mRNA expression levels of IL-1β, IL-6, IL-8 and TNF-α in the cells after UVB irradiation (P < 0.001). After intervention with TDM-1 components, compared with the model group, at 100 μg·mL -1 Under the condition of TDM-1, TDM-1 could reduce the transcription levels of IL-1β, IL-8 and TNF-α (P<0.001, P<0.01). When the concentration of TDM-1 was 200 μg·mL -1 Under the same conditions, the levels of IL-1β, IL-6, IL-8, and mRNA can be significantly reduced (P<0.001). The above results indicate that the TDM-1 component can play an anti-skin photoaging role by inhibiting the expression of cellular inflammatory factors.

[0121] (4) Effect of TDM-1 on the transcriptional levels of MMP-1 and MMP-3 in cells after UVB irradiation

[0122] Collagen is the most important structural protein in the skin and is widely distributed in the dermis. It plays an important role in maintaining the tension, elasticity and structural stability of the skin. The extracellular matrix (ECM) in the dermis is mainly composed of type I and type III collagen, which together constitute the supporting structure of the skin, giving the skin good elasticity and strength. In addition, collagen also works synergistically with ECM components such as hyaluronic acid, elastin, and fibronectin to ensure the repair and regeneration of the skin barrier and maintain its stability. When skin cells are exposed to ultraviolet light (UVB), the expression of matrix metalloproteinases (MMPs) increases significantly. MMPs are key enzymes for degrading collagen. They are zinc-dependent proteases that are mainly secreted by fibroblasts, keratinocytes, and inflammatory cells. And their activity is regulated by multiple factors. The results are as follows Figure 13 As shown in the figure, compared with the control group, the transcriptional expression of MMP-1 and MMP-3 mRNA in the model group was increased (P<0.001); after intervention with different concentrations of TDM-1 components, the expression of MMP-1 and MMP-3 mRNA in the model group was significantly increased compared with the model group at 100 μg·mL -1 Under the condition of TDM-1, the transcriptional expression levels of MMP-1 and MMP-3 mRNA can be reduced (P < 0.001), and with the increase of TDM-1 component concentration, the inhibitory effect on the expression levels of MMP-1 and MMP-3 mRNA is more obvious.

[0123] (5) Effects of TDM-5 and TDM-1 on the MAPK / AP-1 signaling pathway

[0124] When UVB irradiates skin cells, p38, JNK and ERK1 / 2 in the MAPK pathway undergo phosphorylation reactions, causing overexpression of inflammatory factors (IL-1β, IL-6, TNF-α) and MMPs (matrix metalloproteinases), leading to ECM damage, aggravating wrinkle formation and skin barrier damage. Figure 14 As shown, there was no significant difference in total p38 levels among the groups, but p-p38 was significantly increased in the model group, indicating that UVB irradiation leads to activation of the p38 MAPK signaling pathway. In the experimental groups with the addition of TDM-5 and TDM-1 components, the expression level of p-p38 was significantly reduced, indicating that these two components have the effect of inhibiting the p38-mediated inflammatory signaling pathway.

[0125] While total JNK protein levels did not change significantly, p-JNK and the p-JNK / JNK ratio increased significantly in the model group, indicating that UVB irradiation activates the JNK signaling pathway. This indicator decreased significantly after treatment with TDM-5 and TDM-1. The results show that both components effectively inhibit JNK phosphorylation, thereby reducing UVB-induced oxidative stress and inflammatory responses.

[0126] There was no statistical difference in the total protein of ERK1 / 2 among the groups, but p-ERK1 / 2 was significantly increased in the model group (P<0.01), indicating that UVB can promote the phosphorylation of ERK1 / 2. -1 The levels of p-ERK1 / 2 were reduced at concentrations of 1:1, 2:1, and 3:1, indicating that TDM-1 may play a regulatory role in the ERK1 / 2-mediated signaling pathway.

[0127] The AP-1 complex (c-Jun / c-Fos) plays a key role in UVB-induced matrix degradation and inflammatory response. In addition, the JNK downstream transcription factor c-Jun and its binding protein c-Fos also showed phosphorylation activation. Among them, pc-Jun and pc-Fos and their phosphorylation ratios (pc-Jun / c-Jun, pc-Fos / c-Fos) were significantly increased in the model group. After the addition of TDM-5 and TDM-1 components, these indicators were significantly decreased, indicating that both components can inhibit the activation of the AP-1 complex. In terms of component dosage, TDM-1 and TDM-5 at 200 μg·mL -1 The effects were more significant at concentrations of , further verifying their reversal effects on the p38 MAPK and JNK MAPK pathways.

[0128] Since the p38 and JNK signaling pathways are closely related to the AP-1 complex, activated AP-1 can enhance the expression of matrix metalloproteinases (MMPs), thereby inducing the release of inflammatory factors IL-1β, IL-6, IL-8 and TNF-α. Therefore, based on the experimental results, it is speculated that the inhibitory effects of TDM-5 and TDM-1 on the expression of MMPs and inflammatory factors are related to the regulation of the p38 and JNK signaling pathways by the two components.

