Composite hydrogels, methods of making and using the same

Abstract

The application provides a composite hydrogel and a preparation method and application thereof, and the preparation method comprises the following steps: S1, dissolving dendrobium fimbriatum polysaccharide DDAM-3 in water and heating and stirring; S2, under the stirring state, adding an aqueous solution of dendrobium officinale polysaccharide DOMOS; S3, after cooling to room temperature, adding nicotinamide and hyaluronic acid and uniformly mixing; and S4, standing to form a stable composite hydrogel. The dendrobium polysaccharide (DDAM-3 and DOMOS), hyaluronic acid and nicotinamide are prepared into the composite hydrogel through intermolecular self-assembly, and the anti-aging effect of the hydrogel is significantly improved.

Description

Technical Field

[0001] This invention relates to the field of hydrogel technology, and in particular to a composite hydrogel, its preparation method, and its application. Background Technology

[0002] Skin aging is a complex physiological process, mainly manifested in extracellular matrix degradation, collagen loss, increased oxidative stress, and chronic inflammation. This drives the continuous research and development demand for anti-aging active ingredients and advanced delivery systems. Natural products, especially medicinal plant polysaccharides, show great promise in the field of anti-aging cosmetics and functional skincare products due to their good biocompatibility, multi-target mechanism of action, and low side effects. Among them, Dendrobium polysaccharides have been proven to have multiple biological activities such as antioxidant, anti-inflammatory, and collagen synthesis promotion, making them potential anti-aging active ingredients; hyaluronic acid, as a classic moisturizer, can improve skin hydration; and niacinamide can regulate cellular energy metabolism and inhibit pigmentation. However, how to efficiently integrate these multiple active ingredients to construct a dosage form with synergistic effects, stability, controllability, and ease of application remains a technical problem that urgently needs to be solved in this field.

[0003] Currently, physically mixing multiple natural active ingredients is a common formulation method. However, this method often struggles to achieve molecular-level synergy and stable binding between components, potentially leading to problems such as easy inactivation of active ingredients, uncontrollable release, and low transdermal absorption efficiency. Hydrogels, as hydrophilic materials with a three-dimensional network structure, can serve as ideal carriers for active ingredients. However, their construction often relies on chemical cross-linking agents or complex synthetic processes, which may introduce biosafety risks and insufficiently utilize the functional structures of natural polysaccharides. Therefore, developing a hydrogel system based on the inherent properties of natural ingredients, formed through a gentle self-assembly process, and possessing excellent stability, injectability, and multiple synergistic anti-aging functions is of great significance for improving the efficacy and safety of anti-aging products. Summary of the Invention

[0004] The purpose of this invention is to propose a composite hydrogel, its preparation method, and its application. The aim is to prepare a self-contained hydrogel that carries DOMOS, NMN, and HA, providing new ideas for the application of Dendrobium polysaccharides in anti-aging.

[0005] In a first aspect, the present invention provides a method for preparing a composite hydrogel, the method comprising: Step S1: Dissolve Dendrobium nobile polysaccharide DDAM-3 in water and heat while stirring; Step S2: While stirring, add an aqueous solution of Dendrobium officinale polysaccharide DOMOS; Step S3: After cooling to room temperature, add nicotinamide and hyaluronic acid, and mix well; Step S4: Let stand to form a stable composite hydrogel.

[0006] In some embodiments, in step S1, the concentration of DDAM-3 is 30-70 mg / mL, and the heating temperature is 60-90°C.

[0007] In some embodiments, in step S2, the concentration of the DOMOS solution is 30-70 mg / mL, and the weight ratio of the added DOMOS to DDAM-3 is 0.5-2:1.

[0008] In some embodiments, the weight ratio of DDAM-3, DOMOS, nicotinamide, and hyaluronic acid is 50-80:50-80:5-10:50-80.

[0009] In some embodiments, the weight ratio of DDAM-3, DOMOS, nicotinamide, and hyaluronic acid is 250:250:23:250.

[0010] In some embodiments, the settling time in step S4 is 1-3 hours.

