A collagen delivery system, and a preparation method and application thereof

Abstract

The application discloses a collagen delivery system and a preparation method and application thereof, and raw materials of the collagen delivery system include an oil phase, an aqueous phase and a crosslinking agent, wherein the oil phase includes liquid paraffin oil and Span 80; the aqueous phase includes a collagen solution and a safflower polysaccharide solution; and the crosslinking agent includes an FeCl3 solution. The safflower polysaccharide microgel loaded collagen delivery system constructed by the application improves oral bioavailability of collagen, expands double application scenarios, realizes efficient anti-photoaging, and has the advantages of simple preparation process, safety controllability and scaleable production. The application solves the problems of poor repair effect of existing external anti-photoaging products, low oral collagen bioavailability, complex existing delivery system process, poor delivery efficiency and stability and the like.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a collagen delivery system, its preparation method, and its application. Background Technology

[0002] Skin anti-aging is a crucial research area in the beauty, skincare, and biomedical fields. Photoaging is the most significant exogenous factor contributing to skin aging, prevalent across various population groups, particularly those with prolonged sun exposure. Photoaging primarily manifests as a series of skin aging problems caused by long-term UV radiation, including impaired skin barrier function, breakage of elastic fibers, accelerated collagen degradation, and exacerbated oxidative stress damage. Externally, this results in rough skin, increased wrinkles, pigmentation, and decreased elasticity. Currently, photoaging repair focuses on beauty and skincare products and medical skincare. However, traditional skincare products struggle to penetrate the dermis, failing to reach the core repair sites of photodamage; their formulas are often limited, lacking adaptability to different skin types and prone to causing irritation or allergic reactions; and the absorption rate of active ingredients is limited, hindering deep repair and limiting overall intervention effectiveness. As people's demand for skin health and anti-aging continues to rise, technological research and product innovation in related fields are constantly advancing. How to efficiently and safely delay photoaging has become one of the core research directions in the industry.

[0003] Based on the traditional health concept of internal regulation and external nourishment, oral administration has become a research hotspot in the field of skin anti-aging in recent years due to its convenience and safety. Among them, collagen has become a key research object due to its unique advantages. Collagen has a wide range of sources, whether animal or plant-based, making it easy to obtain. It also has good biological activity, can participate in the repair and regeneration of skin tissue, and is characterized by high safety, ease of use, and cost-effectiveness. It shows great application potential in the field of anti-photoaging of the skin, but key technical bottlenecks still exist in practical applications. The bioavailability of directly orally administered collagen is low, and the proportion that can actually be absorbed by skin tissue and exert anti-photoaging effects is low. If the absorption is increased simply by increasing the intake of collagen, it will not only increase the metabolic burden of free amino acids in the body, but the excess amino acids will also undergo oxidative decomposition in the body, leading to the dysfunction of the electron transport chain, inducing abnormal accumulation of reactive oxygen species, and ultimately increasing the risk of oxidative stress in the body, which is detrimental to skin health and normal metabolism. Summary of the Invention

[0004] To overcome the technical bottlenecks in existing research, this invention provides a collagen delivery system, its preparation method, and its applications. This system uses Artemisia annua polysaccharide as a carrier, which not only possesses excellent biocompatibility but also exhibits intestinal responsiveness. It can swell in the intestinal environment and achieve targeted release of active ingredients, thereby improving the gastrointestinal retention rate of collagen. Simultaneously, the numerous hydrophilic groups in the polysaccharide molecules can non-covalently bind to the intestinal mucus layer, prolonging the retention time of the delivery system in the intestine and providing favorable conditions for collagen absorption and functional activity. This invention can improve the oral bioavailability of collagen, achieving its efficient anti-photoaging effect, and the preparation process is simple, safe, controllable, and suitable for large-scale production.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a collagen delivery system, comprising an oil phase, an aqueous phase, and a cross-linking agent, wherein: The oil phase comprises liquid paraffin oil and Span 80; The aqueous phase includes a collagen solution and an Artemisia annua polysaccharide solution; The crosslinking agent includes a FeCl3 solution.

[0006] Furthermore, the concentration of Span80 is 2%, and the mass ratio of liquid paraffin oil to Span80 is (40~50):(1~1.2); the mass concentration of the collagen solution is 2%~5%, and the mass concentration of the Artemisia annua polysaccharide solution is 1.5%~2%; the volume ratio of the oil phase to the water phase is (10~15):(1~1.5); the mass concentration of the FeCl3 solution is 2%~3%, and the volume ratio of the FeCl3 solution to the water phase is 1:1.

[0007] The present invention also provides a product for resisting skin photoaging, specifically a topical composition and / or oral collagen drink prepared using the above-mentioned collagen delivery system.

[0008] Furthermore, in preparing oral collagen drinks, the reconstituted collagen delivery system is dispersed in a solvent, wherein the solvent is deionized water or sterile saline.

[0009] Furthermore, the oral collagen drink has an oral relative bioavailability of greater than 143.58%.

[0010] Furthermore, the oral collagen drink can alleviate UV-induced photoaging of the skin, specifically by: improving the macroscopic condition of the skin, restoring skin moisture content and oil barrier, reducing skin oxidative stress and inflammatory response, and promoting the synthesis and deposition of extracellular matrix components in the dermal layer of the skin.

[0011] The present invention also provides a method for preparing the above-mentioned collagen delivery system, the specific steps of which are as follows: Liquid paraffin oil and Span80 are mixed and heated to a set temperature to obtain the oil phase; The collagen solution and the Artemisia annua polysaccharide solution were mixed to obtain the aqueous phase; The aqueous phase is added dropwise to the oil phase, stirred at a set speed, and emulsified for a set time to obtain the emulsified system. FeCl3 solution was added dropwise to the emulsion system, the system was adjusted to the set pH value, stirred at the set speed, crosslinked for the set time, centrifuged at the set speed for the set time, washed, and reconstituted to obtain the collagen delivery system.

[0012] Furthermore, the liquid paraffin oil and Span80 are mixed and heated to 35°C~40°C to obtain the oil phase.

[0013] Furthermore, the aqueous phase is added dropwise to the oil phase, and emulsification is carried out at a stirring speed of 1500 rpm to 2000 rpm for 1 to 2 hours to obtain an emulsified system.

