Composition for delaying cell senescence and preparation method and application thereof

Abstract

The invention provides a composition for delaying cell senescence as well as a preparation method and application thereof, and belongs to the technical field of medicines. The preparation method comprises the following steps: loading tea polyphenol on a dog bone marrow mesenchymal stem cell exosome, and modifying the surface of the dog bone marrow mesenchymal stem cell exosome with arginine nonamer to prepare a modified tea polyphenol-loaded exosome; the preparation method comprises the following steps of: modifying cholesterol by using arginine nonamer, and preparing lipidosome by using the cholesterol modified by the arginine nonamer, DSPE-PEG2000, phospholipid embedded resveratrol and epigallotein gallate; polysaccharide is extracted from rhizoma polygonati, fructus lycii and ginseng, and rhizoma polygonati / fructus lycii / ginseng polysaccharide is prepared; the preparation method comprises the following steps: mixing an acellular dermal matrix with polygonatum sibiricum / medlar / ginseng polysaccharide, adding lipidosome and a modified tea polyphenol-loaded exosome, co-incubating, and freeze-drying to prepare the composition for delaying cell aging, the composition has a better drug embedding effect, can improve the stability and transdermal absorption effect of the drug, has better antioxidant, antibacterial and anti-inflammatory effects, and can be used for preparing the medicine for delaying cell aging. The effect of delaying skin cell senescence can be better achieved through multiple ways, and safety and degradability are achieved.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a composition for delaying cell aging, its preparation method, and its application. Background Technology

[0002] Population aging and the high incidence of age-related diseases are major challenges facing the world, and aging is the greatest risk factor for chronic diseases in humans. Cellular senescence and stem cell depletion are important triggers for aging and the development of various age-related diseases. Programmed changes in epigenetics are considered key factors determining the aging process of cells and stem cells. Cellular senescence is one of the main characteristics and driving forces of tissue functional decline and even overall bodily degeneration, and it is also a key factor in inducing chronic diseases. Promoting cell renewal, delaying cellular senescence, and thus promoting tissue rejuvenation is an important strategy for slowing down or even reversing aging.

[0003] Mesenchymal stem cells (MSCs) have shown great promise in regenerative medicine, tissue engineering, and the treatment of immune-related diseases due to their multi-lineage differentiation potential, tissue repair capabilities, and immunomodulatory properties. However, during in vitro expansion, MSCs inevitably undergo replicative senescence, leading to a significant decline in their proliferative capacity, differentiation potential, and immunomodulatory function. This senescence is primarily driven by multiple mechanisms, including activation of cell cycle repressors, telomere length depletion, mitochondrial dysfunction, accumulation of oxidative stress, and the release of senescence-associated secretory phenotypes (SASPs). SASPs contain various pro-inflammatory factors such as IL-6, IL-8, and MMPs, which further induce surrounding cells into a senescent state through paracrine effects. Studies have shown that the SA-β-gal positivity rate in MSCs cultured to passage 5 (P5) can reach over 40%, indicating not only significant senescence accumulation but also a substantial decrease in homing ability, tissue repair efficacy, and immunomodulatory potency. Furthermore, the endocrinological changes in senescent MSCs exacerbate the inflammatory microenvironment, weakening their therapeutic safety and stability in vivo.

[0004] Currently, intervention strategies for MSC aging mainly include the following categories: ① Antioxidants (NACs), which reduce oxidative stress by scavenging reactive oxygen species (ROS), but cannot repair existing DNA damage or restore telomere length; ② Gene editing methods (such as overexpression of telomerase reverse transcriptase TERT), which can prolong cell lifespan, but have safety risks such as insertion mutations and gene instability; ③ Small molecule inhibitors (such as rapamycin), which delay aging by regulating aging-related pathways such as mTOR, but may interfere with normal metabolic regulation and bring potential side effects.

[0005] However, there are currently no good stem cell preparations that can effectively delay cell aging. Summary of the Invention

[0006] The purpose of this invention is to propose a composition for delaying cell aging, its preparation method, and its application. It has a good drug encapsulation effect, which can improve the stability and transdermal absorption of the drug. At the same time, it has good antioxidant, antibacterial, and anti-inflammatory effects. It can better delay the aging of skin cells through multiple pathways. It is also safe and biodegradable, and has good application prospects.

[0007] The technical solution of this invention is implemented as follows: This invention provides a method for preparing a composition that delays cell aging. The method involves loading tea polyphenols onto canine bone marrow mesenchymal stem cell exosomes, then modifying the surface with arginine nonamers to obtain modified tea polyphenol-loaded exosomes. Arginine nonamers are then modified with cholesterol and combined with DSPE-PEG2000, phospholipid-encapsulated resveratrol, and epigallocatechin gallate to prepare liposomes. Polysaccharides are extracted from Polygonatum, Lycium barbarum, and ginseng to prepare Polygonatum / Lycium barbarum / ginseng polysaccharide. Decellularized dermal matrix is ​​mixed with Polygonatum / Lycium barbarum / ginseng polysaccharide, and liposomes and modified tea polyphenol-loaded exosomes are added. After co-incubation, the mixture is freeze-dried to obtain the composition that delays cell aging.

