Phosphatidylated urolithin composition for resisting fibroblast aging as well as preparation method and application of phosphatidylated urolithin composition

Abstract

The invention discloses a phospholipid urolithin composition for resisting fibroblast aging as well as a preparation method and application of the phospholipid urolithin composition. Belongs to the technical field of biological medicine, nutritious food and health food. The composition consists of the following raw materials in parts by weight: 10-50 parts of urolithin, 5-30 parts of phospholipid, 1-10 parts of polyglycerol fatty acid ester, 1-10 parts of fatty acid monoglyceride, 2-10 parts of Arabic gum, 2-10 parts of trehalose, 10-20 parts of starch sodium octenylsuccinate and 0.5-2 parts of vitamin E. The composition is prepared by combining high-speed shearing emulsification with a high-pressure homogenization technology. The problems that the urolithin A is poor in water solubility and low in absorption efficiency are effectively solved. In-vitro experiments prove that compared with free urolithin A, the phospholipid urolithin composition prepared by the invention can more effectively inhibit fibroblast aging, shows good anti-aging activity, and can be widely applied to the fields of anti-aging medicines, nutritional foods and health foods.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine, nutritional food and health food technology, and more specifically to a phospholipid-modified urolithin composition for anti-fibroblast senescence, its preparation method and application. Background Technology

[0002] Fibroblast senescence is a common driver of aging in multiple organs. It disrupts the tissue microenvironment by secreting aging-associated secretory phenotype (SASP) factors, such as IL-6 and MMPs, inducing chronic inflammation and extracellular matrix (ECM) degradation, thereby accelerating functional decline and structural remodeling in organs such as the skin, muscles, liver, brain, cardiovascular system, and ovaries. For example, in the skin, senescent fibroblasts degrade collagen, leading to wrinkles and loss of elasticity; in the liver and heart, their pro-fibrotic effects exacerbate organ hardening and functional decline; and in the brain and ovaries, they may disrupt neuroendocrine balance through inflammatory signaling, promoting cognitive decline and reproductive function reduction. Therefore, fibroblast senescence is not only a marker of local tissue aging but also an important cellular basis for systemic organ aging.

[0003] Ellagic acid (EA), also known as gallic acid, is a class of natural polyphenols with important physiological activities, existing in the form of trans-gallic acid tannins. Among the urolithin metabolites (urolithin A, urolithin B, urolithin C, and isourolithin) produced by ellagic acid, urolithin A is considered to be the main substance exerting anti-inflammatory, antioxidant, and anti-aging biological activities in the body. Studies have shown that urolithin A (Uro-A) maintains mitochondrial function by regulating mitophagy, thus delaying aging. Daily oral administration of 10-1000 mg of Uro-A can effectively improve age-related diseases such as muscle aging, cognitive function, metabolic dysfunction, and cardiovascular diseases.

[0004] Direct dietary supplementation of urolithin A often fails to achieve the desired efficacy. Urolithin A itself has significant technical defects that severely limit its bioavailability and practical application effects: First, it has extremely low water solubility, making it difficult to disperse evenly in body fluids or conventional preparations; it is only slightly soluble in ethanol, resulting in low bioavailability from direct supplementation. Second, oral or transdermal absorption is poor, with most ingested urolithin A failing to effectively reach target cells to exert its effects. These shortcomings mean that even with high in vitro activity, it is difficult to achieve the expected anti-aging effects in practical applications. Therefore, improving the bioavailability of urolithin A is currently a hot research topic.

[0005] Liposomes are microcapsules composed of a phospholipid bilayer, exhibiting good biocompatibility and biodegradability. They can serve as ideal carriers for hydrophobic drugs, improving their solubility, stability, and targeting. However, there are few studies on applying phospholipidation technology to urolithin A to enhance its anti-aging bioactivity.

