Anti-aging composition based on hexadecanedioic acid and application of anti-aging composition

Abstract

The invention relates to the technical field of anti-aging intervention, in particular to an anti-aging composition based on hexadecanedioic acid, which is prepared from the following raw materials in parts by weight: 10 to 30 parts of hexadecanedioic acid, 5 to 15 parts of N-acetylcysteine, 3 to 10 parts of vitamin E, 2 to 8 parts of curcumin, 1 to 6 parts of resveratrol, 5 to 20 parts of astragalus polysaccharide, 2 to 7 parts of berberine and 30 to 60 parts of liposome nano-carrier. By using hexadecanedioic acid as a main active component, the expression of senescence markers p16 and p21 and SASP related proteins can be down-regulated, and the proliferation and osteogenic differentiation of BMSCs (bone marrow mesenchymal stem cells) are promoted; n-acetylcysteine enhances the oxidation resistance by improving the level of glutathione, and the bioavailability of each component is improved by the lipidosome nano-carrier. All the components cooperate through multiple channels, and the anti-BMSCs aging effect is remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of anti-aging intervention technology, specifically to an anti-aging composition based on hexadecanoic acid and its application. Background Technology

[0002] With the increasing aging of the population, the incidence of age-related diseases (such as osteoporosis and impaired fracture healing) is rising year by year, seriously affecting the quality of life of the elderly. Studies have shown that the occurrence of these diseases is closely related to the functional decline of bone marrow mesenchymal stem cells (BMSCs). BMSC aging is mainly manifested by decreased proliferation capacity, osteogenic differentiation disorder, and cell cycle arrest. Its underlying mechanisms involve multiple aspects such as oxidative stress accumulation, mitochondrial dysfunction, metabolic disorders, and chronic inflammation.

[0003] Currently, intervention strategies for BMSC aging mainly rely on various anti-aging drugs, such as NMN, resveratrol, and metformin. However, existing drugs have many limitations: some drugs have unstable efficacy and are greatly affected by the internal microenvironment; some drugs have poor water solubility and low bioavailability; and some drugs are expensive or have certain toxic side effects with long-term use, making it difficult to meet the needs of clinical application.

[0004] Hexadecanedioic acid (HDA) is a naturally occurring medium-chain dicarboxylic acid that participates in fatty acid metabolism pathways in the body. However, to date, no literature or patents have reported its application in regulating stem cell aging. This invention is the first to discover that HDA has a significant anti-aging ability in BMSCs, and further enhances its anti-aging effect through synergistic effects with other active ingredients, providing a new approach to address the shortcomings of existing technologies. Summary of the Invention

[0005] This invention aims to overcome the shortcomings of existing anti-aging drugs in anti-BMSCs aging applications, such as unstable efficacy, poor solubility, and weak synergistic effects, and provides an anti-aging composition based on hexadecanoic acid. This composition can effectively delay or reverse BMSCs aging through multi-pathway synergistic effects, and has good biocompatibility and application prospects.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution: an anti-aging composition based on hexadecanoic acid, comprising the following raw materials in parts by weight: 10-30 parts of hexadecanoic acid, 5-15 parts of N-acetylcysteine, 3-10 parts of vitamin E, 2-8 parts of curcumin, 1-6 parts of resveratrol, 5-20 parts of astragalus polysaccharide, 2-7 parts of berberine, and 30-60 parts of liposome nanocarrier.

[0007] Preferably, the liposome nanocarrier has a particle size of 50-200 nm, which can enhance the cell membrane permeability of the carrier and increase the intracellular concentration of the loaded drug.

[0008] Preferably, the molecular weight of the astragalus polysaccharide is 10,000-50,000 Da, astragalus polysaccharides in this molecular weight range have better metabolic regulation and anti-inflammatory activity.