[0011] Secondly, the present invention also proposes a composite hydrogel prepared according to the above-described method for preparing composite hydrogels.

[0012] Thirdly, the present invention also proposes the use of the above-described composite hydrogel in the preparation of pharmaceutical compositions or skin care products having any one or more of the following functions: Inhibits elastase and / or collagenase; Promotes the secretion of type I collagen in human dermal fibroblasts; Clearing intracellular reactive oxygen species and / or reducing SA-β-gal activity; Downregulate the expression of IL-6 and / or MMP-3; Promotes collagen deposition in the skin; Increase the thickness of the dermis; Improve D-galactose induction.

[0013] Fourthly, the present invention also provides a pharmaceutical composition comprising: Physiologically or pharmaceutically acceptable excipient formulations; And the composite hydrogels described above.

[0014] Fifthly, the present invention also proposes a skin care product comprising: Cosmetic base solvent; And the composite hydrogels described above.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention prepares a composite hydrogel by intermolecular self-assembly of Dendrobium polysaccharides (DDAM-3 and DOMOS), hyaluronic acid, and nicotinamide, which significantly enhances the anti-aging efficacy of the hydrogel. This includes effectively inhibiting the activity of elastase and collagenase, promoting the secretion of type I collagen, scavenging excess reactive oxygen species, and reducing the expression of aging-related secretory phenotypic factors (such as IL-6 and MMP-3). Thus, it exhibits a strong anti-skin aging effect in both in vivo and in vitro experiments, providing a new and efficient solution for the development of anti-aging cosmetics and pharmaceutical compositions. Attached Figure Description

[0016] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 The diagram shows the preparation of the composite hydrogel. Figure 1 a shows the incomplete hydrogel morphology during the preparation of DDAM-3. Figure 1 b is a morphological diagram of the successfully prepared composite hydrogel. Figure 1 c and Figure 1 d shows schematic diagrams of film formation properties before and after successful preparation of the composite hydrogel; Figure 2 Cryo-SEM images of the composite hydrogel, where, Figure 2 a and Figure 2 Figures b show the Cryo-SEM analysis results before and after successful preparation of the composite hydrogel. Figure 3 The diagrams show the G'G'' graph and viscosity and shear stress of the composite hydrogel. Figure 3 a is the storage modulus diagram of the composite hydrogel. Figure 3 b is the viscosity diagram of the composite hydrogel. Figure 3 c is the shear stress curve of the composite hydrogel; Figure 4 The image shows the ITC of the composite hydrogel, where... Figure 4 Figure a shows a standard calorimetric titration experiment using DOMOS / NMN. Figure 4 b is a graph of DOMOS / NMN's ITC data; Figure 4 c is a diagram of the standard calorimetric titration experiment of HA / NMN. Figure 4 d represents the ITC data for HA / NMN; Figure 4 e is a graph from a standard calorimetric titration experiment of DDAM-3 / NMN. Figure 4 f is the ITC data plot for DDAM-3 / NMN; where ΔG represents the Gibbs free energy of the binding reaction (blue), ΔH represents the enthalpy change (green), and -TΔS represents the entropy change (red). Figure 5 This is a diagram of molecular docking in a composite hydrogel, where... Figure 5 a is a diagram showing the integration between DOMOS and NMN. Figure 5 b is the diagram of HA and NMN docking. Figure 5 c shows the docking diagram of DDAM-3 and NMN; Figure 6 The graph shows the anti-aging activities of DDAM-3, DOMOS, HA, and NMN. Figure 6 a is a graph showing the inhibitory activity of DDAM-3 on elastase. Figure 6 b shows the inhibitory activity of DDAM-3 against collagenase. Figure 6 c is a graph showing the activity of DDAM-3 in the secretion of type I collagen. Figure 6 d is a graph showing the inhibitory activity of DOMOS on elastase. Figure 6 e represents the DOMOS inhibitory activity against collagenase. Figure 6 f is a graph showing the activity of DOMOS in the secretion of type I collagen. Figure 6 g represents the inhibitory activity of HA on elastase. Figure 6 h represents the inhibitory activity of HA on collagenase. Figure 6 i represents the activity diagram of HA on type I collagen secretion. Figure 6 j represents the graph of NMN's inhibitory activity against elastase. Figure 6 k represents the inhibitory activity of NMN on collagenase. Figure 6 l is a graph showing the activity of NMN on the secretion of type I collagen; Figure 7 This is a graph showing the anti-aging activity of the composite hydrogel, where... Figure 7 a is a graph showing the inhibitory activity of the composite hydrogel elastase. Figure 7 b is a graph showing the inhibitory activity of the composite hydrogel on collagenase. Figure 7 c is a graph showing the activity of the composite hydrogel on the secretion of type I collagen; Figure 8 This diagram illustrates the inhibitory effect of the composite hydrogel on β-galactosidase in HSF cells. Figure 8 a is a staining image of β-galactosidase. Figure 8 b is a quantitative map of β-galactosidase-positive cells; Figure 9 This diagram illustrates the inhibitory effect of the composite hydrogel on ROS in HSF cells. Figure 9 a is the ROS fluorescence staining image. Figure 9 b is a quantitative map of ROS-positive cells stained with fluorescent dyes; Figure 10 The diagram shows the inhibitory effect of the composite hydrogel on SASP, where, Figure 10 Figure a shows the effect of the composite hydrogel on MMP-3 secretion in HSF cells. Figure 10b shows the effect of the composite hydrogel on IL-6 secretion in HSF cells; Figure 11 The image shows the pathological changes in mouse skin improved by composite hydrogel. Figure 12 The image shows the effect of composite hydrogel on promoting collagen expression in mouse skin. Figure 12 Image a shows Sirius red staining of mouse skin. Figure 12 b is a quantitative diagram of cells stained with Sirius red; Figure 13 This diagram illustrates how composite hydrogels inhibit the expression of MMP-3 in mouse skin. Detailed Implementation