[0014] Further, FeCl3 solution was added dropwise to the emulsion system, the pH of the system was adjusted to 4.5~5.0, and the mixture was stirred at 1000rpm~1500rpm for 10min~20min for crosslinking. The mixture was then centrifuged at 1500rpm~2000rpm for 5min~10min, washed, and reconstituted to obtain the collagen delivery system.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects: This invention provides a collagen delivery system using Artemisia annua polysaccharide microgel as the collagen delivery system, which effectively solves the problem of poor stability of existing delivery systems. The raw materials are widely available, cost-effective, and have excellent biocompatibility with no obvious toxic side effects. This avoids the problems of allergies and irritation associated with existing topical products. At the same time, it simplifies the raw material system, provides support for subsequent large-scale production, and meets the safety and efficiency requirements of anti-skin photoaging products.

[0016] This invention, by limiting the specific concentrations and proportions of each raw material, ensures stable emulsification performance of the oil phase, stable collagen loading in the aqueous phase, and a mild and sufficient cross-linking reaction, effectively solving the problem of poor delivery efficiency in existing delivery systems. The specific ratio gives the collagen delivery system excellent intestinal responsiveness, providing a guarantee for improving the oral bioavailability of collagen, while avoiding raw material waste, optimizing preparation costs, ensuring the structural stability of the collagen delivery system, and enabling it to stably exert anti-photoaging related effects.

[0017] This invention clarifies that the collagen delivery system can be used to prepare topical compositions and / or oral collagen drinks, breaking the limitation of traditional anti-photoaging products being limited to topical use. It effectively solves the problems of poor repair effects of existing topical products and low bioavailability of oral products; it achieves a combination of internal regulation and external nourishment, adapting to the usage needs of different groups and different scenarios. The oral form is convenient and safe, while the topical form can directly act on the skin surface, significantly improving the overall effect of anti-photoaging of the skin.

[0018] This invention uses deionized water or sterile saline as a solvent, which is highly safe and non-irritating. It can make the collagen delivery system uniformly dispersed and stably reconstituted, avoiding the aggregation of the collagen delivery system that affects the release and absorption of collagen, and solving the problem of poor delivery stability of existing oral products. At the same time, it simplifies the preparation process of oral beverages, facilitates large-scale production, and ensures that it can stably exert its anti-skin photoaging effect after oral administration.

[0019] The relative bioavailability of the oral collagen drink of this invention is greater than 143.58%, which is significantly higher than the bioavailability of existing direct oral collagen. It precisely solves the core problem of low bioavailability of existing oral collagen. It can achieve excellent anti-photoaging effects without increasing collagen intake, reduce the metabolic burden of free amino acids in the body, reduce the risk of oxidative stress, and at the same time enhance the anti-aging efficacy and market competitiveness of the product.

[0020] The oral collagen drink of this invention can specifically alleviate UV-induced photoaging of the skin and repair deep skin damage from multiple dimensions, effectively solving the problem that existing products cannot fundamentally solve the problem of deep skin damage caused by photoaging. Specifically, it can improve the macroscopic condition of the skin, restore the skin's moisture content and oil barrier, reduce oxidative stress and inflammatory response, and promote the synthesis and deposition of extracellular matrix in the dermis. Its repair effect is significantly better than that of existing oral collagen products.

[0021] This invention provides a simple, convenient, mild, and controllable method for preparing a collagen delivery system. It requires no complex equipment and effectively solves the problems of complex processes and difficulties in large-scale application of existing delivery systems. The preparation process does not introduce toxic or harmful reagents, which can effectively preserve the bioactivity of collagen and Artemisia annua polysaccharides, ensuring the safety and anti-photoaging efficacy of the collagen delivery system. The continuous preparation steps can obtain a uniformly dispersed and structurally stable collagen delivery system, ensuring its intestinal targeted sustained-release performance and providing reliable process support for improving the oral bioavailability of collagen.

[0022] This invention limits the oil phase heating temperature to 35℃~40℃, allowing liquid paraffin oil and Span80 to fully mix and form a stable oil phase. This avoids excessively high temperatures damaging the activity of the raw materials and excessively low temperatures causing uneven mixing, solving the problems of poor oil phase stability and affecting subsequent emulsification effects in existing preparation processes. It lays a stable foundation for aqueous emulsification and collagen delivery system formation, ensuring the stability and consistency of the collagen delivery system. Limiting the rotation speed and time during the emulsification stage ensures uniform dispersion of the aqueous phase in the oil phase, forming a stable emulsion system. This avoids aqueous phase aggregation leading to uneven particle size and unstable structure in the collagen delivery system, solving the problem of poor stability in existing collagen delivery systems. Appropriate rotation speed and time ensure sufficient emulsification while avoiding excessive stirring that damages collagen activity, ensuring the loading capacity and subsequent intestinal responsiveness of the collagen delivery system. By defining specific parameters for the cross-linking and centrifugation stages, the cross-linking reaction can be ensured to be sufficient and gentle, forming a stable collagen delivery system precipitate. This avoids excessive cross-linking, which would prevent the collagen delivery system from achieving sustained intestinal release, thus solving the problem of poor delivery efficiency in existing delivery systems. Appropriate centrifugation parameters can efficiently separate the collagen delivery system precipitate, reduce impurity residue, and improve the purity of the collagen delivery system, ensuring that it can stably exert its anti-skin photoaging effects after oral administration, further guaranteeing the stability of the preparation process and product quality. Attached Figure Description

[0023] Figure 1 The images show the morphological characteristics of the collagen delivery system in Example 1 of this invention, where: a-polarized light microscope image of the collagen delivery system; b-scanning electron microscope image of the collagen delivery system; c-particle size distribution diagram of the collagen delivery system.

[0024] Figure 2 This is a graph showing the release rate of hydroxyproline (Hyp) from simulated gastrointestinal digestion in the collagen delivery system of Example 1 of the present invention.

[0025] Figure 3 The pharmacokinetic diagrams for various collagen-related samples orally administered to KM mice are shown below: a - Schematic diagram of the experimental procedure; b - Changes in plasma Hyp content over time in the control group; c - Changes in plasma Hyp content over time in the Gly-Pro-Hyp tripeptide reference reagent group; d - Changes in plasma Hyp content over time in the Col group; e - Changes in plasma Hyp content over time in the Col / ASKP-MG group; f - Changes in plasma peptide-bound Hyp content over time in the control, Col, Col / ASKP-MG, and Gly-Pro-Hyp tripeptide reference reagent groups; g - Relative oral bioavailability in the Col and Col / ASKP-MG groups; h - Hyp content in feces in the control, Col, and Col / ASKP-MG groups.