[0006] Therefore, how to develop a novel formulation that can significantly improve the solubility, absorption efficiency and bioactivity of urolithin A is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides a phospholipid-modified urolithin composition for anti-fibroblast senescence, its preparation method, and its application. This composition, through specific liposome encapsulation technology, significantly improves the water solubility and cellular absorption efficiency of urolithin A, exhibiting a good anti-fibroblast senescence effect.

[0008] To solve the above-mentioned technical problems, this application adopts the following technical solution: A phospholipid-modified urolithin composition for inhibiting fibroblast growth comprises the following components in parts by weight: 10-50 parts urolithin, 5-30 parts phospholipid, 1-10 parts polyglycerol fatty acid ester, 1-10 parts mono-fatty acid glyceride, 2-10 parts gum arabic, 2-10 parts trehalose, 10-20 parts sodium octenyl succinate starch, and 0.5-2 parts vitamin E.

[0009] Furthermore, a phospholipid-modified urolithin composition for inhibiting fibroblast growth comprises the following components in parts by weight: 10-20 parts urolithin, 25-30 parts phospholipid, 8-10 parts polyglycerol fatty acid ester, 3-5 parts mono-fatty acid glyceride, 8-10 parts gum arabic, 10 parts trehalose, 15-20 parts sodium octenyl succinate starch, and 1 part vitamin E.

[0010] Furthermore, the urolithin is urolithin A.

[0011] Furthermore, the phospholipid is one or a combination of two of soybean phospholipid, sunflower phospholipid, peanut phospholipid, and corn phospholipid.

[0012] Furthermore, the polyglycerol fatty acid ester is one or a combination of two of polyglycerol-10 laurate, polyglycerol-10 oleate, and sucrose glyceride.

[0013] Furthermore, it includes the following components in parts by weight: 20 parts urolithin A, 25 parts sunflower phospholipids, 8 parts polyglycerol-10 laurate, 5 parts monoacylglycerol, 10 parts gum arabic, 10 parts trehalose, 20 parts sodium octenyl succinate starch, and 1 part vitamin E.

[0014] Furthermore, it includes the following components in parts by weight: 10 parts urolithin A, 30 parts soybean lecithin, 10 parts sucrose glycerides, 3 parts monofatty acid glycerides, 8 parts gum arabic, 10 parts trehalose, 15 parts sodium octenyl succinate starch, and 1 part vitamin E.

[0015] The method for preparing the above composition includes the following steps: (1) Weigh out urolithin, phospholipids, polyglycerol fatty acid esters and monoglycerides according to the formula, add deionized water and process by high-speed shearing to obtain crude emulsion a of phospholipid-modified urolithin composition; (2) Add gum arabic, trehalose, sodium octenyl succinate starch and vitamin E to crude emulsion a of phospholipid composition, and then perform high-speed shearing to obtain crude emulsion b of phospholipid composition; (3) Take the crude emulsion b of the phospholipid urolithin composition and perform high pressure homogenization to obtain the homogenized solution of the phospholipid urolithin composition; (4) After drying the homogenized solution of phospholipid urolithin composition, the phospholipid urolithin composition is obtained.

[0016] Furthermore, in step (1), the mass ratio of the material to deionized water is 1:5 to 1:10.

[0017] Furthermore, the high-speed shearing parameters mentioned in steps (1) and (2) are 12000 r / min, and the treatment time is 20~40 min.

[0018] Furthermore, the high-pressure homogenization conditions described in step (3) are 60~100 MPa, and the cycle is repeated 3 times.

[0019] Furthermore, the drying method described in step (4) is spray drying or freeze drying.

[0020] Furthermore, the parameters for spray drying are an inlet air temperature of 160~180℃.

[0021] The above-described compositions and compositions prepared by the above-described methods are used in the preparation of drugs and foods that combat fibroblast aging.

[0022] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: ① Simple process and easy to industrialize: This invention uses water as a solvent to prepare phospholipid-modified urolithiasis composition through high-speed shearing, high-pressure homogenization, spray drying or freeze drying. No processing aids are required in the production process, and no organic solvents or solvents with pollution or health hazards are used. The shearing and homogenization technologies used have mature industrial equipment. The process is simple, the conditions are easy to control, which is conducive to large-scale production and has the characteristics of being green and environmentally friendly.