[0017] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. It should be noted that, unless otherwise specified, the embodiments and features described herein can be combined with each other.

[0018] The following detailed description is exemplary and intended to provide further detailed explanation of the invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used in this invention is for describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention.

[0019] Dendrobium, a precious medicinal plant with a history of over a century, is a resource used for both medicinal and edible purposes. It is renowned for its ability to nourish yin and stomach, promote body fluid production, moisten the lungs, relieve coughs, reduce internal heat, and enhance yin energy. The main components of Dendrobium are polysaccharides, flavonoids, alkaloids, phenols, and amino acids, which possess significant physiological activities such as enhancing immunity, lowering blood sugar, inhibiting cancer, alleviating inflammation, and reducing oxidative stress.

[0020] Dendrobium devonianum, also known as purple-skinned Dendrobium, is mainly distributed in southern my country and is a high-quality health food used for immune regulation and enhancing physical fitness. Polysaccharides are the main component of Dendrobium devonianum. The applicant discovered that the skeleton of the Dendrobium devonianum polysaccharide DDAM-3 is acetylated mannan. The acetylated polysaccharide undergoes a change in spatial conformation. Combined with electron microscopy analysis, the applicant found that DDAM-3 possesses excellent carrier properties and is a good carrier for preparing hydrogels.

[0021] Dendrobium officinale, whose medicinal use was first recorded in the "Shennong's Classic of Materia Medica", has high health value. The applicant's research found that it has a variety of biological activities such as anti-aging, hypoglycemic, immune regulation, and anti-oxidation. At the same time, the research found that the oligosaccharide DOMOS in Dendrobium officinale can be used to treat skin aging and its complications, indicating its potential therapeutic efficacy for clinical age-related diseases.

[0022] In summary, based on the carrier characteristics of Dendrobium officinale polysaccharide DDAM-3, the applicant attempts to invent a self-contained hydrogel carrying DOMOS, NMN, and HA, providing new ideas for the application of Dendrobium officinale polysaccharide in anti-aging.