[0026] Figure 4 The flowcharts and growth index graphs for the anti-photoaging experiment of mouse skin are shown for each experimental group, where: a- Schematic diagram of the experimental process; b- Weight change rate graph; c- Liver index graph; d- Spleen index graph; e- Kidney index graph.

[0027] Figure 5 This is a diagram of an ultraviolet irradiation device.

[0028] Figure 6 The graphs show the skin's moisture and oil content, where: a - moisture content measured by the drying method; b - moisture content measured by low-field NMR; c - triglyceride content; d - low-field NMR moisture distribution waterfall plot; e - low-field NMR moisture distribution percentage packing plot.

[0029] Figure 7 H&E stained full-dermis image of a skin tissue section.

[0030] Figure 8 H&E stained epidermal layer of skin tissue section.

[0031] Figure 9 A statistical graph of epidermal thickness stained with H&E on skin tissue sections.

[0032] Figure 10 Masson-stained dermis of a skin tissue section.

[0033] Figure 11 The graphs show oxidation-related indicators in skin tissue, including: a) T-SOD activity; b) GSH content; c) GSH-PX activity; and d) MDA content.

[0034] Figure 12 This is a graph showing the levels of inflammation-related factors in skin tissue, including: a-IL-10 content; b-IL-1β content; c-TNF-α content; and d-IL-6 content.

[0035] Figure 13 Immunohistochemical image of type I collagen in the dermis of a skin tissue section.

[0036] Figure 14 This is a graph showing the content of extracellular matrix components in skin tissue, including: a-collagen content; b-elastin content; c-hyaluronic acid content; d-Hyp content. Detailed Implementation

[0037] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0038] This invention provides a collagen delivery system, the preparation method of which is as follows: Step 1: Mix liquid paraffin oil with a mass ratio of (40~50):(1~1.2) and Span80 with a concentration of 2%, and heat to 35~40℃ to obtain the oil phase; Step 2: Mix a collagen solution with a mass concentration of 2%~5% and an Artemisia annua polysaccharide solution with a mass concentration of 1.5%~2% as the aqueous phase; The preparation of Artemisia annua polysaccharide was as follows: Crude Artemisia annua gum powder was prepared into a 0.7% (w / w) solution and magnetically stirred at room temperature for 12 hours until fully swollen. Subsequently, it was vacuum filtered, and the filtrate was concentrated to 20% of its original volume by rotary evaporation at 50℃ and 0.1 MPa. The resulting concentrate was continuously dialyzed for 72 hours to thoroughly remove small molecule impurities. The dialyzed solution was collected, concentrated again by rotary evaporation, and then freeze-dried under vacuum to obtain Artemisia annua polysaccharide.

[0039] Step 3: Add the aqueous phase dropwise to the oil phase, with a volume ratio of oil phase to aqueous phase of (10~15):(1~1.5), and stir at 1500~2000 rpm for 1~2 h to emulsify. Step 4: Add a 2%~3% FeCl3 solution dropwise to the oil phase, adjust the pH to 4.5~5.0, stir at 1000~1500 rpm, and crosslink for 10min~20min to obtain the collagen delivery system precipitate; the volume of the FeCl3 solution is the same as the volume of the aqueous phase added in Step 3; Step 5: Centrifuge at 1500 rpm to 2000 rpm for 5 to 10 minutes, collect the collagen delivery system precipitate, wash it several times with physiological saline containing 1% Tween 80 to obtain the collagen delivery system, and reconstitute it with sterile physiological saline for later use.

[0040] Furthermore, the reconstituted sterile saline solution was obtained by autoclaving (121℃, 15~20min) and the pH was adjusted to 4.5~5.0.

[0041] The collagen delivery system prepared by this invention is specifically a collagen-loaded Artemisia annua polysaccharide microgel. This microgel can be used to prepare collagen drinks that resist skin photoaging. The relative bioavailability of collagen in this collagen drink during oral administration is 143.58%, which is significantly higher than the relative bioavailability of 85.92% of existing oral collagen. Moreover, its anti-photoaging effect in mouse photoaging models is significantly better than that of existing oral collagen.

[0042] Example 1 This embodiment provides a collagen delivery system, the preparation method of which is as follows: First, liquid paraffin oil and 2% Span80 were mixed at a mass ratio of 40:1 and heated to 40°C to form the oil phase. Then, 5% collagen and 1.5% Artemisia annua polysaccharide were mixed to form the aqueous phase. Subsequently, the aqueous phase was added dropwise to the oil phase at a volume ratio of 10:1 and emulsified at 2000 rpm for 2 h. Then, an equal volume of FeCl3 solution with a mass concentration of 2% and a pH of 4.5 was added dropwise, and crosslinking was carried out at 1500 rpm for 15 min. Finally, the precipitate was collected by centrifugation at 2000 rpm for 5 min, washed three times with physiological saline containing 1% Tween80 to obtain microgels, and reconstituted with sterile physiological saline at pH 4.5 for later use, thus obtaining the collagen delivery system.

[0043] Example 2 The method is the same as in Example 1, except that the collagen protein concentration is 2%.

[0044] Example 3 The method is the same as in Example 1, except that the collagen protein concentration is 3%.

[0045] Experimental Example 1 The collagen delivery system dispersion from Example 1, after being reconstituted, was appropriately diluted with deionized water. A suitable amount was then dropped onto a clean glass slide, and a coverslip was gently placed over it to prevent air bubbles. Subsequently, the macroscopic morphology, dispersion state, and approximate size range of the collagen delivery system were observed and images were captured under an optical microscope. To observe the fine surface structure of the collagen delivery system, the sample was freeze-dried to maintain its original morphology. The dried sample was placed on a sample stage using conductive adhesive, and a metal coating with a thickness of approximately 10 nm was sputtered onto the surface of the collagen delivery system using an ion sputtering instrument to enhance its conductivity. Under an accelerating voltage of 15 kV, the surface morphology images of the collagen delivery system were observed and captured using a scanning electron microscope. Based on the multiple images captured, the particle size of the collagen delivery system was statistically analyzed using the professional image analysis software ImageJ. The diameter of no less than 100 collagen delivery system particles was measured, and the average particle size and particle size distribution of each region were calculated using the software's data processing function after Gaussian distribution fitting. Finally, the statistical results were presented in the form of a particle size distribution histogram.