[0008] As a further improvement to the present invention, the following steps are included: S1. Preparation of tea polyphenol-loaded exosomes: Canine bone marrow mesenchymal stem cell exosomes and tea polyphenols were added to PBS solution, co-incubated on ice by sonication, purified, concentrated by ultrafiltration tube, and freeze-dried to obtain tea polyphenol-loaded exosomes; S2. Preparation of modified tea polyphenol-loaded exosomes: Tea polyphenol-loaded exosomes were added to PBS solution, NHS and EDC were added, the mixture was stirred and activated, arginine nonamer was added, the mixture was stirred and reacted, dialyzed, and freeze-dried to obtain modified tea polyphenol-loaded exosomes; S3. Modified cholesterol: Arginine nonamer was dissolved in water, and a dimethylformamide solution of ditert-butyl dicarbonate was added. The pH of the solution was adjusted, and the reaction was stirred. Tween was added, and the mixture was stirred until homogeneous. A dimethylformamide solution containing cholesterol, dicyclohexylcarbodiimide, and 4-dimethylaminopyridine was added. The reaction was heated and stirred under inert gas protection. Stirring was stopped, trifluoroacetic acid was added to eliminate BOC protection, and the mixture was dialyzed and freeze-dried to obtain modified cholesterol. S4. Preparation of liposomes: Resveratrol and epigallocatechin gallate were dissolved in tetrahydrofuran, modified cholesterol, DSPE-PEG2000 and phospholipids were added, the mixture was stirred and mixed evenly, and the film was formed by rotary evaporation under reduced pressure. Ultrapure water was added for hydration, followed by sonication, filtration, washing and drying to obtain liposomes. S5. Polysaccharide extraction: Mix and pulverize Polygonatum, Lycium barbarum and ginseng, add water, heat to boiling and extract, filter, repeat extraction 1-2 times, combine filtrates, concentrate, add ethanol to precipitate, filter, wash solid and dry to obtain Polygonatum / Lycium barbarum / ginseng polysaccharide; S6. Preparation of a composition for delaying cell aging: Pepsin and decellularized dermal matrix fragments were added to hydrochloric acid solution, stirred and digested, the pH of the solution was adjusted, diluted, enzymes were inactivated, and Polygonatum sibiricum / Lycium barbarum / ginseng polysaccharide, modified loaded tea polyphenol exosomes and liposomes were added and stirred evenly, heated and incubated, and freeze-dried to obtain a composition for delaying cell aging.

[0009] As a further improvement of the present invention, the mass ratio of canine bone marrow mesenchymal stem cell exosomes and tea polyphenols in step S1 is 10:2-3, the pH value of the PBS solution is 6.5-7, the power of the ultrasound is 30-50W, and the ultrasound co-incubation time is 30-50min.

[0010] As a further improvement of the present invention, the pH value of the PBS solution in step S2 is 6.5-7, and the mass ratio of the loaded tea polyphenol exosomes, NHS, EDC and arginine nonamer is 10:0.5-1:1-2:2-3.

[0011] As a further improvement of the present invention, the mass ratio of arginine nonamer, ditert-butyl dicarbonate, Tween, cholesterol, dicyclohexylcarbodiimide and 4-dimethylaminopyridine in step S3 is 15-25:12-17:300-500:20-40:25-35:2-3, wherein the Tween is selected from at least one of Tween-20, Tween-40, Tween-60, Tween-80 and Tween-85, and the heating and stirring reaction temperature is 25-35°C and the time is 40-50 h.

[0012] As a further improvement of the present invention, the mass ratio of resveratrol, epigallocatechin gallate, modified cholesterol, DSPE-PEG2000 and phospholipids in step S4 is 1-2:0.5-1:10-12:3-5:50-70.

[0013] As a further improvement of the present invention, the mass ratio of Polygonatum sibiricum, Lycium barbarum and Ginseng in step S5 is 4-7:2-5:1-2.

[0014] As a further improvement of the present invention, the mass ratio of pepsin, decellularized dermal matrix fragments, Polygonatum / Lycium barbarum / ginseng polysaccharide, modified tea polyphenol-loaded exosomes, and liposomes in step S6 is 0.1-0.12:3-5:1-2:0.3-0.5:0.4-0.7; the concentration of the hydrochloric acid solution is 0.01-0.15 mol / L; the dilution to a protein concentration of 1.5-2.5 mg / mL is performed; and the heating and incubation temperature is 35-40℃ for 30-50 min.

[0015] The present invention further protects a composition for delaying cell aging prepared by the above-described preparation method.

[0016] The present invention further protects the use of the above-mentioned composition for delaying cell aging in the preparation of anti-aging pharmaceuticals.

[0017] The present invention has the following beneficial effects: Arginine nonamers are cell-penetrating peptides. Modified exosomes based on these peptides are novel carriers that do not cause permanent damage to cell membranes. Containing positively charged arginine and lysine, these peptides carry a large amount of positive charge, allowing them to form hydrogen bonds with negatively charged salts in cells. This enables them to adsorb onto the membrane surface, breaking the tight bonds between epithelial cells, lowering the membrane potential, and promoting intercellular or intracellular transport of the drug, thereby improving the efficiency of penetrating the skin barrier. This overcomes the shortcomings of traditional exosomes, which remain only in the epidermis, failing to reach the deep dermis or even subcutaneous tissue, resulting in weaker drug efficacy. In addition, unmodified exosomes mainly rely on clathrin-mediated endocytosis, which is inefficient and easily degraded by lysosomes. Modified exosomes can significantly increase intracellular drug concentration. After penetrating the cell membrane, the repair factors such as miR-21 and miR-126 in the exosomes act rapidly on the cells. At the same time, the tea polyphenols loaded on them can be released rapidly. Through antioxidant and anti-inflammatory mechanisms, they can significantly delay skin aging. The loaded and encapsulated tea polyphenols also solve the problems of poor stability and easy decomposition.

[0018] Arginine nonamers can also be used to modify liposomes, improving their transdermal absorption. Furthermore, inserting DSPE-PEG2000 during liposome preparation gives the liposomes long-cycle characteristics, improves their physical stability, reduces the interaction between liposomes and plasma proteins, and allows them to function better and for a longer duration.

[0019] Goji berry polysaccharides are one of the main active ingredients of goji berries. Synergistically, they work with polygonatum polysaccharides and ginseng polysaccharides to have antioxidant and anti-aging effects on the skin. Resveratrol and epigallocatechin gallate work synergistically to combat oxidation, inflammation, and inhibit collagenase, hyaluronidase, and tyrosinase, thereby improving signs of aging such as collagen degradation, dull skin, and inflammation. The decellularized matrix mimics the natural three-dimensional structure and various bioactive components of the extracellular matrix, mainly composed of fibrin (collagen, elastin) and glycoproteins (fibronectin, proteoglycans, laminin). The formation mechanism of the composite hydrogel is based on collagen self-assembly and the formation of a hydrogen-bonded network structure with hydroxyl groups in polygonatum / goji berry / ginseng polysaccharides. Simultaneously, the rich collagen content significantly enhances skin's collagen uptake, improves skin elasticity, restores cell vitality, and exhibits good biocompatibility and low immunogenicity, making it safer and more biodegradable.