[0023] Example 1: Preparation and Characterization of Composite Hydrogels (1) Preparation of solution: Weigh 1g of Dendrobium officinale polysaccharide DDAM-3, place it in a 100 mL beaker, add water to dissolve and make up to 20 mL, i.e. 50mg / mL for later use; take Dendrobium officinale polysaccharide DOMOS, place it in a 50 mL centrifuge tube, add water to dissolve and make up to 20 mL, i.e. 50mg / mL for later use; take 92.3077mg of nicotinamide NMN and 1g of hyaluronic acid HA for later use.

[0024] (2) Preparation of composite hydrogel: 50 mg / mL DDAM-3 solution was placed in a beaker and heated and stirred on an electric furnace. At the same time, 20 mL of 50 mg / mL DOMOS solution was slowly added and mixed well. After cooling to room temperature, weighed NMN and HA were added, mixed well, and allowed to stand for 2 h to observe the morphology of the composite hydrogel. The composite hydrogel is morphologically stable and can be stored for a long time at room temperature or 4 °C.

[0025] (3) Characterization of the composite hydrogel: Cryo-scanning electron microscopy (Cryo-SEM) analysis—The microstructure of DDAM-3 and the composite hydrogel was observed using Cryo-SEM. After pre-freezing the hydrogel with liquid nitrogen, it was sublimated at -70℃ for 15 min, sliced, and its morphological characteristics were observed. Rheological properties analysis of the composite hydrogel—The shear stress (Pa), viscosity (Pa·s), storage modulus (G´), and loss modulus (G´´) of the hydrogel were tested using an MCR rheometer. The test conditions were as follows: rotor was CP25; test module was viscosity curve; measurement interval was 0.1 mm; shear rate was 0.01-100 1 / s; normal force was 10 N; shear frequency was 0.1-10 Hz; and measurement temperature was 25℃.

[0026] Experimental Results: By adjusting the concentration of DDAM-3, the optimal preparation conditions for the hydrogel were determined as follows: Add 20 mL of 50 mg / mL DOMOS to every 20 mL of 50 mg / mL DDAM-3, heat and stir on an 80℃ electric furnace until completely dissolved, then cool to room temperature. Add 92.3077 mg of nicotinamide (NMN) and 1 g of hyaluronic acid (HA), stirring until completely dissolved. Let stand for at least 2 hours to obtain the hydrogel. Figure 1 As shown, Figure 1 Image a shows the incomplete hydrogel morphology during the preparation of DDAM-3, exhibiting fluidity. Figure 1b is an image of the successfully prepared composite hydrogel. Figure 1 c and Figure 1 d shows the film-forming properties before and after successful hydrogel preparation. (See figure d.) Figure 2 As shown, Cryo-SEM analysis results indicate that DDAM-3 exhibits a loose chain-like structure. Figure 2 a), and after being prepared into a hydrogel, it exhibits a regular three-dimensional porous network structure, with a significantly improved degree of crosslinking ( Figure 2 b). For example Figure 3 As shown in Figure a, throughout the entire frequency scanning range, the storage modulus (G') of DDAM-3 and the composite hydrogel ranges from 560 to 700 Pa, consistently exceeding the loss modulus (G'') by 100 to 240 Pa. Furthermore, the storage modulus of the composite system is more than 200 Pa higher than that of DDAM-3, confirming the formation of a stable elastic network structure. Figure 3 b and Figure 3 As shown in Figure c, the apparent viscosity decreases with increasing shear rate, from 38000 mP·s to 5000 mP·s, exhibiting shear-thinning behavior, indicating that the hydrogel possesses good injectability. Combined rheological results show that a denser, more stable, and robust three-dimensional cross-linked network has formed within the composite hydrogel.