[0046] Depend on Figure 1 Observation revealed that the collagen delivery system exhibited plump, yellow spherical morphology and was uniformly dispersed. Further scanning electron microscopy revealed that... Figure 1 b. Observation of individual collagen delivery systems revealed that their surface roughness increased the specific surface area, which is beneficial for improving adhesion and enhancing the potential for active ingredient utilization. Statistical analysis of the particle size of the collagen delivery system was conducted... Figure 1 c shows that the measured average particle size is 29.26 ± 9.25 μm.

[0047] Experimental Example 2 Simulated gastric fluid stage: 2 g / L NaCl and 7 mL / L HCl were dissolved in ultrapure water, the pH was adjusted to 2.0, and 3.2 g of pepsin was added. The mixture was thoroughly mixed to prepare simulated gastric fluid (SGF). Then, 1 g of the collagen delivery system from Example 1 was placed in 40 mL of SGF and incubated at 37°C and 100 rpm for 2 h. Simulated intestinal fluid stage: After the simulated gastric fluid stage, the simulated gastric fluid was replaced with simulated intestinal fluid (SIF), and then simulated intestinal digestion experiments were performed. The SIF solution was prepared as follows: KCl (0.514 g / L), KH₂PO₄ (0.1224 g / L), NaHCO₃ (7.14 g / L), MgCl₂·6H₂O (0.067 g / L), CaCl₂(H₂O)₂ (0.088 g / L), NaCl (0.02 g / L), trypsin (0.1 g / L), and bile salts (0.025 g / L). All reagents were dissolved in ultrapure water, and the solution was adjusted to pH 7.0 with 0.1 M NaOH. The solution was incubated at 37°C and 100 rpm for 4 h. Samples were taken at 0, 1, 2, 3, 4, 5, and 6 h, and the Hyp content was determined using the chloramine-T method (unless otherwise specified, all Hyp content determinations were performed using the chloramine-T method).

[0048] Because hyp is a characteristic component of collagen and is present in most collagen's active peptides, it is linked to the active ingredients of collagen; therefore, its release rate is measured. Figure 2 It can be seen that Hyp release is very slow in the gastric juice stage, with a cumulative release rate of only 13.83% within 2 hours. After the digestive environment switches to intestinal juice, the release rate of Hyp accelerates. The release rate gradually increases over the following 4 hours, eventually reaching 71.56%. This indicates that the collagen delivery system carrier formed by Artemisia annua polysaccharide plays an effective protective role. Under the acidic environment of the stomach and the action of pepsin, it slows down the dissolution and decomposition of internal collagen, thereby protecting the active structure of collagen from rapid destruction by the gastric environment. In the intestine, the collagen delivery system gradually swells over time, thus efficiently and centrally releasing the active collagen components, demonstrating the intestinal responsiveness of the collagen delivery system.

[0049] Experimental Example 3 KM mice aged 6-8 weeks (20±2g) were used in the experiment. They were acclimatized under standard laboratory conditions and divided into four groups: Control group (0.2 mL sterile saline via gavage); Collagen solution group (0.2 mL collagen solution prepared with sterile saline via gavage); Collagen delivery system group (Col / ASKP-MG) (0.2 mL Artemisia annua polysaccharide microgel suspension loaded with collagen prepared in Example 1 via gavage); and Tripeptide reference group (Gly-Pro-Hyp) (0.2 mL Gly-Pro-Hyp tripeptide solution via gavage). Except for the control group, all drug-treated samples were uniformly prepared to ensure a Hyp concentration of 12.0 mg / mL. The experimental procedure is as follows: Figure 3 a. After acclimatization, mice in each group were fasted for 12 hours but allowed free access to water, followed by a single gavage administration of the drug. Blood samples were collected via tail vein at 0 h and at 0.5, 1, 2, 4, 8, 12, and 24 h after administration. The samples were placed in centrifuge tubes containing heparin sodium, vortexed, and incubated at 4°C for 30 min. Then, the samples were centrifuged at 4°C and 3000 rpm for 10 min to separate the plasma, which was then stored at -80°C for analysis. The total and free Hyp content in plasma were determined. The plasma peptide-bound Hyp content was calculated using the following formula: Plasma peptide-bound Hyp = Total plasma Hyp - Free plasma Hyp Pharmacokinetic curves were plotted with blood collection time on the x-axis and plasma peptide-bound Hyp content on the y-axis. Using validated data processing software and a non-compartmental mathematical model analysis method, the area under the curve (AUC) of the drug concentration-time curve for each group from 0 h to 24 h was calculated. The incremental area under the curve (iAUC) for the test formulation group and the oral reference formulation group was calculated according to the following formula: iAUC 受试制剂 =AUC 受试制剂 -AUC 空白对照 iAUC 口服参比 =AUC 口服参比 -AUC 空白对照 In the formula: iAUC 受试制剂 —iAUC of the test formulation group; iAUC 口服参比 —iAUC of the oral reference formulation group; AUC 空白对照 —AUC of the blank control group; AUC 受试制剂 —AUC of the test formulation group; AUC 口服参比 —AUC of the oral reference formulation group.

[0050] The relative bioavailability was calculated using Gly-Pro-Hyp tripeptide as a reference, according to the following formula: Fr= iAUC 受试制剂 / iAUC 口服参比 ×100% In the formula: Fr—Relative bioavailability; iAUC 受试制剂 —iAUC of the test formulation group; iAUC 口服参比 —iAUC of the oral reference formulation group.

[0051] Twenty-four hours after administration, all fecal samples from mice in each group were collected, their weights were recorded, and the samples were freeze-dried, ground into a uniform powder, and the Hyp content was determined.