[0020] The composition for delaying cell aging prepared by this invention has a good drug encapsulation effect, which can improve the stability and transdermal absorption of the drug. At the same time, it has good antioxidant, antibacterial and anti-inflammatory effects, and can better exert the effect of delaying skin cell aging through multiple pathways. It is also safe and biodegradable, and has good application prospects. Detailed Implementation

[0021] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] NHS, N-hydroxysuccinimide; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; decellularized dermal matrix fragment, 4×4cm in size.

[0023] The preparation method of canine bone marrow mesenchymal stem cell exosomes is as follows: (1) Vitamin C, linoleic acid, interleukin-2 and rapamycin were added to DMEM / F12 medium to prepare stem cell culture medium; the content of vitamin C in the culture medium was 25 μg / mL, the content of linoleic acid was 1.5 μg / mL, the content of interleukin-2 was 1.5 μg / mL and the content of rapamycin was 7 ng / mL. (2) Canine bone marrow mesenchymal stem cells were seeded into a culture medium at a density of 5 × 10⁻⁶. 4Canine bone marrow mesenchymal stem cell exosomes were obtained by culturing at a concentration of 1.5 v / v% under 15 W ultrasound conditions and 808 nm xenon lamp irradiation for 52 h. The stem cells were isolated, the supernatant was collected, and the cells were purified by ultracentrifugation and freeze-dried. The specific ultracentrifugation purification method was as follows: centrifugation at 2000×g, 4℃ for 30 min; the supernatant was transferred to a new centrifuge tube and centrifuged again at 10,000×g, 4℃ for 45 min to remove larger vesicles; the supernatant was collected, filtered through a 0.45 μm filter membrane, and the filtrate was collected; the filtrate was transferred to a new centrifuge tube and centrifuged at 4℃, 100,000×g for 70 min; the supernatant was removed, and the cells were resuspended in 10 mL of pre-chilled 1×PBS and ultracentrifuged again at 4℃, 100,000×g for 70 min; the supernatant was removed, and the precipitate was resuspended in approximately 400 μL of pre-chilled 1×PBS and stored at -20℃. Example 1

[0024] This embodiment provides a method for preparing a composition that delays cell aging, comprising the following steps: S1. Preparation of tea polyphenol-loaded exosomes: 1g of canine bone marrow mesenchymal stem cell exosomes and 0.2g of tea polyphenols were added to 100mL of PBS solution with a pH of 6.5, and incubated on ice at 30W for 30min by sonication. The mixture was purified by qEV original column, concentrated by 100kD ultrafiltration tube, and freeze-dried to obtain tea polyphenol-loaded exosomes. S2. Preparation of modified tea polyphenol-loaded exosomes: 1 g of tea polyphenol-loaded exosomes were added to 100 mL of PBS solution with a pH of 6.5, along with 0.05 g of NHS and 0.1 g of EDC. The mixture was stirred and activated for 30 min, then 0.2 g of arginine nonamer was added. The mixture was stirred and reacted for 10 h. The mixture was dialyzed through a 5 kDa dialysis bag for 24 h and then freeze-dried to obtain modified tea polyphenol-loaded exosomes. S3. Modified cholesterol: Dissolve 0.15g of arginine nonamer in 100mL of water, add 20mL of dimethylformamide solution containing 0.12g of ditert-butyl dicarbonate, adjust the pH of the solution to 9, stir and react for 24h, add 3g of Tween-60, stir and mix for 10min, add 100mL of dimethylformamide solution containing 0.2g cholesterol, 0.25g of dicyclohexylcarbodiimide and 0.02g of 4-dimethylaminopyridine, heat to 25℃ under nitrogen protection, stir and react for 50h, stop stirring, add 1wt% trifluoroacetic acid to eliminate BOC protection, dialyze through a 5kDa dialysis bag for 24h, freeze dry to obtain modified cholesterol; S4. Preparation of liposomes: 0.1g resveratrol and 0.05g epigallocatechin gallate were dissolved in 150mL tetrahydrofuran, 1g modified cholesterol, 0.3g DSPE-PEG2000 and 5g soybean lecithin were added, and the mixture was stirred and mixed evenly. The mixture was then heated under reduced pressure and rotary evaporated to form a thin film. 20mL of ultrapure water was added for hydration for 50min, and the mixture was sonicated at 1000W for 10min. The mixture was then filtered through a 0.45μm microporous membrane, washed, and dried to obtain liposomes. S5. Polysaccharide extraction: Mix and pulverize 4g Polygonatum, 2g Lycium barbarum and 1g Ginseng, add to 150mL of water, heat to boiling and extract for 3h, filter, repeat extraction once, combine the filtrates, concentrate to 100mL, add ethanol to the system ethanol concentration to 80wt%, precipitate for 1h, filter, wash the solid, dry, and obtain Polygonatum / Lycium barbarum / Ginseng polysaccharide. S6. Preparation of the composition for delaying cell senescence: 0.1g of pepsin and 3g of decellularized dermal matrix fragments were added to 150mL of 0.01mol / L hydrochloric acid solution, stirred and digested, the pH of the solution was adjusted to neutral, diluted to a protein concentration of 1.5mg / mL, enzyme was inactivated, 1g of Polygonatum sibiricum / Lycium barbarum / ginseng polysaccharide, 0.3g of modified loaded tea polyphenol exosomes and 0.4g of liposomes were added and stirred and mixed evenly, heated to 35℃, incubated for 30min, and freeze-dried to obtain the composition for delaying cell senescence. Example 2