[0129] In summary, TDM-5 and TDM-1 can effectively inhibit UVB-induced activation of the p38 and JNK signaling pathways, reduce the phosphorylation of the AP-1 complex, and reduce the expression of MMPs and inflammatory factors, thereby slowing down photoaging damage to the skin.

[0130] (6) Effects of TDM-5 and TDM-1 on the NF-kB signaling pathway

[0131] NF-κB is a core regulator of inflammatory signaling pathways. UVB-induced oxidative stress activates IκBα phosphorylation (p-IκBα), leading to its degradation, allowing NF-κB (p65) to enter the cell nucleus and induce inflammatory gene transcription. Figure 15 As shown in the results, compared with the control group, the expression levels of p-IκBα, p-IκBα / IκBα and p-p65 / NF-κB in the model group were significantly increased after UV irradiation (P<0.001), indicating that IκBα phosphorylation was enhanced and NF-κB was in an activated state.

[0132] When TDM-5 and TDM-1 were added, all relevant indicators decreased, suggesting that TDM-5 and TDM-1 can reduce NF-κB activation by decreasing IκB phosphorylation. Since IκBα is ubiquitinated and degraded after phosphorylation, TDM-5 and TDM-1 inhibited IκBα phosphorylation and reduced its degradation, leading to a rebound in total IκBα protein levels. These results suggest that TDM-5 and TDM-1 can inhibit the sustained activation of NF-κB signaling by stabilizing IκBα.

[0133] Studies have confirmed that excessive NF-κB activation can induce the transcriptional expression of multiple inflammatory factors (IL-1β, IL-6, IL-8, TNF-α) and MMPs, thereby exacerbating inflammatory responses and matrix degradation. Therefore, in this example, TDM-5 and TDM-1 inhibit UVB-induced IκBα phosphorylation, reducing NF-κB activation levels and thus reducing the overexpression of inflammatory factors and MMPs, helping to mitigate UV-induced photoaging damage and enhance skin barrier function.

[0134] (7) Effects of TDM-5 and TDM-1 on the TGF signaling pathway

[0135] The TGF-β / Smad axis plays an important role in ECM metabolic balance and skin barrier stability. When UVB irradiates skin cells, the expression levels of TGF-β and Smad2 / 3, p-Smad2 / 3, and Smad4 decrease, while Smad7 is significantly upregulated, leading to increased ECM degradation. Figure 16 As shown in the figure, compared with the control group, after UVB irradiation, the expression levels of TGF-β, Smad2 / 3, p-Smad2 / 3, and Smad4 in the model group were significantly decreased (P<0.001), while the expression of Smad7 was significantly increased (P<0.001). This indicates that UVB irradiation weakens the activity of the TGF-β signaling pathway, while activating the MAPK and NF-κB signaling pathways, triggering an inflammatory response, exacerbating ECM degradation and skin barrier damage.

[0136] After the addition of TDM-5 and TDM-1, the expression levels of TGF-β, Smad2 / 3, p-Smad2 / 3, and Smad4 all rebounded, while Smad7 expression slightly decreased. This suggests that TDM-5 and TDM-1 may restore the expression level of the TGF-β signaling pathway by inhibiting the competitive antagonism of Smad7, thereby enhancing the cells' ability to regulate ECM metabolism and reducing UVB-induced inflammatory responses.

[0137] In summary, TDM-5 and TDM-1 components play a protective role against UVB-induced photoaging by restoring the function of the TGF-β / Smad axis, reducing NF-κB-mediated inflammatory response, reducing ECM degradation, and improving skin barrier integrity.

[0138] In summary, TDM-5 and TDM-1 can synergistically inhibit UVB-induced inflammatory responses, reduce ECM degradation, enhance skin barrier function, and exhibit promising anti-photoaging activity. TDM-1 (≤10 kDa) focuses on transdermal absorption and inflammation regulation, inhibiting UVB-induced MAPK / NF-κB pathway activation and reducing inflammatory damage. TDM-5 (10 kDa < molecular weight ≤50 kDa) enhances epidermal barrier function (e.g., by upregulating FLG and HAS-2 expression) and promoting ECM repair by promoting TGF-β signaling.