[0052] pass Figure 3 b~ Figure 3 The plasma Hyp concentration-time curve shows that... Figure 3 b is the blank control group: the plasma Hyp concentration remained at a very low level throughout the experiment without significant fluctuations, serving as a baseline control and eliminating the interference of endogenous Hyp on the experimental results; after oral administration of the Gly-Pro-Hyp tripeptide reference reagent, both free Hyp and peptide-bound Hyp in the plasma rapidly increased and were quickly metabolized, exhibiting typical characteristics of rapid absorption and elimination. Figure 3 c). In contrast, while direct oral administration of collagen solution resulted in a sustained increase in total plasma Hyp levels, both its peak concentration and rate of increase were significantly lower than those of the tripeptide group. Figure 3 d). Crucially, the collagen delivery system (Col / ASKP-MG) exhibited superior pharmacokinetic characteristics: its total plasma Hyp concentration increased steadily and continuously after administration, and remained at a relatively stable plateau for a longer period, indicating that the Artemisia annua polysaccharide microgel carrier achieved responsive release and slow absorption of collagen in the intestine. Figure 3 e). Further analysis of peptide-bound Hyp in plasma. Figure 3 The results showed that the peptide-bound Hyp concentration in the Col / ASKP-MG group was higher than that in the Col group at multiple time points, suggesting that the collagen delivery system delivered potentially bioactive collagen peptides more effectively. Oral relative bioavailability calculations were performed. Figure 3 g directly confirmed that the relative bioavailability of the Col / ASKP-MG group (143.58%) was significantly higher than that of the Col group (85.92%). Furthermore, the detection of Hyp content in feces... Figure 3The study found that the fecal Hyp excretion in both the Col group and the Col / ASKP-MG group was significantly higher than that in the control group, and there was a difference between the two groups. This indirectly reflects the difference in the overall retention and absorption efficiency of the two formulations in the digestive tract. In summary, the collagen delivery system of this invention effectively improves the in vivo absorption kinetics of collagen and enhances its oral bioavailability through its intestinal targeted sustained-release characteristics.

[0053] Test Example 4 (I) Test Methods 1. Grouping KM mice aged 6-8 weeks, weighing 20±2g, were used in the experiment. They were acclimatized and tested under standard laboratory conditions. Specific grouping and experimental procedures are shown in Table 1 and [Table data missing]. Figure 4 a. Among them, Control was the blank control group, which was administered an equal volume of physiological saline by gavage without irradiation; UV-Mod was the model control group, which was administered an equal volume of physiological saline by gavage and irradiated; ASKP-MG group was a microgel sample without collagen, Col group was a collagen sample prepared with sterile physiological saline, Col / ASKP-MG-L group was a microgel sample of Example 2, and Col / ASKP-MG-H group was a microgel sample of Example 1.

[0054] Table 1 Animal Experiment Grouping

[0055] 2. Construction and processing of photoaging model Ultraviolet irradiation equipment ( Figure 5 The entire structure is framed by 8.5mm diameter fiberglass tubing connected by T-joints. The overall dimensions are approximately 44.5×31.5×31.5cm. The light source consists of two UVA lamps (313nm 15W) and one UVB lamp (313nm 15W), alternately fixed to the frame in a UVA-UVB-UVA configuration. The lamps simulate an ultraviolet irradiation environment. During use, a light-shielding curtain is used to protect the safety of the experimenters. Before each experiment, the UV lamps are turned on and stabilized for 10 minutes. Then, an ultraviolet irradiance meter is placed at the same height and under the same conditions as the sample to measure the ultraviolet radiation intensity.

[0056] Mice were placed in a restraint bag and secured with elastic bands to ensure that the skin on the back of each mouse was fully and evenly exposed to ultraviolet light. The lamp was 30 cm away from the mouse's back. The dose required to observe the appearance of visible erythema on the mouse's back skin 24 hours after irradiation was defined as the minimum erythema dose (MED) (1200 mJ / cm²). 2Irradiation was performed three times a week, with at least one day between each irradiation session. The dose was 1 MED per session in the first week, 2 MED per session in the second week, 3 MED per session in the third week, and 4 MED per session in the fourth week. The dose was maintained at 4 MED per session until the end of the experiment, for a total of 5 weeks. Except for the control group, all participants were required to undergo UV modeling. If severe erosion, bleeding, blisters, or other adverse reactions occurred, irradiation was immediately stopped, and the experiment was resumed only after recovery.

[0057] After the experiment, mice were fasted for 12 hours but allowed free access to water. They were then anesthetized with isoflurane and euthanized by cervical dislocation. Blood was collected, and after clotting at room temperature for 40 minutes, the blood was centrifuged at 3000 rpm and 4°C for 10 minutes. The serum was collected and stored at -80°C for later use. Skin from the back of the mice was soaked in pre-cooled physiological saline at 4°C to remove blood. The surface liquid was blotted dry with filter paper, and the skin tissue was divided into three parts. One part was flash-frozen in liquid nitrogen and then stored at -80°C, one part was fixed in 4% paraformaldehyde, and the last part was used to determine the skin water content.

[0058] 3. Growth Index Measurement Observe the growth and health status of mice daily, including their mental state, appetite, and gait. Weigh and record their weight weekly, and adjust the gavage volume according to their weight. The gavage volume is 10 mL / kg. Prepare the gavage sample fresh with physiological saline as the solvent.

[0059] After euthanasia, the liver, spleen, and kidneys were dissected and rinsed thoroughly with pre-cooled physiological saline. They were then dried with filter paper and weighed. The organ index of the mouse was calculated using the following formula:

[0060] 4. Skin macroscopic condition score Before the sample was taken, a photo of the back was taken. After the experiment, the participants who did not participate in the experiment were asked to rate the sample according to the scoring criteria. The control group was scored out of 10, and the worse the skin appearance, the lower the score.

[0061] Table 2 Scoring criteria for photodamage to the skin on the back of mice

[0062] 5. Measurement of skin oil content and water content The water binding state is determined using a low-field nuclear magnetic resonance (LF-NMR) with a multi-exponential fitting analysis (T-invfit) inversion program. After transferring the skin sample to a sample vial, place it in a transparent cylindrical glass tube (30 mm in diameter), and use the CPMG sequence to measure the transverse relaxation time (T2), with sampling frequency SW=100 kHz, radio frequency delay 0.08 ms, accumulation number 8 times, pre-amplifier gear 1, echo time 0.3 ms, number of echoes 3,000, scanning interval 10,000 ms, and pulse width 14.00 μm. The T2 distribution can be obtained through multi-exponential fitting, where each relaxation peak represents a certain distribution of hydrogen protons, from which T2i can be obtained; i respectively represents the signal amplitude and transverse relaxation time of the i-th hydrogen proton state. According to the value of T2, they are recorded as T2b, T21, T22 respectively (T2b<T21<T22). A shorter relaxation time indicates that water is more tightly bound to the substrate; a longer relaxation time indicates a greater degree of freedom of water.