[0025] This embodiment provides a method for preparing a composition that delays cell aging, comprising the following steps: S1. Preparation of tea polyphenol-loaded exosomes: 1g of canine bone marrow mesenchymal stem cell exosomes and 0.3g of tea polyphenols were added to 100mL of PBS solution with a pH of 7, and the mixture was incubated on ice at 50W for 50min by sonication. The mixture was purified by qEV original column, concentrated by 100kD ultrafiltration tube, and freeze-dried to obtain tea polyphenol-loaded exosomes. S2. Preparation of modified tea polyphenol-loaded exosomes: 1g of tea polyphenol-loaded exosomes were added to 100mL of PBS solution with a pH of 7, along with 0.1g of NHS and 0.2g of EDC. The mixture was stirred and activated for 30min, then 0.3g of arginine nonamer was added. The mixture was stirred and reacted for 10h, dialyzed through a dialysis bag with a pore size of 5kDa for 24h, and then freeze-dried to obtain modified tea polyphenol-loaded exosomes. S3. Modified cholesterol: Dissolve 0.25g of arginine nonamer in 100mL of water, add 20mL of dimethylformamide solution containing 0.17g of ditert-butyl dicarbonate, adjust the pH of the solution to 10, stir and react for 24h, add 5g of Tween-80, stir and mix for 10min, add 100mL of dimethylformamide solution containing 0.4g cholesterol, 0.35g of dicyclohexylcarbodiimide and 0.03g of 4-dimethylaminopyridine, heat to 35℃ under nitrogen protection, stir and react for 50h, stop stirring, add 1wt% trifluoroacetic acid to eliminate BOC protection, dialyze through a 5kDa dialysis bag for 24h, freeze dry to obtain modified cholesterol; S4. Preparation of liposomes: 0.2g resveratrol and 0.1g epigallocatechin gallate were dissolved in 150mL tetrahydrofuran, 1.2g modified cholesterol, 0.5g DSPE-PEG2000 and 7g soybean lecithin were added, and the mixture was stirred and mixed evenly. The mixture was then heated under reduced pressure and rotary evaporated to form a thin film. 20mL of ultrapure water was added for hydration for 50min, and the mixture was sonicated at 1000W for 10min. The mixture was filtered through a 0.45μm microporous membrane, washed, and dried to obtain liposomes. S5. Polysaccharide extraction: Mix and pulverize 7g Polygonatum, 5g Lycium barbarum and 2g Ginseng, add to 150mL of water, heat to boiling and extract for 3h, filter, repeat extraction twice, combine the filtrates, concentrate to 200mL, add ethanol to the system ethanol concentration to 80wt%, precipitate for 1h, filter, wash the solid, dry, and obtain Polygonatum / Lycium barbarum / Ginseng polysaccharide. S6. Preparation of the composition for delaying cell senescence: 0.12g of pepsin and 5g of decellularized dermal matrix fragments were added to 150mL of 0.15mol / L hydrochloric acid solution, stirred and digested, the pH of the solution was adjusted to neutral, diluted to a protein concentration of 2.5mg / mL, enzyme was inactivated, 2g of Polygonatum sibiricum / Lycium barbarum / ginseng polysaccharide, 0.5g of modified loaded tea polyphenol exosomes and 0.7g of liposomes were added and stirred and mixed evenly, heated to 40℃, incubated for 50min, and freeze-dried to obtain the composition for delaying cell senescence. Example 3

[0026] This embodiment provides a method for preparing a composition that delays cell aging, comprising the following steps: S1. Preparation of tea polyphenol-loaded exosomes: 1g of canine bone marrow mesenchymal stem cell exosomes and 0.25g of tea polyphenols were added to 100mL of PBS solution with a pH of 6.7, and incubated on ice at 40W for 40min by sonication. The mixture was purified by qEV original column, concentrated by 100kD ultrafiltration tube, and freeze-dried to obtain tea polyphenol-loaded exosomes. S2. Preparation of modified tea polyphenol-loaded exosomes: 1 g of tea polyphenol-loaded exosomes were added to 100 mL of PBS solution with a pH of 6.7, along with 0.07 g of NHS and 0.15 g of EDC. The mixture was stirred and activated for 30 min, then 0.25 g of arginine nonamer was added. The mixture was stirred and reacted for 10 h. The mixture was dialyzed through a 5 kDa dialysis bag for 24 h and then freeze-dried to obtain modified tea polyphenol-loaded exosomes. S3. Modified cholesterol: Dissolve 0.2g of arginine nonamer in 100mL of water, add 20mL of dimethylformamide solution containing 0.15g of ditert-butyl dicarbonate, adjust the pH of the solution to 9.5, stir and react for 24h, add 4g of Tween-85, stir and mix for 10min, add 100mL of dimethylformamide solution containing 0.3g cholesterol, 0.3g dicyclohexylcarbodiimide and 0.025g 4-dimethylaminopyridine, heat to 30℃ under nitrogen protection, stir and react for 45h, stop stirring, add 1wt% trifluoroacetic acid to eliminate BOC protection, dialyze through a 5kDa dialysis bag for 24h, freeze dry to obtain modified cholesterol; S4. Preparation of liposomes: 0.15g resveratrol and 0.07g epigallocatechin gallate were dissolved in 150mL tetrahydrofuran, 1.1g modified cholesterol, 0.4g DSPE-PEG2000 and 6g soybean lecithin were added, and the mixture was stirred and mixed evenly. The mixture was then heated under reduced pressure and rotary evaporated to form a thin film. 20mL of ultrapure water was added for hydration for 50min, and the mixture was sonicated at 1000W for 10min. The mixture was then filtered through a 0.45μm microporous membrane, washed, and dried to obtain liposomes. S5. Polysaccharide extraction: Mix and pulverize 6g Polygonatum, 3g Lycium barbarum, and 1.5g Ginseng, add to 150mL of water, heat to boiling and extract for 3h, filter, repeat extraction twice, combine the filtrates, concentrate to 200mL, add ethanol to the system ethanol concentration to 80wt%, precipitate for 1h, filter, wash the solid, dry, and obtain Polygonatum / Lycium barbarum / Ginseng polysaccharide; S6. Preparation of the composition for delaying cell senescence: 0.11g of pepsin and 4g of decellularized dermal matrix fragments were added to 150mL of 0.012mol / L hydrochloric acid solution, stirred and digested, the pH of the solution was adjusted to neutral, diluted to a protein concentration of 2mg / mL, enzyme inactivation was performed, 1.5g of Polygonatum sibiricum / Lycium barbarum / ginseng polysaccharide, 0.4g of modified loaded tea polyphenol exosomes and 0.55g of liposomes were added and stirred until homogeneous, heated to 37℃ and incubated for 40min, and then freeze-dried to obtain the composition for delaying cell senescence.