[0009] The present invention also provides a method for preparing the above-mentioned anti-aging composition based on hexadecanoic acid, comprising the following steps: (1) Weigh each raw material according to the weight parts, mix hexadecanoic acid with curcumin and resveratrol to obtain the mixture to be loaded; (2) Preparation of liposome nanocarriers by thin film dispersion method: Liposome material is dissolved in organic solvent, organic solvent is removed by rotary evaporation to form a lipid film, buffer solution is added for hydration, and ultrasonic treatment is performed to obtain blank liposomes; (3) Add the mixture to be loaded in step (1) to the blank liposome in step (2), stir and incubate to obtain drug-loaded liposome nanocarriers; (4) Dissolve N-acetylcysteine, vitamin E, astragalus polysaccharide and berberine in appropriate amounts of solvent, and add them sequentially to the drug-loaded liposome nanocarrier in step (3). Stir and mix evenly to obtain the anti-aging composition.

[0010] Preferably, in step (2), the liposome material is phospholipid and cholesterol in a mass ratio of (3-5):1; the organic solvent is chloroform or dichloromethane; the rotary evaporation temperature is 35-45℃ and the time is 20-40 min; the ultrasonic treatment power is 200-400W and the time is 5-15 min.

[0011] Preferably, in step (3), the stirring incubation temperature is 25-37℃, the time is 1-3h, and the stirring rate is 100-300r / min.

[0012] Preferably, the present invention also protects the use of the above-mentioned anti-aging composition in the preparation of products for anti-BMSCs aging, said products including pharmaceuticals, health products or cell culture additives; when said products are pharmaceuticals, their dosage forms may be injections, capsules, tablets or powders.

[0013] This invention provides an anti-aging composition based on hexadecanoic acid and its application. It possesses the following beneficial effects: 1. In the composition of this invention, hexadecanoic acid, as the main active ingredient, can downregulate the expression of aging markers p16, p21, and SASP-related proteins (IL-1, IL-6, pai-1), promoting BMSC proliferation and osteogenic differentiation; N-acetylcysteine ​​enhances antioxidant capacity by increasing glutathione levels; vitamin E inhibits lipid peroxidation chain reactions; curcumin activates the Nrf2 / HO-1 pathway to strengthen antioxidant defense; resveratrol promotes mitochondrial biosynthesis; astragalus polysaccharide regulates glucose and lipid metabolism and has anti-inflammatory effects; berberine induces autophagy and improves gut microbiota; and liposome nanocarriers improve the bioavailability of each component. Through multi-pathway synergy, these components significantly enhance the anti-BMSC aging effect.

[0014] 2. The core component of the composition of this invention, hexadecanoic acid, exhibits effects comparable to NMN and superior to resveratrol and metformin in regulating p21 expression, reducing the proportion of β-galactosidase-positive cells, and enhancing osteogenic potential. Furthermore, these effects are further enhanced through synergistic effects with the composition. Hexadecanoic acid maintains over 90% cell viability in BMSCs within a concentration range of 5-200 μM, with no significant apoptosis or necrosis. Combined with other natural components and biocompatible carriers, the overall composition demonstrates high safety. Attached Figure Description