[0063] Take the skin tissue and place it into an uncovered clean sample vial, and accurately weigh its wet weight. Place the sample vial into an oven, dry it at 80°C to constant weight, and calculate the percentage of water content in mouse skin according to the following formula: Percentage of skin water content (%) = (wet weight - dry weight) / wet weight × 100.

[0064] 6. Staining of skin tissue sections Take the back skin of mice and immerse it in 4% paraformaldehyde for fixation for 24 h, rinse with running water for 12 h, then dehydrate it in 75% ethanol, after wax impregnation, prepare a wax block by paraffin embedding, cut it into 8 μm thin tissue sections with a microtome, gently attach it to a glass slide, and bake the slide in a constant temperature oven (62°C) for 60 min, fully immerse in xylene for 10 min for dewaxing. After two dewaxing steps, place the slide in 100% ethanol for 5 min to wash off xylene, then sequentially place it in gradient ethanol (soak in 95%, 85% and 70% ethanol for 5 min each), soak and wash 3 times with PBS, then add 100 μL of hematoxylin staining solution dropwise (each 3 L of hematoxylin staining solution contains 6 g of hematoxylin, 100 mL of anhydrous ethanol, 150 g of aluminum potassium sulfate, 1.2 g of sodium iodate, 120 mL of glacial acetic acid, 900 mL of glycerol) for staining for 10 min, then wash off the hematoxylin staining solution with distilled water, differentiate with 1% hydrochloric acid ethanol, rinse the slide with double distilled water, then stain with 0.5% eosin staining solution for 3 min, then dehydrate with gradient ethanol respectively (dehydrate with 80% ethanol for 5 s, 95% ethanol for 2 min and 100% ethanol for 2 min). Then immerse the sections in xylene for transparency twice, 4 min each time, then use neutral resin and a coverslip to seal the tissue sample section. Finally, a microscope is used for image acquisition and analysis.

[0065] Take the skin tissue sections dewaxed to water from the previous step, stain with Weigert iron hematoxylin staining solution from the Masson staining kit for 5 min, wash off the dye with distilled water, differentiate with 1% hydrochloric acid ethanol for 10 seconds, rinse with double distilled water, and then rinse with running water for 3 min to regain blue color. Then stain with Ponceau S acid fuchsin solution (containing 0.7 g Ponceau S, 0.3 g acid fuchsin, and 1 mL glacial acetic acid per 100 mL) for 5-10 min, and rinse off the dye with distilled water. Treat the sections with 1% phosphomolybdic acid solution for 5 min, counterstain with 2% aniline blue solution for 5 min, treat with 1% glacial acetic acid for 1 min for differentiation, then dehydrate with graded ethanol, clear with xylene, and mount with neutral resin and coverslips. Finally, use a microscope for image acquisition and analysis.

[0066] 7. Measurement of antioxidant markers and oxidative stress damage in the skin Take about 0.1 g of back skin and add it to 0.9 mL of pre-cooled physiological saline. Grind it thoroughly under ice bath to make a 10% tissue homogenate. Centrifuge at 3000 rpm / min and 4℃ for 15 min. Take the supernatant and use a kit to determine the antioxidant-related indicators (SOD, GSH, GSH-Px and MDA).

[0067] 8. Measurement of skin endogenous components and inflammatory factor markers Approximately 0.2g of skin tissue was weighed, and the Hyp content was determined using the chloramine T method.

[0068] Take the skin tissue sections dewaxed to water from the previous experiment and perform heat antigen retrieval (96-98℃) for 15 min using antigen retrieval solution (citrate buffer, pH=6.1). After cooling to room temperature, rinse twice each with PBS and distilled water. Circle the tissue section with an immunohistochemical pen, place it in a humidified chamber, add 3% hydrogen peroxide, and incubate for 10 min to block endogenous peroxidase and reduce non-specific background staining. Rinse three times each with PBS and distilled water for 5 min each time, blot dry, and incubate with 5% goat serum blocking solution at 37℃ for 30 min to block non-specific sites and remove serum. Then, directly add an appropriate amount of primary antibody working solution and incubate overnight at 4℃. The next day, after returning to room temperature, the sample was rinsed three times with PBS, then washed with distilled water for 3 minutes. After drying, it was incubated with 50 μL of secondary antibody working solution at room temperature for 10 minutes, rinsed three times with PBS, then washed with distilled water for 3 minutes, and then incubated with 50 μL of streptomycin peroxidase solution at room temperature for 10 minutes. The sample was rinsed three times with PBS, then washed with distilled water for 3 minutes, and then stained with 100 μL of diaminobenzidine (DAB) solution for 3 minutes. After rinsing with tap water, it was counterstained with hematoxylin and differentiated with 1% hydrochloric acid ethanol. Then, it was dehydrated using a gradient of ethanol, cleared with xylene, and sealed with neutral resin and coverslips. Finally, images were acquired and analyzed using a microscope.

[0069] Weigh an appropriate amount of skin tissue into a 2 mL EP tube and shear the tissue into small fragments on crushed ice. Add lysis buffer at a ratio of 1 mL RIPA tissue lysis buffer per 100 mg of tissue, along with a cocktail and PMSF protease inhibitor, each at 1 / 100 of the tissue lysis buffer volume, and vortex to mix. Then, add three 2 mm tissue grinding beads to each EP tube and grind the tissue using a high-speed refrigerated homogenizer at a frequency of 90 Hz, a time of 120 seconds, and a temperature of 4°C. After grinding, place the tissue in a 4°C freezer for further digestion for 30 min. After digestion, centrifuge the tissue at 13000 rpm / min at 4°C for 40 min. Immediately after centrifugation, the tissue supernatant was transferred to another 1.5 mL EP tube, and a portion of the supernatant was separated for use in an ELISA kit to determine the levels of HA (hyaluronic acid), ELN (elastin), TNF-α (tumor necrosis factor-α), IL-1β (interleukin-1β), IL-6 (interleukin-6), and IL-10 (interleukin-10).