[0027] Comparative Example 1 The difference from Example 3 is that step S2 was not performed.

[0028] Includes the following steps: S1. Preparation of tea polyphenol-loaded exosomes: 1g of canine bone marrow mesenchymal stem cell exosomes and 0.25g of tea polyphenols were added to 100mL of PBS solution with a pH of 6.7, and incubated on ice at 40W for 40min by sonication. The mixture was purified by qEV original column, concentrated by 100kD ultrafiltration tube, and freeze-dried to obtain tea polyphenol-loaded exosomes. S2. Modified cholesterol: 0.2g of arginine nonamer was dissolved in 100mL of water, and 20mL of dimethylformamide solution containing 0.15g of ditert-butyl dicarbonate was added. The pH of the solution was adjusted to 9.5, and the mixture was stirred for 24h. 4g of Tween-85 was added, and the mixture was stirred for 10min. 100mL of dimethylformamide solution containing 0.3g of cholesterol, 0.3g of dicyclohexylcarbodiimide, and 0.025g of 4-dimethylaminopyridine was added. Under nitrogen protection, the mixture was heated to 30℃ and stirred for 45h. Stirring was stopped, and 1wt% trifluoroacetic acid was added to eliminate BOC protection. The mixture was dialyzed through a 5kDa dialysis bag for 24h and then freeze-dried to obtain modified cholesterol. S3. Preparation of liposomes: 0.15g resveratrol and 0.07g epigallocatechin gallate were dissolved in 150mL tetrahydrofuran, 1.1g modified cholesterol, 0.4g DSPE-PEG2000 and 6g soybean lecithin were added, and the mixture was stirred and mixed evenly. The mixture was then heated under reduced pressure and rotary evaporated to form a thin film. 20mL of ultrapure water was added for hydration for 50min, and the mixture was sonicated at 1000W for 10min. The mixture was then filtered through a 0.45μm microporous membrane, washed, and dried to obtain liposomes. S4. Polysaccharide extraction: Mix and pulverize 6g Polygonatum, 3g Lycium barbarum and 1.5g Ginseng, add to 150mL of water, heat to boiling and extract for 3h, filter, repeat extraction twice, combine the filtrates, concentrate to 200mL, add ethanol to the system ethanol concentration to 80wt%, precipitate for 1h, filter, wash the solid, dry, and obtain Polygonatum / Lycium barbarum / Ginseng polysaccharide; S5. Preparation of the composition for delaying cell senescence: 0.11g of pepsin and 4g of decellularized dermal matrix fragments were added to 150mL of 0.012mol / L hydrochloric acid solution, stirred and digested, the pH of the solution was adjusted to neutral, diluted to a protein concentration of 2mg / mL, enzyme inactivation was performed, 1.5g of Polygonatum sibiricum / Lycium barbarum / ginseng polysaccharide, 0.4g of tea polyphenol exosomes loaded with 0.55g of liposomes were added and stirred until homogeneous, heated to 37℃ and incubated for 40min, and then freeze-dried to obtain the composition for delaying cell senescence.

[0029] Comparative Example 2 The difference from Example 3 is that step S3 was not performed.

[0030] Includes the following steps: S1. Preparation of tea polyphenol-loaded exosomes: 1g of canine bone marrow mesenchymal stem cell exosomes and 0.25g of tea polyphenols were added to 100mL of PBS solution with a pH of 6.7, and incubated on ice at 40W for 40min by sonication. The mixture was purified by qEV original column, concentrated by 100kD ultrafiltration tube, and freeze-dried to obtain tea polyphenol-loaded exosomes. S2. Preparation of modified tea polyphenol-loaded exosomes: 1 g of tea polyphenol-loaded exosomes were added to 100 mL of PBS solution with a pH of 6.7, along with 0.07 g of NHS and 0.15 g of EDC. The mixture was stirred and activated for 30 min, then 0.25 g of arginine nonamer was added. The mixture was stirred and reacted for 10 h. The mixture was dialyzed through a 5 kDa dialysis bag for 24 h and then freeze-dried to obtain modified tea polyphenol-loaded exosomes. S3. Preparation of liposomes: 0.15g resveratrol and 0.07g epigallocatechin gallate were dissolved in 150mL tetrahydrofuran, 1.1g cholesterol, 0.4g DSPE-PEG2000 and 6g soybean lecithin were added, and the mixture was stirred and mixed evenly. The mixture was then heated under reduced pressure and rotary evaporated to form a thin film. 20mL of ultrapure water was added for hydration for 50min, and the mixture was sonicated at 1000W for 10min. The mixture was then filtered through a 0.45μm microporous membrane, washed, and dried to obtain liposomes. S4. Polysaccharide extraction: Mix and pulverize 6g Polygonatum, 3g Lycium barbarum and 1.5g Ginseng, add to 150mL of water, heat to boiling and extract for 3h, filter, repeat extraction twice, combine the filtrates, concentrate to 200mL, add ethanol to the system ethanol concentration to 80wt%, precipitate for 1h, filter, wash the solid, dry, and obtain Polygonatum / Lycium barbarum / Ginseng polysaccharide; S5. Preparation of the composition for delaying cell senescence: 0.11g of pepsin and 4g of decellularized dermal matrix fragments were added to 150mL of 0.012mol / L hydrochloric acid solution, stirred and digested, the pH of the solution was adjusted to neutral, diluted to a protein concentration of 2mg / mL, enzyme was inactivated, 1.5g of Polygonatum sibiricum / Lycium barbarum / ginseng polysaccharide, 0.4g of modified loaded tea polyphenol exosomes and 0.55g of liposomes were added and stirred and mixed evenly, heated to 37℃, incubated for 40min, and freeze-dried to obtain the composition for delaying cell senescence.