[0015] Figure 1 This is a schematic diagram showing the effects of different concentrations of the composition on the toxicity and proliferation of BMSCs. In this diagram, A represents the activity rate of BMSCs after treatment with each concentration of the composition, and B represents the promoting effect of each concentration of the composition on the proliferation of BMSCs. Figure 2 This is a schematic diagram of SA-β-Gal staining results, where A shows a comparison of staining results for different concentrations of the composition, and B shows a comparison of the anti-aging effects of different concentrations of the composition. Figure 3 The figure shows the results of qPCR detection of aging-related gene expression. In the figure, A shows the expression of p16 and p21 genes in total RNA of BMSCs after treatment with different concentrations of the composition, and B shows the expression of SASP-related genes in total RNA of BMSCs after treatment with different concentrations of the composition. Figure 4 The images show the results of ALP and ARS staining, where A is a schematic diagram of ALP staining for each concentration of the composition, B is a schematic diagram of ARS staining for each concentration of the composition, C is a comparison of ALP activity for each concentration of the composition, and D is a comparison of the amount of calcium nodules formed for each concentration of the composition. Figure 5 This is a graph showing the results of qPCR detection of osteogenic-related genes. Figure 6The figure shows the qPCR comparison results of the composition of the present invention and existing drugs, where A represents the expression of p16 and p21 genes in each experimental group, and B represents the expression of IL-1, IL-6, and pai-1 in each experimental group. Figure 7 The image shows the SA-β-Gal staining results of the composition of the present invention compared with existing drugs, where A is a comparison of the SA-β-Gal staining results of each experimental group, and B is a comparison of the positive cells of each experimental group. Figure 8 The figure shows the osteogenic differentiation experiment results of the composition of the present invention compared with existing drugs. In the figure, A is a comparison of ALP staining results of each experimental group, B is a comparison of ARS staining results of each experimental group, C is a comparison of ALP activity of each experimental group, and D is a comparison of calcium nodule formation of each experimental group. Figure 9 The images show a comparison of animal experimental results. In the images, A shows the hair growth of mice after injection of different drugs, B shows the comparison of the volume of bone trabeculae in mice after injection of different drugs, C shows the comparison of the thickness of bone trabeculae after injection of different drugs, and D shows the comparison of the separation degree of bone trabeculae after injection of different drugs. Detailed Implementation

[0016] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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.

[0017] Example 1: This invention provides an anti-aging composition based on hexadecanoic acid, comprising the following raw materials in parts by weight: 30 parts hexadecanoic acid, 15 parts N-acetylcysteine, 10 parts vitamin E, 8 parts curcumin, 6 parts resveratrol, 20 parts astragalus polysaccharide, 7 parts berberine, and 60 parts liposome nanocarriers. The liposome nanocarriers have a particle size of 200 nm; the astragalus polysaccharide has a molecular weight of 50,000 Da.

[0018] A method for preparing an anti-aging composition based on hexadecanoic acid includes the following steps: (1) Weigh each raw material according to the weight parts, mix hexadecanoic acid with curcumin and resveratrol to obtain the mixture to be loaded; (2) Preparation of liposome nanocarriers by thin film dispersion method: Liposome material was dissolved in an organic solvent and removed by rotary evaporation at 45°C for 40 min to form a lipid film; hydration was achieved by adding buffer solution and then ultrasonic treatment at 400W power for 15 min to obtain blank liposomes; the liposome material was phospholipid and cholesterol in a mass ratio of 5:1, and the organic solvent was chloroform or dichloromethane; (3) Add the mixture to be loaded in step (1) to the blank liposome in step (2), stir and incubate at 37°C at a rate of 300 r / min for 3 h to obtain drug-loaded liposome nanocarriers. (4) Dissolve N-acetylcysteine, vitamin E, astragalus polysaccharide and berberine in appropriate solvents (such as water and ethanol), and add them to the drug-loaded liposome nanocarrier in step (3) in sequence. Stir and mix evenly to obtain the anti-aging composition.

[0019] Example 2: This invention provides an anti-aging composition based on hexadecanoic acid, comprising the following raw materials in parts by weight: 10 parts hexadecanoic acid, 5 parts N-acetylcysteine, 3 parts vitamin E, 2 parts curcumin, 1 part resveratrol, 5 parts astragalus polysaccharide, 2 parts berberine, and 30 parts liposome nanocarriers. The liposome nanocarriers have a particle size of 50 nm; the astragalus polysaccharide has a molecular weight of 10000 Da.