[0023] ② Significantly improves solubility: By encapsulating urolithin A inside liposomes through a specific phospholipid and emulsifier system, the problem of poor water solubility of urolithin A is effectively solved, the absorption efficiency of urolithin A is improved, and the stability of urolithin A preparations during storage and use is also improved.

[0024] ③ Significantly enhanced anti-aging effect: Cell experiments have confirmed that, compared with the same dose of free urolithin A, the urolithin A liposome composition prepared in this invention exhibits a significantly enhanced effect in inhibiting fibroblast senescence, providing a new solution for developing highly effective anti-aging products. Detailed Implementation

[0025] 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.

[0026] Example 1 A phospholipid-modified urolithin composition for anti-fibroblast senescence, comprising the following raw materials in parts by weight: 20 parts urolithin A, 25 parts sunflower phospholipid, 8 parts polyglycerol-10 laurate, 5 parts mono-fatty acid glycerides, 10 parts gum arabic, 10 parts trehalose, 20 parts sodium octenyl succinate starch, and 1 part vitamin E.

[0027] It is obtained through the following preparation method: (1) Weigh out urolithin A, sunflower phospholipid, polyglycerol-10 laurate and monoacylglycerol according to the formula, add 6 times the mass of deionized water and treat with high-speed shearing at 12000r / min for 30min to obtain crude emulsion a of phospholipid composition.

[0028] (2) Add gum arabic, trehalose, sodium octenyl succinate starch and vitamin E to crude emulsion a of phospholipid composition in step (1), and perform high-speed shearing treatment under the conditions of step (1) to obtain crude emulsion b of phospholipid composition.

[0029] (3) Take the crude emulsion b of the phospholipid urolithin composition from step (2) and perform high-pressure homogenization and circulation three times under conditions of 80 MPa to obtain the homogenized solution of the phospholipid urolithin composition.

[0030] (4) After the homogenized liquid of the phospholipid urolithin composition from step (3) is spray-dried at an air inlet temperature of 160°C, the phospholipid urolithin composition is obtained. The moisture content of the composition is 3.86%.

[0031] Example 2 A phospholipid-modified urolithin composition for resisting fibroblast senescence, comprising the following raw materials in parts by weight: 10 parts urolithin A, 30 parts soybean lecithin, 10 parts sucrose glycerides, 3 parts mono-fatty acid glycerides, 8 parts gum arabic, 10 parts trehalose, 15 parts sodium octenyl succinate starch, and 1 part vitamin E.

[0032] It is obtained through the following preparation method: (1) Weigh out urolithin A, soybean lecithin, sucrose glycerol and monofatty acid glycerol according to the formula, add 10 times the mass of deionized water and treat with high-speed shearing at 12000r / min for 30min to obtain crude emulsion a of phospholipid urolithin composition.

[0033] (2) Add gum arabic, trehalose, sodium octenyl succinate starch and vitamin E to crude emulsion a of phospholipid composition in step (1), and perform high-speed shearing treatment under the conditions of step (1) to obtain crude emulsion b of phospholipid composition.

[0034] (3) Take the crude emulsion b of the phospholipid urolithin composition from step (2) and perform high-pressure homogenization and circulation three times under the condition of 100 MPa to obtain the homogenized liquid of the phospholipid urolithin composition.

[0035] (4) After freeze-drying the homogenized phospholipid urolithin composition from step (3), a phospholipid urolithin composition is obtained, the moisture content of which is 5.35%.

[0036] Comparative Example 1 Free urolithiasis A suspension Urolithin A (purity ≥99%) was directly dispersed in DMEM cell culture medium to a final concentration of 50 μg / mL. It was then placed in a 150W ultrasonic cleaner and sonicated for 30 minutes to ensure it was suspended as uniformly as possible for subsequent cell experiments.