[0031] Comparative Example 3 The difference from Example 3 is that DSPE-PEG2000 was not added in step S4.

[0032] Includes the following steps: S1. Preparation of tea polyphenol-loaded exosomes: 1g of canine bone marrow mesenchymal stem cell exosomes and 0.25g of tea polyphenols were added to 100mL of PBS solution with a pH of 6.7, and incubated on ice at 40W for 40min by sonication. The mixture was purified by qEV original column, concentrated by 100kD ultrafiltration tube, and freeze-dried to obtain tea polyphenol-loaded exosomes. S2. Preparation of modified tea polyphenol-loaded exosomes: 1 g of tea polyphenol-loaded exosomes were added to 100 mL of PBS solution with a pH of 6.7, along with 0.07 g of NHS and 0.15 g of EDC. The mixture was stirred and activated for 30 min, then 0.25 g of arginine nonamer was added. The mixture was stirred and reacted for 10 h. The mixture was dialyzed through a 5 kDa dialysis bag for 24 h and then freeze-dried to obtain modified tea polyphenol-loaded exosomes. S3. Modified cholesterol: Dissolve 0.2g of arginine nonamer in 100mL of water, add 20mL of dimethylformamide solution containing 0.15g of ditert-butyl dicarbonate, adjust the pH of the solution to 9.5, stir and react for 24h, add 4g of Tween-85, stir and mix for 10min, add 100mL of dimethylformamide solution containing 0.3g cholesterol, 0.3g dicyclohexylcarbodiimide and 0.025g 4-dimethylaminopyridine, heat to 30℃ under nitrogen protection, stir and react for 45h, stop stirring, add 1wt% trifluoroacetic acid to eliminate BOC protection, dialyze through a 5kDa dialysis bag for 24h, freeze dry to obtain modified cholesterol; S4. Preparation of liposomes: 0.15g resveratrol and 0.07g epigallocatechin gallate were dissolved in 150mL tetrahydrofuran, 1.5g modified cholesterol and 6g soybean lecithin were added, and the mixture was stirred and mixed evenly. The mixture was then heated under reduced pressure and rotary evaporated to form a thin film. 20mL of ultrapure water was added for hydration for 50min, and the mixture was sonicated at 1000W for 10min. The mixture was then filtered through a 0.45μm microporous membrane, washed, and dried to obtain liposomes. S5. Polysaccharide extraction: Mix and pulverize 6g Polygonatum, 3g Lycium barbarum, and 1.5g Ginseng, add to 150mL of water, heat to boiling and extract for 3h, filter, repeat extraction twice, combine the filtrates, concentrate to 200mL, add ethanol to the system ethanol concentration to 80wt%, precipitate for 1h, filter, wash the solid, dry, and obtain Polygonatum / Lycium barbarum / Ginseng polysaccharide; S6. Preparation of the composition for delaying cell senescence: 0.11g of pepsin and 4g of decellularized dermal matrix fragments were added to 150mL of 0.012mol / L hydrochloric acid solution, stirred and digested, the pH of the solution was adjusted to neutral, diluted to a protein concentration of 2mg / mL, enzyme inactivation was performed, 1.5g of Polygonatum sibiricum / Lycium barbarum / ginseng polysaccharide, 0.4g of modified loaded tea polyphenol exosomes and 0.55g of liposomes were added and stirred until homogeneous, heated to 37℃ and incubated for 40min, and then freeze-dried to obtain the composition for delaying cell senescence.

[0033] Comparative Example 4 The difference from Example 3 is that in step S6, the modified loaded tea polyphenol exosomes are replaced by an equal mass of tea polyphenols.

[0034] Includes the following steps: S1. Modified cholesterol: 0.2g of arginine nonamer was dissolved in 100mL of water, and 20mL of dimethylformamide solution containing 0.15g of ditert-butyl dicarbonate was added. The pH of the solution was adjusted to 9.5, and the mixture was stirred for 24h. 4g of Tween-85 was added, and the mixture was stirred for 10min. 100mL of dimethylformamide solution containing 0.3g of cholesterol, 0.3g of dicyclohexylcarbodiimide, and 0.025g of 4-dimethylaminopyridine was added. Under nitrogen protection, the mixture was heated to 30℃ and stirred for 45h. Stirring was stopped, and 1wt% trifluoroacetic acid was added to eliminate BOC protection. The mixture was dialyzed through a 5kDa dialysis bag for 24h and then freeze-dried to obtain modified cholesterol. S2. Preparation of liposomes: 0.15g resveratrol and 0.07g epigallocatechin gallate were dissolved in 150mL tetrahydrofuran, 1.1g modified cholesterol, 0.4g DSPE-PEG2000 and 6g soybean lecithin were added, and the mixture was stirred and mixed evenly. The mixture was then heated under reduced pressure and rotary evaporated to form a thin film. 20mL of ultrapure water was added for hydration for 50min, and the mixture was sonicated at 1000W for 10min. The mixture was then filtered through a 0.45μm microporous membrane, washed, and dried to obtain liposomes. S3. Polysaccharide extraction: Mix and pulverize 6g Polygonatum, 3g Lycium barbarum and 1.5g Ginseng, add to 150mL of water, heat to boiling and extract for 3h, filter, repeat extraction twice, combine the filtrates, concentrate to 200mL, add ethanol to the system ethanol concentration to 80wt%, precipitate for 1h, filter, wash the solid, dry, and obtain Polygonatum / Lycium barbarum / Ginseng polysaccharide; S4. Preparation of the composition for delaying cell senescence: 0.11g of pepsin and 4g of decellularized dermal matrix fragments were added to 150mL of 0.012mol / L hydrochloric acid solution, stirred and digested, the pH of the solution was adjusted to neutral, diluted to a protein concentration of 2mg / mL, enzyme inactivation was performed, 1.5g of Polygonatum sibiricum / Lycium barbarum / ginseng polysaccharide, 0.4g of tea polyphenols and 0.55g of liposomes were added and stirred until homogeneous, heated to 37℃ and incubated for 40min, and then freeze-dried to obtain the composition for delaying cell senescence.