[0027] Example 2: Study on particle size distribution, zeta potential, and intermolecular interactions of hydrogels Experimental methods: (1) ITC: The intermolecular interaction forces of molecules in the hydrogel were tested by isothermal titration calorimetry (ITC). DDAM-3, DOMOS, and HA were prepared into 5 mg / mL aqueous solutions, and small molecule NMN was prepared into 1 mg / mL aqueous solution. The solutions were filtered and mixed. 260 μL of the prepared DDAM-3, DOMOS, and HA aqueous solutions were added to the sample cell, and 40 μL of NMN mixed aqueous solution was injected into the syringe. The sample cell was titrated with the syringe. The titration experiment was performed 17 times in total, with a titration volume of 2 μL each time, an interval of 150 s, a temperature of 25℃, and a rotation speed of 750 rpm. Deionized water was titrated with deionized water to perform a blank experiment. After the titration, MicroCal PEAQ-ITC analysis software was used for data analysis. The "one-sided mode" fitting model was used to analyze the data in combination with the isotherm. (2) Molecular docking: The three-dimensional structures of hyaluronic acid and nicotinamide mononucleotide (NMN) were obtained from the PubChem database and the energy minimization geometry was optimized by the MM2 force field of Chem3D software. Hydrogen atoms were added to all molecules and Gasteiger charges were calculated using the AutoDockTools software package, with NMN as the ligand using the default rotatable bond setting and polysaccharide molecules (HA, DOMOS, DDAM-3) as rigid acceptors. Semi-flexible molecular docking was performed using the AutoDock Vina program

[30] , with the docking grid set at the geometric center of each polysaccharide molecule, and the binding free energy (ΔG) of the resulting complex was calculated.

[0028] Experimental results: such as Figure 4 a to Figure 4 f、 Figure 5 As shown in Table 1, the ITC experimental results indicate that DDAM-3, DOMOS, HA, and NMN can interact, and the pathways of these interactions are investigated. For ITC, the KD value of DDAM-3 / NMN (158e) is... -6 ), KD value of DOMOS / NMN (89.5e) -6 ), KD value of HA / NMN (248e) -6 The KD value reflects the ability of the interaction to proceed; the smaller the KD value, the stronger the interaction between the two. Based on the experimental results, ΔG(158e) of DDAM-3 / NMN... -6 kJ / mol), ΔG of DOMOS / NMN (89.5e -6 kJ / mol), ΔG of HA / NMN (248e -6The kJ / mol value indicates a spontaneous interaction between them. The negative ΔH value and positive -TΔS value suggest that the reaction between NMN and DDAM-3 / DOMOS / HA is an enthalpy-driven process. This thermodynamic behavior indicates that the co-assembly of polysaccharides and NMN is primarily driven by hydrogen bonds or van der Waals forces, rather than entropy-dominated hydrophobic interactions, reflecting the formation of a specific and stable intermolecular bond.

[0029] Table 1. Thermodynamic parameters of the interaction between DOMOS / DDAM-3 / HA and NMN

[0030] Example 3: In vitro anti-aging activity study of the complex (1) Elastase inhibition assay: 96-well plates were divided into a normal control group, a low-dose composite hydrogel group (100 μg / mL) (hereinafter referred to as Mix), and a high-dose composite hydrogel group (400 μg / mL). For each group, 50 μL of diluted sample solution, 50 μL of porcine pancreatic elastase solution (0.171 U / mL), and 100 μL of substrate were added sequentially. The mixture was shaken and incubated at room temperature for 5 min. The absorbance at 420 nm was measured using a microplate reader. In the control group, 50 μL of distilled water replaced the sample in the blank wells, and 100 μL of distilled water replaced the substrate solution in the sample control wells. The assay method was the same as above, and each sample was repeated three times. The substrate solution was N-succinyl-alanine-alanine-p-nitroaniline (AAAPVN), prepared to 2 mM using HCl (0.1 M, pH=8.0) buffer. The inhibition rate is calculated according to formula (1): Inhibition rate (%) = 1 - (An - An') / (A0 - A0') × 100% (1).