[0139] Example 6 Synergistic anti-photoaging effect of TDM-1 and TDM-5

[0140] We investigated whether the combination of Dendrobium officinale proteolytic products TDM-1 and TDM-5 within a certain mass ratio had a synergistic effect on UVB-induced photoaging damage in HaCaT cells. HaCaT cell viability was assessed using the CCK-8 assay, and the expression levels of key genes (IL-1β, MMP-1, FLG, and HAS-2) were measured using fluorescent quantitative RT-qPCR to clarify the correlation between the combination ratio and efficacy. The experimental groups and treatments are shown in Table 2:

[0141] Table 2 Experimental groups and treatments

[0142]

[0143] Cell survival results are shown in Table 3. The model group had a cell survival rate of 62.5±4.1%, significantly lower than that of the control group (100±3.2%, *P<0.001). The survival rates of the TDM-1 and TDM-5 groups alone increased to 78.3±3.5% and 75.6±2.8%, respectively (P<0.01). The effects of the combined groups were significantly enhanced: the survival rates of the 1:0.5 group were 84.2±3.1% (*P<0.001), the 1:1 group reached 92.7±2.9% (P<0.001), and the 1:2 group were 81.5±3.0% (**P<0.001). Gene expression results, shown in Table 4, further support a synergistic effect: IL-1β and MMP-1 expression levels in the 1:1 combination group were 1.08±0.1 and 0.89±0.1, respectively, which were 48.8% to 64.6% lower than those in the TDM-1 group (2.11±0.2, 1.95±0.3) and the TDM-5 group (3.05±0.3, 2.87±0.2) (*P<0.001). Concomitantly, FLG and HAS-2 expression levels in the 1:1 combination group (1.15±0.2, 1.02±0.2) were 40.2% to 34.2% higher than those in the TDM-5 group (0.82±0.2, 0.76±0.1) (P<0.01). The 1:0.5 and 1:2 groups showed less pronounced effects, but were still significantly superior to either component alone.

[0144] Table 3 Cell survival rate

[0145]

[0146] Table 4 Gene expression levels (relative mRNA expression)

[0147]

[0148] These results demonstrate that when TDM-1 and TDM-5 are combined in a mass ratio of 1:0.5 to 2, they exhibit a synergistic effect by synergistically inhibiting inflammatory factors (IL-1β, MMP-1) and enhancing the expression of barrier repair genes (FLG, HAS-2), significantly enhancing anti-photoaging efficacy. A 1:1 ratio achieves the most effective effect. This result is consistent with the previously revealed mechanisms of MAPK / NF-κB pathway inhibition (TDM-1 effect) and TGF-β pathway activation (TDM-5 effect), confirming that TDM-1 and TDM-5 components can achieve multi-target synergistic anti-aging effects, providing a core raw material for the development of anti-photoaging cosmetics.

Claims

1. A Dendrobium officinale enzymatic hydrolysate composition, characterized in that: The invention is composed of Dendrobium officinale protein hydrolysis products TDM-1 and TDM-5, with a mass ratio of 1:(0.5-2); TDM-1 and TDM-5 are peptide components with a molecular weight of ≤10 kDa and a peptide component with a molecular weight of 10 kDa < ≤50 kDa, respectively, obtained by enzymatic hydrolysis of Dendrobium officinale protein using neutral protease and papain.

2. The Dendrobium officinale enzymatic hydrolysate composition according to claim 1, wherein The mass ratio of Dendrobium officinale enzymatic hydrolysis products TDM-1 and TDM-5 is 1:(0.5~1).

3. The Dendrobium officinale enzymatic hydrolysate composition according to claim 1, characterized in that The Dendrobium officinale protein is extracted by adopting the alkali extraction and acid precipitation method.

4. The Dendrobium officinale enzymatic hydrolysate composition according to claim 3, characterized in that The extraction temperature of the alkali extraction and acid precipitation is 55-65° C., the extraction time is 1.0-2.0 h, the alkali solution concentration is 0.08-0.12 mol / L, the material-liquid ratio is 1.45-1:65 g / mL; and the acid precipitation pH is 3.0-4.

5.

5. The Dendrobium officinale enzymatic hydrolysate composition according to claim 1, characterized in that: During the combined enzymatic hydrolysis, the mass ratio of neutral protease to papain is 1:1, the enzymatic hydrolysis time is 2 to 5 hours, the enzymatic hydrolysis pH is 6.0 to 8.0, the enzymatic hydrolysis temperature is 55 to 65° C., and the total enzyme addition amount is 6000 to 8000 U / g of Dendrobium officinale protein.

6. The Dendrobium officinale enzymatic hydrolysate composition according to claim 1, characterized in that The separation is carried out by gradient ultrafiltration, using 50 kDa and 10 kDa ultrafiltration membranes for separation in sequence to obtain TDM-1 with a molecular weight ≤ 10 kDa and TDM-5 with a molecular weight of 10 kDa < ≤ 50 kDa.

7. Use of the Dendrobium officinale enzymatic hydrolysate composition according to any one of claims 1 to 6 in the preparation of anti-aging cosmetics.

8. A cosmetic with anti-aging effect, characterized in that: A composition containing the Dendrobium officinale enzymatic hydrolysate according to any one of claims 1 to 6.

9. The use according to claim 7 or the cosmetic according to claim 8, characterized in that: The dosage form of the cosmetic is essence, cream or mask.

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