[0020] A method for preparing an anti-aging composition based on hexadecanoic acid includes the following steps: (1) Weigh each raw material according to the weight parts, mix hexadecanoic acid with curcumin and resveratrol to obtain the mixture to be loaded; (2) Preparation of liposome nanocarriers by thin film dispersion method: Liposome material was dissolved in an organic solvent, and the organic solvent was removed by rotary evaporation at 35°C for 20 min to form a lipid film; hydration was achieved by adding buffer solution, and then ultrasonic treatment was performed at 200W power for 5 min to obtain blank liposomes; the liposome material was phospholipid and cholesterol in a mass ratio of 3:1, and the organic solvent was chloroform or dichloromethane; (3) Add the mixture to be loaded in step (1) to the blank liposome in step (2), stir and incubate at 25°C at a rate of 100 r / min for 1 h to obtain drug-loaded liposome nanocarriers. (4) Dissolve N-acetylcysteine, vitamin E, astragalus polysaccharide and berberine in appropriate solvents (such as water and ethanol), and add them to the drug-loaded liposome nanocarrier in step (3) in sequence. Stir and mix evenly to obtain the anti-aging composition.

[0021] Experiment 1: Assessment of Cytotoxicity and Proliferative Capacity Experimental materials: P5 generation elderly-derived BMSCs, anti-aging composition prepared in Example 1 (diluted to contain 5-200 μM hexadecanoic acid), and CCK-8 assay kit.

[0022] Experimental method: BMSCs were seeded into 96-well plates and cultured for 24 h. Different concentrations of the composition were added, with 3 replicates per group. After 1, 3 and 5 days of culture, 10 μL of CCK-8 reagent was added to each well and cultured for another 2 h. The absorbance (OD value) at 450 nm was measured.

[0023] Result: As Figure 1 As shown, the activity of BMSCs remained above 90% after treatment with each concentration of the composition, with no significant toxicity. Among them, the composition containing 25 μM hexadecanoic acid had the best promoting effect on BMSC proliferation, and its OD value was significantly higher than that of other concentration groups at 5 days.

[0024] Experiment 2: Verification of Anti-aging Effects β-galactosidase (SA-β-Gal) staining assay: BMSCs were seeded in 6-well plates, and a combination containing 0, 10, 25, and 50 μM hexadecanoic acid was added. After culturing for 7 days, SA-β-Gal staining was performed, and the proportion of positive cells was counted. Results are as follows: Figure 2 As shown, the 25μM concentration group had the lowest proportion of positive cells, indicating that it had the best effect in reversing aging.

[0025] qPCR detection: Total RNA was extracted from BMSCs treated with different concentrations of the combined assay, and the expression of p16, p21, and SASP-related genes (IL-1, IL-6, pai-1) was detected. Results are as follows: Figure 3 As shown, the expression levels of the above-mentioned genes were significantly downregulated in the 25μM concentration group, further confirming its anti-aging effect.

[0026] Experiment 3: Osteogenic Differentiation Capacity Detection ALP and ARS staining: BMSCs were seeded into 6-well plates, and osteogenic induction medium and a combination containing 0, 10, 25, and 50 μM hexadecanoic acid were added. ALP staining was performed after 14 days of culture, and ARS staining was performed after 21 days. The staining intensity was quantitatively analyzed. Results are as follows: Figure 4 As shown, the 25 μM concentration group had the highest ALP activity and calcium nodule formation, and the strongest osteogenic differentiation capacity.

[0027] Osteogenesis-related gene detection: qPCR was used to detect the expression of osteogenic markers runx2, opn, and ocn. Results are as follows: Figure 5 As shown, the expression levels of the above genes were significantly increased in the 25 μM concentration group, indicating that it can effectively restore the osteogenic function of BMSCs.

[0028] Experiment 4: Comparison with existing anti-aging drugs Experimental groups: control group (untreated), NMN group (150μM), resveratrol group (50μM), metformin group (1mM), and the composition of this invention group (containing 25μM hexadecanoic acid).