[0031] (2) Collagenase inhibition assay: 96-well plates were divided into a normal control group, a low-dose complex group (100 μg / mL), and a high-dose complex group (400 μg / mL). For each administration group, 50 μL of diluted sample solution, 50 μL of type I collagenase solution, 60 μL of Tris-HC1 (0.1M, pH=8.0) buffer, and 50 μL of water were added sequentially. The mixture was shaken and incubated at 37℃ for 30 min. Then, 50 μL of substrate was added, shaken, and reacted at 37℃ for 30 min. The absorbance at 335 nm was measured using a microplate reader. In the control group, 50 μL of distilled water replaced the sample in the blank wells, and 50 μL of distilled water replaced the substrate solution in the sample control wells. The assay method was the same as above, and each sample was repeated three times. Wherein: Substrate solution: 1 mmol / L N-[3-(2-furanyl)acryloyl]leucine-glycine-proline-alanine (FALGPA) was prepared using Tris-HC1 buffer, and the inhibition rate was calculated according to formula (1).

[0032] (3) Collagen secretion-promoting assay: 500 μL of HSF cells (5 × 10⁻⁶ cells) were added to a 24-well plate. 4 After incubation for 24 h, the supernatant was discarded. The drug group was given different concentrations of drug diluted with basal medium, 500 μL was added to each well, and a blank control group was set up at the same time. Five replicates were set up for each group. After incubation for 24 h, the supernatant was collected, and the OD value was measured at 450 nm using the human PINP enzyme-linked immunosorbent assay kit according to the manufacturer's instructions. The amount of collagen secreted was calculated according to the standard curve provided by the reagent manufacturer.

[0033] Experimental results: such as Figures 6 to 7As shown, DOMOS exhibited average inhibition rates of 10.78% and 6.63% against elastase and collagenase, respectively; DDAM-3 showed average inhibition rates of 15.39% and 2.64% against elastase and collagenase, respectively; HA showed average inhibition rates of 13.9% and 5.49% against elastase and collagenase, respectively; and NMN showed average inhibition rates of 18.08% and 6.92% against elastase and collagenase, respectively. Compared with the single components, the composite hydrogel showed improved average inhibition rates against elastase and collagenase, at 21.21% and 19.04%, respectively, indicating its good enzyme inhibition ability. The promotion rates of type I collagen secretion by HSF cells by DOMOS, DDAM-3, HA, and NMN were 88.33%, 2.33%, 94.66%, and 40.00%, respectively. Compared with the single components, HSF cells treated with the composite hydrogel showed a significant increase in type I collagen secretion at all three test concentrations, with an average promotion rate of 129.33%. In summary, compared with single components, the composite hydrogel exhibits superior performance in multiple anti-aging indicators, indicating that the integration of components achieves a synergistic enhancement effect, and the composite system demonstrates significantly stronger anti-aging potential.

[0034] Example 4: Study on the in vitro anti-aging effect of the complex (1) β-galactosidase staining: HSF cells were seeded in 6-well plates (4 × 10⁻⁶ cells / well). 4 Cells were divided into 5 groups (number of cells / mL): normal control group, model control group, low-dose composite hydrogel group (100 μg / mL), and high-dose composite hydrogel group (400 μg / mL). The control group was cultured in normal culture medium, the model group was cultured in culture medium containing 40 g / L D-gal for 24 h, and the drug-treated groups were cultured in a drug containing 40 g / L D-gal for another 24 h. After washing twice with PBS, the cells were stained using the SA-β-gal staining kit according to the manufacturer's instructions, incubated overnight at 37°C, and then observed for cell senescence under a microscope. The positive rate of cells was assessed using ImageJ software.

[0035] (2) Reactive oxygen species (ROS) detection: HSF cells were seeded in 6-well plates (4 × 10⁻⁶ cells / well). 4HSF cells were divided into 5 groups and treated with the drug for 24 hours. After washing the cells twice with PBS, 10 μM 2′,7′-dichlorofluorescein diacetate (DCFH-DA) was added for 30 minutes. After that, the cells were washed twice with PBS. Finally, the cells were examined with a fluorescence inverted microscope with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The fluorescence intensity of positive cells was evaluated using ImageJ software. (3) SASP detection: HSF cells were cultured in 24-well culture dishes (5 × 10⁻⁶ cells / mL). 4 After 24 hours of treatment with the drug, the supernatant was collected 24 hours later. The levels of IL-6 and MMP-3 in the HSF cell supernatant were assessed using an enzyme-linked immunosorbent assay (ELISA) kit, and the absorbance was measured at 450 nm using an ELISA reader.