[0037] Experiment 1 Solubility test of phospholipid-modified urolithin composition Test method: HPLC: WATERS Alliance E2695, column temperature: 30℃±1℃, detection wavelength: 210 nm, flow rate: 1.0 mL / min, column: C18 250 mm × 4.6 mm × 5 μm, mobile phase A: 0.1% phosphoric acid, mobile phase B: acetonitrile, injection volume: 100 μL, runtime: 32 minutes. Blank solution (diluent): methanol.

[0038] Table 1 Procedure

[0039] Standard curve preparation: Accurately weigh 10 mg of urolithiasis A reference standard (CAS: 1143-70-0) into a 50 mL volumetric flask, dissolve completely in methanol, and dilute to volume to obtain a 200 μg / mL Uro-A reference standard stock solution. Dilute the Uro-A reference standard stock solution sequentially to obtain Uro-A reference standard solutions with concentrations of 100, 50, 25, 12.5, and 6.25 μg / mL, and analyze them using the method described above. Perform linear regression analysis on peak area (A) against concentration (C) to plot the standard curve, obtaining y = 291554x + 506817, R0. 2 =0.9999.

[0040] Solubility test: Take 0.02g of urolithin A raw material and the phospholipid urolithin composition prepared in Examples 1 and 2 respectively, add 2mL of water, vortex and sonicate for 5-10min to aid dissolution, incubate at 37℃ on a shaker for 24h, centrifuge at 12000 r / min for 10min, take an appropriate amount of supernatant and filter through a 0.22μm membrane, take an appropriate amount of supernatant and dilute with 4 times the amount of methanol, and test according to the above method. Substitute the peak area into the above standard curve to calculate the content of urolithin A in the supernatant, which is the solubility.

[0041] The solubility results are shown in the table below.

[0042] Table 2 Solubility Results

[0043] As shown in Table 2, the solubility of free urolithin A is 7.88 μg / mL. The solubility of the phospholipid-modified urolithin compositions prepared in Examples 1 and 2 of this invention is 38.13 μg / mL and 27.50 μg / mL, respectively, proving that the phospholipid-modified urolithin compositions prepared in this invention can significantly improve the solubility of urolithin A.

[0044] Experiment 2 In vitro absorption evaluation of phospholipid-modified urolithin compositions Experimental methods: Establishment of the urolithin A standard curve: Following the method described in the reference (Hu Y, Zhang L, Wei L, et al. Liposomes encapsulation by pH driven improves the stability, bioaccessibility and bioavailability of urolithin A: A comparative study[J]. Internationaljournal of biological macromolecules, 2023, 253: 127554.), the ultraviolet-visible spectrophotometry method was used for testing. Specifically, 10 mg of urolithin A standard was accurately weighed and placed in a 50 mL volumetric flask, completely dissolved in methanol, and then diluted to volume to obtain a urolithin A stock solution with a concentration of 200 μg / mL. This stock solution was then successively diluted to prepare urolithin A standard solutions with concentrations of 50, 25, 12.5, 6.25, and 3.125 μg / mL. The absorbance was measured at 280 nm using an ultraviolet spectrophotometer. The standard regression curve was y = 0.0783x + 0.0572, R0. 2 =0.9993.

[0045] In vitro absorption evaluation method: Validation was performed using an in vitro simulated digestion experiment. The digestive characteristics of the phospholipid-modified urolithiasis composition were studied by simulating gastric and small intestinal digestion. The specific steps are as follows: (1) Gastric digestion: Weigh 2 g NaCl and dissolve it in 500 mL of ultrapure water. Then slowly add 7 mL of concentrated HCl solution and dilute to 1 L with ultrapure water to obtain a simulated gastric digestion solution. Add and dissolve pepsin (3.2 mg / mL, freshly prepared) in the simulated gastric digestion solution to obtain a simulated gastric digestion solution for testing. Add 6 mL of the simulated gastric digestion solution containing dissolved pepsin, preheated to 37 °C, to 0.02 g of sample (urolithin A raw material, phospholipid-modified urolithin composition prepared in Example 1 or Example 2). Adjust the pH to 2.5 and incubate in a shaker at 100 r / min for 2 h.