[0070] (II) Experimental Results 1. Effects of different experimental groups on mouse growth index Results of weight change rate monitoring, such as Figure 4 b indicates that body weight increased significantly over time in all experimental groups. The change rate was approximately 157% in the Control group and approximately 142% in the UV-Mod group at 5 weeks. The change rates for ASKP-MG, Col, Col / ASKP-MG-L, and Col / ASKP-MG-H in other groups ranged from 142% to 148%, suggesting slight differences in body weight gain among the different intervention groups. To assess the effects of ultraviolet radiation and oral administration on various organ indices in mice, liver, spleen, and kidney indices were measured experimentally. The results are as follows: Figure 4 c~ Figure 4 The results showed that the organ indices of all treatment groups varied within a narrow range compared to the control group, and different interventions did not significantly change the organ indices of mice.

[0071] 2. Macroeconomic Evaluation To assess the effects of ultraviolet radiation and oral administration on the macroscopic condition of the dorsal skin in mice, clinical severity scores (0-10 points) and skin appearance were assessed through photography and macroscopic evaluation by non-experimental personnel. Results showed that the Control group had the highest score (approximately 10 points), with relatively light and uniform skin appearance; the UV-Mod group scored approximately 4 points, with obvious erythema; the ASKP-MG group scored approximately 4 points; other groups, such as the Col group (approximately 6 points), Col / ASKP-MG-L group (approximately 6 points), and Col / ASKP-MG-H group (approximately 8 points), exhibited varying skin conditions, including mild erythema or slight pigmentation, suggesting that different interventions had significantly different effects on the degree of skin damage.

[0072] 3. Skin moisture and oil content To assess the effects of ultraviolet radiation and oral administration interventions on skin moisture content in mice, relative skin moisture content was measured using the drying method and low-field NMR spectroscopy. Figure 6 a, Figure 6 The results showed that the Control group had the highest moisture content, while the UV-Mod and ASKP-MG groups had the lowest moisture content. The moisture content of other treatment groups was between that of the Control and UV-Mod groups. Among them, the low-field NMR measured the moisture content of the Col / ASKP-MG-H group, which was significantly higher than that of the other intervention groups.

[0073] To assess the effects of ultraviolet radiation and oral administration on skin water status in mice, the T2 relaxation time distribution (reflecting the degrees of freedom of water molecules) and water state ratio (A) were measured using low-field nuclear magnetic resonance spectroscopy. 2b Combined water, A 21 Binding water, A 22 (Free water). Figure 6 d、 Figure 6 The results showed that, compared with the Control group, the main peak of the T2 relaxation spectrum in the UV-Mod group was significantly shifted to the right (shifted to a longer relaxation time), while A 22 The proportion increased significantly, A 2b The proportion was significantly reduced, indicating that UV radiation led to increased skin water freedom, decreased bound water, and increased free water; compared with the UV-Mod group, the main peak in the Col / ASKP-MG-H group shifted significantly to the left (towards a shorter relaxation time), A 2b The increase in the proportion indicates that the intervention can effectively restore the skin's moisture status and enhance its water-holding capacity.

[0074] Triglycerides are a major component of skin sebum and an important part of the skin barrier, especially the lipid barrier of the stratum corneum. Changes in their content can reflect the integrity of skin barrier function, sebaceous gland activity, and certain pathological conditions, such as xerosis and atopic dermatitis. To assess the effects of ultraviolet radiation and oral administration on triglyceride concentrations in mouse skin, skin triglyceride concentrations (mg / ml) were measured experimentally. The results are as follows: Figure 6 c indicates that the highest concentration was approximately 8 mg / ml in the Control group, while the UV-Mod group showed a significant decrease of approximately 4 mg / ml. Concentrations in other treatment groups were intermediate with no significant differences. The Col / ASKP-MG-H group showed the best improvement compared to the UV-Mod group. This suggests that ultraviolet radiation significantly reduces skin triglyceride levels, and oral interventions can effectively repair the lipid barrier.

[0075] 4. Effects on mouse skin structure Hematoxylin and eosin (HE) staining is a classic histological staining technique widely used in the observation of pathological changes in skin tissues. It clearly reveals the dynamic changes in epidermal and dermal thickness, cell morphology, and distribution characteristics, providing a reliable basis for assessing skin structural integrity. Skin structural integrity is highly dependent on collagen fibers, which play a crucial role not only in maintaining skin elasticity and firmness but also in the integrity of the skin barrier function. According to... Figure 7 H&E staining results showed that the stratum corneum, epidermis, and dermis of the skin in the control group mice were clearly defined. In the model group mice, the epidermis was significantly thickened, with localized hyperkeratosis. The dermis showed fibrous tissue proliferation accompanied by inflammatory cell infiltration, and the boundary between the dermis and epidermis was blurred. In the other oral intervention groups, the epidermal thickening and localized hyperkeratosis were alleviated. Simultaneously, fibrous tissue proliferation was inhibited, inflammatory cell infiltration decreased, and the boundary between the dermis and epidermis became clearer. Further statistical analysis of epidermal thickness using software was performed. Figure 8 , Figure 9 It can be seen that compared with the control group, the epidermal thickness of UV-Mod and ASKP-MG was significantly increased, and the remaining intervention groups were all improved compared with UV-Mod, among which Col / ASKP-MG-H showed the best improvement. Masson staining is a highly sensitive and specific staining method for collagen fibers, which can quantitatively assess the arrangement of collagen fibers in the dermis and their pathological changes. Masson staining, such as... Figure 10 The results showed that the collagen fibers in the control group were dense and orderly arranged, while the collagen fibers in UV-Mod and ASKP-MG were relatively loose and disordered. The looseness of collagen fibers in other intervention groups was improved, with the collagen fibers in the Col, Col / ASKP-MG-L and Col / ASKP-MG-H groups being relatively orderly arranged.

[0076] 5. Effects on oxidation-related indicators in mouse skin tissue To evaluate the protective effects of each intervention group against UV-induced oxidative stress in mouse skin, the activities of SOD and GSH-Px, as well as the contents of GSH and MDA in mouse skin tissue, were measured experimentally. Results are as follows: Figure 11 a~ Figure 11 The results showed that, compared with the UV-Mod group, each intervention group significantly increased T-SOD and GSH-PX activities, increased GSH content, and decreased MDA content, with statistically significant differences, suggesting that these interventions have a protective effect against UV-induced oxidative stress, with Col / ASKP-MG-H showing the most prominent effect.