[0035] Comparative Example 5 The difference from Example 3 is that in step S6, the liposomes are replaced by an equal mass mixture of resveratrol and epigallocatechin gallate.

[0036] Includes the following steps: S1. Preparation of tea polyphenol-loaded exosomes: 1g of canine bone marrow mesenchymal stem cell exosomes and 0.25g of tea polyphenols were added to 100mL of PBS solution with a pH of 6.7, and incubated on ice at 40W for 40min by sonication. The mixture was purified by qEV original column, concentrated by 100kD ultrafiltration tube, and freeze-dried to obtain tea polyphenol-loaded exosomes. S2. Preparation of modified tea polyphenol-loaded exosomes: 1 g of tea polyphenol-loaded exosomes were added to 100 mL of PBS solution with a pH of 6.7, along with 0.07 g of NHS and 0.15 g of EDC. The mixture was stirred and activated for 30 min, then 0.25 g of arginine nonamer was added. The mixture was stirred and reacted for 10 h. The mixture was dialyzed through a 5 kDa dialysis bag for 24 h and then freeze-dried to obtain modified tea polyphenol-loaded exosomes. S3. Preparation of the mixture: 0.15g of resveratrol and 0.07g of epigallocatechin gallate were mixed evenly to obtain the mixture; S4. Preparation of the composition for delaying cell senescence: 0.11g of pepsin and 4g of decellularized dermal matrix fragments were added to 150mL of 0.012mol / L hydrochloric acid solution, stirred and digested, the pH of the solution was adjusted to neutral, diluted to a protein concentration of 2mg / mL, enzyme was inactivated, 1.5g of Polygonatum sibiricum / Lycium barbarum / ginseng polysaccharide, 0.4g of modified loaded tea polyphenol exosomes and 0.55g of the mixture were added and stirred evenly, heated to 37℃, incubated for 40min, and freeze-dried to obtain the composition for delaying cell senescence.

[0037] Test Example 1: Antioxidant Test The cell senescence-delaying compositions prepared in Examples 1-3 and Comparative Examples 1-5 were added to water to prepare a 0.5 mg / mL sample solution. Then, 500 μL of 6 mmol / L ferrous sulfate solution, 500 μL of polysaccharide sample solutions of different concentrations, and 500 μL of 6 mmol / L H2O2 solution were added sequentially to a 2 mL centrifuge tube. The mixture was stirred and incubated at room temperature for 10 min. Then, 250 μL of 6 mmol / L ethanol-salicylic acid solution was added, and the mixture was shaken well. The tube was then incubated in a 37°C water bath for 30 min, and the absorbance was measured at 510 nm. A vitamin C solution of the same concentration was used as a positive control.

[0038] Hydroxyl radical scavenging rate (%) = [1-( A 1- A 2) / A 0]×100%; in, A 0 represents the absorbance value of the blank control, in which water is used instead of the sample being tested in the reaction system; A 1 represents the absorbance value of the sample; A 2 represents the background absorbance value of the sample. Water is used instead of H2O2 in the reaction system.

[0039] The results are shown in Table 1.

[0040] Table 1

[0041] As can be seen from the table above, the compositions for delaying cell aging prepared in Examples 1-3 of the present invention have good antioxidant properties.

[0042] Test Example 2: Cellular Anti-aging Effect The cell senescence-delaying compositions prepared in Examples 1-3 and Comparative Examples 1-5 were added to water to prepare a sample solution of 0.1 mg / mL.

[0043] Test the expression level of type IV collagen: at 2×10 5 HFF-1 cells were seeded into 24-well plates at a cell density of [number] cells / mL. After 24 hours, siRNA working solution was added and the cells were co-incubated for 6 hours. The siRNA working solution was discarded, and sample solution was added as the culture medium. Deionized water served as the blank control. After co-incubation for 36 hours, the cell culture supernatant was collected, and the content of type IV collagen in the supernatant was measured using a human type IV collagen ELISA kit according to the manufacturer's instructions. The results are shown in Table 2.

[0044] Table 2

[0045] As can be seen from the table above, the compositions for delaying cell aging prepared in Examples 1-3 of the present invention have a good effect on promoting the secretion of type IV collagen in cells.

[0046] Test Example 3: Human Skin Patch Test and Allergy Test Test sample: The cell senescence delaying compositions prepared in Examples 1-3 and Comparative Examples 1-5 of this invention were prepared with distilled water to a concentration of 1 wt% to obtain the sample solution.

[0047] Experimental Methods: Subjects aged 32-54 years were randomly divided into 8 groups of 6 subjects each. A 6cm x 6cm area was marked on the flexor surface of the right forearm of each subject and medically disinfected. The marked area was then treated by introducing sample solution through a 36-needle microneedle for 30 seconds, followed by wiping away any remaining liquid with a saline-soaked cotton ball to keep the marked area dry. The test sample was then placed into the wells of a 1-8 spectrophotometer, with a volume of 5-15 μL. Bathing was prohibited during the test. A negative reaction was considered normal. The test sample was distributed to the subjects and applied to their face, neck, and behind their ears. Skin condition was observed after 10 minutes. The results are shown in Table 3.

[0048] Table 3

[0049] As shown in the table above, the patch reactions of the compositions for delaying cell aging prepared in Examples 1-3 and Comparative Examples 1-5 of the present invention were negative, indicating that they are safe to use and that no allergic reactions occurred.

[0050] Test Example 4: Anti-aging effects on the human body The cell senescence-delaying compositions prepared in Examples 1-3 and Comparative Examples 1-5 were added to water to prepare a sample solution of 1 mg / mL.

[0051] Thirty or more participants aged 18-60 years were randomly selected to conduct a before-and-after comparison of product use. The efficacy on facial skin was verified using instruments after 14 days of use. Qualified participants used the sample solution and control (deionized water) on their left and right faces, respectively. After 14 days of use, facial tests were performed using the Antera 3D imaging system, and the analysis dimensions included skin wrinkle size and depth.