[0036] Experimental results: such as Figure 8 As shown, blue represents positive cells. Compared with the normal control group, the number of SA-β-gal positive cells in the model group significantly increased to 18.64%. The composite hydrogel intervention significantly reduced the SA-β-gal positivity rate in HSF cells, recovering to 6.57% at high doses, indicating its potential to delay HSF cell senescence. Furthermore, as... Figure 9 As shown, intracellular ROS levels were detected using a fluorescent reactive oxygen species probe. Green represents positive cells. The normal control group showed only weak fluorescence, accounting for 9.06% of the total, indicating a low ROS physiological state. In contrast, the model group showed significantly enhanced ROS fluorescence intensity, accounting for 27.49% of the total. Composite hydrogel treatment effectively reversed this trend to 11.62%, significantly reducing ROS levels. Excessive ROS can also affect the secretion of SASP factors, such as… Figure 10 As shown, compared with the model group, the composite hydrogel treatment significantly downregulated the expression levels of MMP-3 and IL-6 in HSF. MMP-3 was downregulated to the normal level of approximately 1.26 ng / mL, and the secretion level of IL-6 under high-concentration complex intervention was 46.20 pg / mL, close to that of the normal group. In conclusion, this composite hydrogel effectively alleviates D-galactose-induced HSF cell senescence by inhibiting excessive ROS accumulation and regulating the expression of key SASP factors.

[0037] Example 5: Study on the in vivo anti-aging effect of the complex Animal Experiment Design: Eight-week-old male C57BL / 6J mice (20±2g) were randomly divided into a normal control group, a model group, a low-dose complex group, and a high-dose complex group, with 10 mice in each group. Mice in the control group received daily intraperitoneal injections of 0.9% saline, while mice in the other groups received daily intraperitoneal injections of 200 mg / kg D-galactose for 8 weeks to induce modeling. Starting from the fourth week of modeling, mice were administered the compound hydrogel via gavage: low-dose group (50 mg / kg / day) and high-dose group (100 mg / kg / day). All groups were fed the same diet, with consistent use of sterile water and bedding. Free access to food and water was provided. After 8 weeks of continuous feeding, followed by a 12-hour fast, mice were euthanized under anesthesia. Dissection was performed according to standard laboratory animal procedures, and the dorsal skin was quickly removed, cleaned with saline, and stored in liquid nitrogen for later use.

[0038] Anti-aging effect study: Experimental methods: HE staining observation and analysis: Skin wounds and surrounding tissues were sectioned. Mouse skin tissue was fixed in 4% paraformaldehyde solution and stained with hematoxylin and eosin (H&E) and Sirius red, respectively. After imaging the stained sections under an optical microscope, ImageJ software was used to quantitatively analyze the morphological changes of the epidermis and dermis and the area of ​​collagen. The level of MMP3 in skin tissue was detected by ELISA: The back skin tissue was dissected and carefully dissected to remove adjacent non-skin components. The lysed samples were centrifuged and the supernatant was collected for subsequent analysis. The total protein concentration was determined by the BCA protein detection kit according to the manufacturer's instructions. All samples were adjusted to a uniform concentration using lysis buffer. The MMP-3 level was quantified by the ELISA kit, and the absorbance measurement was strictly performed according to the kit's operating instructions.