[0046] (2) Intestinal digestion: After gastric digestion, 12 mL of PBS (10 mmol / L, pH 6.5) preheated to 37°C was added, and the mixture was kept in a 37°C water bath for 10 min. The pH was then adjusted to 7.0 with NaOH solution. Artificial small intestinal digestion solution (containing pancreatic enzyme (24 mg / mL, 2 mL) + bile salts (50 mg / mL, 2.8 mL) + salt solution (a mixed solution of 0.5 mol / L CaCl2 and 7.5 mol / L NaCl, 1.2 mL)) was added to the gastric digestion solution. The mixture was kept in a water bath for 2 h to simulate digestion. During this process, the pH of the digestion solution was maintained at 7.0 with NaOH solution.

[0047] (3) Urolithin release rate: Take 3 mL of sample after simulated in vitro digestion and measure the absorbance at 280 nm to calculate the total urolithin A concentration in the digestion solution. At the same time, take a certain amount of digested sample and centrifuge at 12000 r / min for 10 min. Test the urolithin A content in the supernatant according to the above method. The part that can be dissolved in the digestion solution is considered to be the part that can be absorbed in micelles.

[0048] (4) The bioacceptability of urolithin A is calculated using the formula: Bioacceptability / % = Concentration of urolithin A dissolved in micelles / Total concentration of urolithin A in digestion solution × 100%.

[0049] The results of the bioacceptability are shown in the table below.

[0050] Table 3. Results of Bioacceptability

[0051] As shown in Table 3, the bioacceptability of free urolithin A is 3.08%. The bioacceptability of the phospholipid urolithin compositions prepared in Examples 1 and 2 of this invention is 37.61% and 36.63%, respectively, which proves that the phospholipid urolithin compositions prepared in this invention can significantly improve the absorption rate of urolithin A.

[0052] Experiment 3 Evaluation of anti-fibroblast aging effect Experimental Methods: A cell senescence model was established using human skin fibroblasts (HSF) induced by hydrogen peroxide (H2O2). The following groups were set up: ① Blank control group: Normally cultured human skin fibroblasts without any treatment. ② Model group: Cells senescent after H2O2 induction without drug treatment. ③ Comparative Example 1 group: Model cells + 50 μg / mL Comparative Example 1 urolithin A suspension (based on urolithin A content). ④ Examples 1 and 2 groups: Model cells + 50 μg / mL phospholipid-modified urolithin compositions prepared in Examples 1 and 2 (based on urolithin A content).

[0053] Human skin fibroblasts (HSF) were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibody, and incubated at 37°C with 5% CO2. The cell senescence model was established as follows: HSF cells in logarithmic growth phase were seeded at an appropriate density in culture plates. After cell attachment, the original culture medium was discarded, and fresh medium containing 200 μM H2O2 was added for 2 hours to induce cell senescence. The H2O2-containing medium was then discarded, the cells were washed twice with PBS, and the medium was replaced with normal medium for another 48 hours. Experimental groups included a blank control group, a model group, comparative example 1 (50 μg / mL), and Examples 1 and 2 (50 μg / mL phospholipid-modified urolithin composition). All groups were treated for 48 hours before testing.

[0054] After 48 hours of treatment, cell viability was assessed using the CCK-8 assay. The positivity rate of senescent cells was then determined using a β-galactosidase senescence staining kit. The positivity rate of senescent cells was detected using β-galactosidase (SA-β-gal) staining: cells were fixed using the kit and incubated overnight at 37°C with staining solution, then observed and photographed under a conventional optical microscope. Positive cells appeared as blue-green stains. The positivity rate was calculated as follows: five or more fields of view were randomly selected, and the number of positive cells (blue-green) and the total number of cells were counted. The positivity rate (%) = (number of positive cells / total number of cells) × 100%.