[0077] 6. Effects on inflammatory markers in mouse skin tissue To determine whether ultraviolet radiation and oral interventions affect photoaging and anti-photoaging effects by regulating immune balance, and to quantitatively assess the degree of skin inflammation, we conducted an experiment by measuring the levels of TNF-α, IL-1β, IL-6, and IL-10 in mouse skin tissue. The results are as follows: Figure 12 The results showed that, compared with the UV-Mod group, all intervention groups, including ASKP-MG, significantly reduced the levels of pro-inflammatory factors and modulated and increased the content of the anti-inflammatory factor IL-10, suggesting that ASKP may contain anti-inflammatory active ingredients and have an synergistic or combined effect with collagen. Col / ASKP-MG-H showed the best anti-inflammatory effect. This suggests that these oral interventions may exert their anti-photoaging effect by reducing the inflammatory response through regulating immune balance.

[0078] 7. Effects on extracellular matrix components in mouse skin tissue To evaluate the effects of different treatments on type I collagen expression in a UV-induced skin photoaging model, the distribution and content of collagen were detected by immunohistochemistry, and quantitative analysis was performed using MOD values. Results are as follows: Figure 13 and Figure 14 The results showed that the control group exhibited the richest collagen expression, while the UV model group showed a significant reduction in expression, confirming the successful establishment of the model. In the intervention group, Col and Col / ASKP-MG-H showed higher expression levels than in the model group, and the statistical difference indicated that the intervention was effective, suggesting that these treatments have a protective effect against UV-induced collagen degradation.

[0079] Similarly, the contents of other major components in the extracellular matrix, namely elastin, hyaluronic acid, and hydroxyproline, were measured. Figure 14 b~ Figure 14The results showed that, compared with the normal group, the levels of ELN, HA, and Hyp in the skin tissue of the model group mice were significantly reduced (P < 0.01), which is consistent with the changes in the content of extracellular matrix in the skin cells of mice with photoaging. Compared with the model group, the levels of ELN, HA, and Hyp in the skin tissue of the intervention groups, except for the ASKP-MG group, were increased. The changes were more significant in the Col / ASKP-MG-H group. The Hyp content corresponded with the immunohistochemical results, which further validated the findings.

[0080] (III) Conclusion Experimental results showed that collagen-loaded Artemisia annua polysaccharide microgels (especially the high-dose group Col / ASKP-MG-H) effectively alleviated UV-induced photoaging in mouse skin. Specifically, this manifested as improved macroscopic skin condition, restoration of moisture content and lipid barrier function, reduced oxidative stress and inflammatory response (decreased pro-inflammatory factors), and promotion of the synthesis and deposition of extracellular matrix components in the dermis, such as type I collagen, elastin, and hyaluronic acid. Compared with direct oral collagen administration, the microgels, through an intestinal-targeted release mechanism, significantly improved skin structural integrity, such as reducing epidermal thickening and collagen fiber disorder, demonstrating their potential in oral anti-photoaging products.

[0081] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A collagen delivery system, characterized in that, The raw materials include an oil phase, an aqueous phase, and a crosslinking agent, wherein: The oil phase comprises liquid paraffin oil and Span 80; The aqueous phase includes a collagen solution and an Artemisia annua polysaccharide solution; The crosslinking agent includes a FeCl3 solution.

2. The collagen delivery system according to claim 1, characterized in that, The concentration of Span80 is 2%, and the mass ratio of liquid paraffin oil to Span80 is (40~50):(1~1.2); the mass concentration of the collagen solution is 2%~5%, and the mass concentration of the Artemisia annua polysaccharide solution is 1.5%~2%; the volume ratio of the oil phase to the water phase is (10~15):(1~1.5); the mass concentration of the FeCl3 solution is 2%~3%, and the volume ratio of the FeCl3 solution to the water phase is 1:

1.

3. A product for combating skin photoaging, characterized in that, Specifically, it refers to topical compositions and / or oral collagen drinks prepared using a collagen delivery system as described in claim 1 or 2.

4. The anti-photoaging product according to claim 3, characterized in that, When preparing oral collagen drinks, the reconstituted collagen delivery system is dispersed in a solvent, which is deionized water or sterile saline.

5. The anti-photoaging product according to claim 3, characterized in that, The oral collagen drink has a relative oral bioavailability of more than 143.58%.

6. The anti-photoaging product according to claim 3, characterized in that, The oral collagen drink can alleviate UV-induced photoaging of the skin, specifically by: improving the macroscopic condition of the skin, restoring skin moisture content and oil barrier, reducing skin oxidative stress and inflammatory response, and promoting the synthesis and deposition of extracellular matrix components in the dermal layer of the skin.

7. A method for preparing a collagen delivery system according to claim 1 or 2, characterized in that, The specific steps are as follows: Liquid paraffin oil and Span80 are mixed and heated to a set temperature to obtain the oil phase; The collagen solution and the Artemisia annua polysaccharide solution were mixed to obtain the aqueous phase; The aqueous phase is added dropwise to the oil phase, stirred at a set speed, and emulsified for a set time to obtain the emulsified system. FeCl3 solution was added dropwise to the emulsion system, the system was adjusted to the set pH value, stirred at the set speed, crosslinked for the set time, centrifuged at the set speed for the set time, washed, and reconstituted to obtain the collagen delivery system.

8. The method for preparing a collagen delivery system according to claim 7, characterized in that, Liquid paraffin oil and Span80 are mixed and heated to 35℃~40℃ to obtain the oil phase.

9. The method for preparing a collagen delivery system according to claim 7, characterized in that, The aqueous phase was added dropwise to the oil phase, and emulsified for 1 to 2 hours at a stirring speed of 1500 rpm to 2000 rpm to obtain the emulsified system.

10. The method for preparing a collagen delivery system according to claim 7, characterized in that, FeCl3 solution was added dropwise to the emulsion system, the pH of the system was adjusted to 4.5~5.0, and the mixture was stirred at 1000rpm~1500rpm for 10min~20min for crosslinking. The mixture was then centrifuged at 1500rpm~2000rpm for 5min~10min, washed, and reconstituted to obtain the collagen delivery system.

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