[0052] The formula for calculating the rate of change is: Rate of change = (T) e -T i ) / T i ×100%; Among them, T i The initial values ​​for wrinkle size and depth of the subjects; T e The values ​​of wrinkle size and wrinkle depth were obtained after the subjects used the product continuously for 14 days.

[0053] The results are shown in Table 4.

[0054]

[0055] As can be seen from the table above, the compositions for delaying cell aging prepared in Examples 1-3 of the present invention can significantly reduce wrinkle depth and wrinkle size.

[0056] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a composition that delays cell senescence, characterized in that, Canine bone marrow mesenchymal stem cell exosomes were loaded with tea polyphenols and then modified with arginine nonamers to obtain modified tea polyphenol-loaded exosomes. Arginine nonamers were modified with cholesterol and then combined with DSPE-PEG2000, phospholipids to encapsulate resveratrol and epigallocatechin gallate to obtain liposomes. Polysaccharides were extracted from Polygonatum, Lycium barbarum, and ginseng to obtain Polygonatum / Lycium barbarum / ginseng polysaccharide. Decellularized dermal matrix was mixed with Polygonatum / Lycium barbarum / ginseng polysaccharide, and liposomes and modified tea polyphenol-loaded exosomes were added. After co-incubation, the mixture was freeze-dried to obtain a composition for delaying cell aging.

2. The preparation method according to claim 1, characterized in that, Includes the following steps: S1. Preparation of tea polyphenol-loaded exosomes: Canine bone marrow mesenchymal stem cell exosomes and tea polyphenols were added to PBS solution, co-incubated on ice by sonication, purified, concentrated by ultrafiltration tube, and freeze-dried to obtain tea polyphenol-loaded exosomes; S2. Preparation of modified tea polyphenol-loaded exosomes: Tea polyphenol-loaded exosomes were added to PBS solution, NHS and EDC were added, the mixture was stirred and activated, arginine nonamer was added, the mixture was stirred and reacted, dialyzed, and freeze-dried to obtain modified tea polyphenol-loaded exosomes; S3. Modified cholesterol: Arginine nonamer was dissolved in water, and a dimethylformamide solution of ditert-butyl dicarbonate was added. The pH of the solution was adjusted, and the reaction was stirred. Tween was added, and the mixture was stirred until homogeneous. A dimethylformamide solution containing cholesterol, dicyclohexylcarbodiimide, and 4-dimethylaminopyridine was added. The reaction was heated and stirred under inert gas protection. Stirring was stopped, trifluoroacetic acid was added to eliminate BOC protection, and the mixture was dialyzed and freeze-dried to obtain modified cholesterol. S4. Preparation of liposomes: Resveratrol and epigallocatechin gallate were dissolved in tetrahydrofuran, modified cholesterol, DSPE-PEG2000 and phospholipids were added, the mixture was stirred and mixed evenly, and the film was formed by rotary evaporation under reduced pressure. Ultrapure water was added for hydration, followed by sonication, filtration, washing and drying to obtain liposomes. S5. Polysaccharide extraction: Mix and pulverize Polygonatum, Lycium barbarum and ginseng, add water, heat to boiling and extract, filter, repeat extraction 1-2 times, combine filtrates, concentrate, add ethanol to precipitate, filter, wash solid and dry to obtain Polygonatum / Lycium barbarum / ginseng polysaccharide; S6. Preparation of a composition for delaying cell aging: Pepsin and decellularized dermal matrix fragments were added to hydrochloric acid solution, stirred and digested, the pH of the solution was adjusted, diluted, enzymes were inactivated, and Polygonatum sibiricum / Lycium barbarum / ginseng polysaccharide, modified loaded tea polyphenol exosomes and liposomes were added and stirred evenly, heated and incubated, and freeze-dried to obtain a composition for delaying cell aging.

3. The preparation method according to claim 2, characterized in that, In step S1, the mass ratio of canine bone marrow mesenchymal stem cell exosomes to tea polyphenols is 10:2-3, the pH of the PBS solution is 6.5-7, the power of the ultrasound is 30-50W, and the ultrasound co-incubation time is 30-50min.

4. The preparation method according to claim 2, characterized in that, The pH of the PBS solution in step S2 is 6.5-7, and the mass ratio of the loaded tea polyphenol exosomes, NHS, EDC, and arginine nonamers is 10:0.5-1:1-2:2-3.

5. The preparation method according to claim 2, characterized in that, In step S3, the mass ratio of arginine nonamer, ditert-butyl dicarbonate, Tween, cholesterol, dicyclohexylcarbodiimide, and 4-dimethylaminopyridine is 15-25:12-17:300-500:20-40:25-35:2-3. The Tween is selected from at least one of Tween-20, Tween-40, Tween-60, Tween-80, and Tween-85. The heating and stirring reaction is carried out at a temperature of 25-35°C for 40-50 hours.

6. The preparation method according to claim 2, characterized in that, The mass ratio of resveratrol, epigallocatechin gallate, modified cholesterol, DSPE-PEG2000, and phospholipids in step S4 is 1-2:0.5-1:10-12:3-5:50-70.

7. The preparation method according to claim 2, characterized in that, The mass ratio of Polygonatum, Lycium barbarum, and Ginseng in step S5 is 4-7:2-5:1-2.

8. The preparation method according to claim 2, characterized in that, In step S6, the mass ratio of pepsin, decellularized dermal matrix fragments, Polygonatum / Lycium / Ginseng polysaccharide, modified tea polyphenol-loaded exosomes, and liposomes is 0.1-0.12:3-5:1-2:0.3-0.5:0.4-0.7; the concentration of the hydrochloric acid solution is 0.01-0.15 mol / L; the dilution to a protein concentration of 1.5-2.5 mg / mL is performed; and the heating incubation temperature is 35-40℃ for 30-50 min.

9. A composition for delaying cell senescence prepared by any one of claims 1-8.

10. The use of the composition for delaying cell aging as described in claim 9 in the preparation of an anti-aging pharmaceutical product.

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