[0029] Detection indicators: qPCR detection of the expression of p16, p21, IL-1, IL-6, and pai-1 ( Figure 6 ), SA-β-Gal staining count of positive cells ( Figure 7 ALP and ARS staining were used to assess osteogenic differentiation. Figure 8 ).

[0030] Results: The composition of the present invention is superior to the resveratrol group and the metformin group in terms of downregulating the expression of aging-related genes, reducing the proportion of positive cells, and promoting osteogenic differentiation, and is comparable to or better than the NMN group.

[0031] Experiment 5: In vivo animal experiments Experimental animals: 18-month-old C57 mice were divided into a control group, an NMN group (150 mg / kg), a resveratrol group (15 mg / kg), a metformin group (100 mg / kg), and a composition group of the present invention (containing 5 mg / kg of HDA), with 10 mice in each group.

[0032] Administration method: Tail vein injection, twice a week, for one month.

[0033] Detection indicators: Observe the hair regeneration of mice; detect bone mineral density and bone microstructure of mouse femur using micro-CT.

[0034] Results: The hair regeneration effect in the combined group was significantly better than that in other groups; micro-CT showed that the bone density of the combined group was significantly higher than that in the control group and other drug groups, and the trabecular bone structure was more intact, suggesting that it had the best anti-aging effect on osteoporosis. Figure 9 ).

[0035] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. An anti-aging composition based on hexadecanoic acid, characterized in that: The ingredients include the following parts by weight: 10-30 parts of hexadecanoic acid, 5-15 parts of N-acetylcysteine, 3-10 parts of vitamin E, 2-8 parts of curcumin, 1-6 parts of resveratrol, 5-20 parts of astragalus polysaccharide, 2-7 parts of berberine, and 30-60 parts of liposome nanocarrier.

2. The anti-aging composition based on hexadecanoic acid according to claim 1, characterized in that: The liposome nanocarrier has a particle size of 50-200 nm.

3. The anti-aging composition based on hexadecanoic acid according to claim 1, characterized in that: The molecular weight of the Astragalus polysaccharide is 10,000-50,000 Da.

4. The method for preparing an anti-aging composition based on hexadecanoic acid according to claim 1, characterized in that: Includes the following steps: (1) Weigh each raw material according to the weight parts, mix hexadecanoic acid with curcumin and resveratrol to obtain the mixture to be loaded; (2) Preparation of liposome nanocarriers by thin film dispersion method: Liposome material is dissolved in organic solvent, organic solvent is removed by rotary evaporation to form a lipid film, buffer solution is added for hydration, and ultrasonic treatment is performed to obtain blank liposomes; (3) Add the mixture to be loaded in step (1) to the blank liposome in step (2), stir and incubate to obtain drug-loaded liposome nanocarriers; (4) Dissolve N-acetylcysteine, vitamin E, astragalus polysaccharide and berberine in appropriate amounts of solvent, and add them sequentially to the drug-loaded liposome nanocarrier in step (3). Stir and mix evenly to obtain the anti-aging composition.

5. The method for preparing an anti-aging composition based on hexadecanoic acid according to claim 1, characterized in that: In step (2), the liposome material is phospholipid and cholesterol in a mass ratio of (3-5):1; the organic solvent is chloroform or dichloromethane; the rotary evaporation temperature is 35-45℃ and the time is 20-40 min; the ultrasonic treatment power is 200-400W and the time is 5-15 min.

6. The method for preparing an anti-aging composition based on hexadecanoic acid according to claim 1, characterized in that: In step (3), the stirring incubation temperature is 25-37℃, the time is 1-3h, and the stirring rate is 100-300r / min.

7. The use of the anti-aging composition prepared by the method according to any one of claims 4-6 in the preparation of products for anti-BMSCs aging.

8. The application according to claim 7, characterized in that: The product is a drug, health product, or cell culture additive.

9. The application according to claim 7, characterized in that: When the product is a drug, its dosage form is injection, capsule, tablet or powder.

Images