[0039] Experimental results: HE staining results are as follows Figure 11 As shown, compared with the normal group, the D-galactose-induced aging model group exhibited irregular and uneven epidermal thickening, dermal thinning, inflammatory cell infiltration, and disordered collagen fiber structure. The mixed hydrogel treatment significantly reversed these age-related pathological changes, resulting in a more regular epidermal morphology and a significant increase in dermal thickness. Sirius red staining results are shown below. Figure 12 As shown, the normal control group's skin tissue exhibited a complete structure with dense and uniform collagen fiber arrangement; in contrast, the model group showed collagen expression at only 23.54%, with loose and disordered fiber arrangement. After treatment with the composite hydrogel, the density and orderly arrangement of collagen fibers were significantly improved to 49.67%, indicating that the hydrogel can effectively promote collagen synthesis and orderly deposition. ELISA results are as follows. Figure 13As shown, MMP-3 expression in the model group was significantly increased to 278.03 ng / mL, while treatment with the composite hydrogel significantly inhibited its expression to 203.41 ng / mL. This indicates that the composite hydrogel may exert its anti-skin aging effect by downregulating MMP expression and reducing collagen degradation, thereby synergistically promoting collagen homeostasis.

[0040] In summary, the successful construction of the self-assembled composite hydrogel provides a novel strategy for addressing the complexities of skin aging. In vitro experiments show that this composite hydrogel significantly inhibits elastase and collagenase activity and effectively promotes the secretion of type I collagen in human dermal fibroblasts. In a D-galactose-induced cellular senescence model, the composite hydrogel exhibits strong cytoprotective effects by effectively scavenging excess reactive oxygen species, reducing SA-β-gal activity, and downregulating the expression of key aging-related secretory phenotypic factors such as IL-6 and MMP-3. Animal models demonstrate that treatment with the composite hydrogel effectively improves age-related histopathological changes, such as increasing epidermal and dermal thickness and enhancing skin collagen fiber density to combat skin aging.

[0041] As is known from common technical knowledge, this invention can be implemented through other embodiments that do not depart from its spirit or essential characteristics. Therefore, the disclosed embodiments described above are merely illustrative in all respects and are not the only ones. All modifications within the scope of this invention or equivalent to the scope of this invention are included in this invention.

[0042] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a composite hydrogel, characterized in that, The preparation method includes: Step S1: Dissolve Dendrobium nobile polysaccharide DDAM-3 in water and heat while stirring; Step S2: While stirring, add an aqueous solution of Dendrobium officinale polysaccharide DOMOS; Step S3: After cooling to room temperature, add nicotinamide and hyaluronic acid, and mix well; Step S4: Let stand to form a stable composite hydrogel.

2. The method for preparing the composite hydrogel according to claim 1, characterized in that, In step S1, the concentration of DDAM-3 is 30-70 mg / mL, and the heating temperature is 60-90℃.

3. The method for preparing the composite hydrogel according to claim 1, characterized in that, In step S2, the concentration of the DOMOS solution is 30-70 mg / mL, and the weight ratio of the added DOMOS to DDAM-3 is 0.5-2:

1.

4. The method for preparing the composite hydrogel according to claim 1, characterized in that, The weight ratio of DDAM-3, DOMOS, nicotinamide, and hyaluronic acid is 50-80:50-80:5-10:50-80.

5. The method for preparing the composite hydrogel according to claim 4, characterized in that, The weight ratio of DDAM-3, DOMOS, nicotinamide, and hyaluronic acid is 65:65:65:

6.

6. The method for preparing the composite hydrogel according to claim 1, characterized in that, In step S4, the settling time is 1-3 hours.

7. A composite hydrogel prepared by the method according to any one of claims 1-6.

8. The use of the composite hydrogel according to claim 7 in the preparation of pharmaceutical compositions or skin care products having any one or more of the following functions: Inhibits elastase and / or collagenase; Promotes the secretion of type I collagen in human dermal fibroblasts; Clearing intracellular reactive oxygen species and / or reducing SA-β-gal activity; Downregulate the expression of IL-6 and / or MMP-3; Promotes collagen deposition in the skin; Increase the thickness of the dermis; Improve D-galactose induction.

9. A pharmaceutical composition, characterized in that, Include: Physiologically or pharmaceutically acceptable excipient formulations; And the composite hydrogel as described in claim 7.

10. A skincare product, characterized in that, Include: Cosmetic base solvent; And the composite hydrogel as described in claim 1.

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