[0055] The results are shown in the table below.

[0056] Table 4. Survival rate and positive rate of senescent fibroblasts

[0057] Note: Compared with the model group, ### is p<0.001; compared with Comparative Example 1, *** is p<0.001.

[0058] The results showed that, compared with the model group, the Example 1 group, Example 2 group, and Comparative Example 1 group all significantly inhibited cell senescence (p<0.01), and the survival rate of senescent fibroblasts in the Example 1 group was higher. The positive rate of senescent cells in the Example 1 group and Example 2 group was significantly lower than that in the Comparative Example 1 group (p<0.01), proving that the phospholipid-modified urolithin composition prepared in this invention can be more effectively taken up by cells and exert a stronger anti-aging effect.

[0059] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0060] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A phospholipid-modified urolithin composition for inhibiting fibroblast growth, characterized in that, It includes the following components in parts by weight: 10-50 parts urolithin, 5-30 parts phospholipids, 1-10 parts polyglycerol fatty acid esters, 1-10 parts monoglycerides, 2-10 parts gum arabic, 2-10 parts trehalose, 10-20 parts sodium octenyl succinate starch, and 0.5-2 parts vitamin E.

2. The phospholipid-modified urolithin composition for inhibiting fibroblast growth as described in claim 1, characterized in that, It includes the following components in parts by weight: 10-20 parts urolithin, 25-30 parts phospholipids, 8-10 parts polyglycerol fatty acid esters, 3-5 parts monoglycerides, 8-10 parts gum arabic, 10 parts trehalose, 15-20 parts sodium octenyl succinate starch, and 1 part vitamin E.

3. The composition according to any one of claims 1 to 2, characterized in that, The urolithin is urolithin A.

4. The composition according to any one of claims 1 to 2, characterized in that, The phospholipids are one or a combination of two of soybean phospholipids, sunflower phospholipids, peanut phospholipids, and corn phospholipids.

5. The composition according to any one of claims 1 to 2, characterized in that, The polyglycerol fatty acid ester is one or a combination of two of polyglycerol-10 laurate, polyglycerol-10 oleate, and sucrose glyceride.

6. The composition according to claim 1, characterized in that, The product comprises the following components in parts by weight: 20 parts urolithin A, 25 parts sunflower phospholipids, 8 parts polyglycerol-10 laurate, 5 parts monoacylglycerol, 10 parts gum arabic, 10 parts trehalose, 20 parts sodium octenyl succinate starch, and 1 part vitamin E.

7. The composition according to claim 1, characterized in that, The product comprises the following components in parts by weight: 10 parts urolithin A, 30 parts soybean lecithin, 10 parts sucrose glycerides, 3 parts monoacylglycerols, 8 parts gum arabic, 10 parts trehalose, 15 parts sodium octenyl succinate starch, and 1 part vitamin E.

8. A method for preparing the composition according to any one of claims 1 to 2, characterized in that, Includes the following steps: (1) Weigh out urolithin, phospholipids, polyglycerol fatty acid esters and monoglycerides according to the formula, add deionized water and process by high-speed shearing to obtain crude emulsion a of phospholipid-modified urolithin composition; (2) Add gum arabic, trehalose, sodium octenyl succinate starch and vitamin E to crude emulsion a of phospholipid composition, and then perform high-speed shearing to obtain crude emulsion b of phospholipid composition; (3) Take the crude emulsion b of the phospholipid urolithin composition and perform high pressure homogenization to obtain the homogenized solution of the phospholipid urolithin composition; (4) After drying the homogenized solution of phospholipid urolithin composition, the phospholipid urolithin composition is obtained.

9. The use of the composition according to any one of claims 1 to 7, or the composition prepared by the method of claim 8, in the preparation of drugs and foods that combat fibroblast aging.

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