A stem cell-targeted oral anti-aging composition and method of making the same

Abstract

The application discloses an oral anti-aging composition based on targeted stem cells and a preparation method thereof, and belongs to the technical field of anti-aging compositions. The oral anti-aging composition based on targeted stem cells comprises three active ingredient groups. The first active ingredient group comprises glycolipid-modified nicotinamide mononucleotide, icariin-II and spermidine. The second active ingredient group comprises mitochondrion-targeted coenzyme Q10, pyrroloquinoline quinone and R-thioctic acid. The third active ingredient group comprises morin, quercetin, astaxanthin and N-acetylcysteine. The oral anti-aging composition based on targeted stem cells is prepared by accurately matching the dosages, glycolipid-modifying nicotinamide mononucleotide and complex matrix, so that the components are mutually enhanced in absorption, distribution, metabolism and action links, multi-level synergistic intervention on stem cell microenvironment, mitochondrial function and aging cell clearance can be realized, physiological age-related functional decline can be effectively delayed, and tissue regeneration capacity can be maintained.

Description

Technical Field

[0001] This invention relates to the field of anti-aging composition technology, specifically to an oral anti-aging composition based on targeted stem cells and its preparation method. Background Technology

[0002] With the accelerating aging of the global population, anti-aging has evolved from a simple concept of antioxidant nutritional supplementation to a multi-target, multi-level systemic intervention targeting core markers of aging such as stem cell depletion, mitochondrial dysfunction, and cellular senescence. Oral anti-aging supplements have become the mainstream consumer form for delaying aging and maintaining tissue regeneration due to their convenience and patient compliance. Active molecules, such as nicotinamide mononucleotide (NMN), senolytic flavonoids (quercetin, rosin), and mitochondrial-targeting antioxidants (MitoQ, PQQ), exhibit significant anti-aging potential through mechanisms such as synergistic activation of the SIRTUIN longevity protein pathway, elimination of senescent cells, and optimization of energy metabolism. However, these multi-component synergistic products face severe biopharmaceutical challenges in actual oral administration: before the active ingredients enter systemic circulation, they encounter multiple barriers of degradation, metabolism, and efflux in the gastrointestinal tract, leading to a common dilemma of high-dose administration and low-efficiency absorption, severely limiting the actual efficacy of the products.

[0003] The core shortcomings of existing oral anti-aging supplements are concentrated in three aspects. First, the stability and absorption efficiency of the key precursor ingredient NMN are extremely low. NMN, as NAD+, has very low absorption efficiency. + Its direct precursor is readily metabolized by CD38 enzyme (NAD28) in the intestinal epithelium and blood after oral administration. + Glycoside hydrolases recognize and degrade these enzymes, breaking their N-glycosidic bonds to produce nicotinamide and phosphoribosyl group, which cannot be effectively converted into NAD. + Meanwhile, free NMN is absorbed via passive diffusion, and its poor permeability to the intestinal epithelium leads to decreased plasma NAD5 levels. +Insufficient efficiency gains force consumers to compensate for losses with extremely high doses (hundreds to thousands of milligrams daily), increasing both costs and gastrointestinal burden. Secondly, there are bottlenecks in the first-pass metabolism and bioavailability of senolytic flavonoids. Quercetin, rosin, and other flavonoid aglycones themselves have poor water solubility and low membrane permeability, resulting in oral bioavailability typically below 5%. More importantly, once in the intestines, they are rapidly metabolized into sulfate esters and glucuronide conjugates by sulfatases and glucuronide transferases within epithelial cells, leading to direct excretion and failing to achieve the effective blood concentrations required to clear senescent cells. Thirdly, there are compatibility and delivery barriers in multi-component synergistic formulations. Traditional tablets, capsules, or simple mixed powders cannot simultaneously accommodate highly polar ingredients (NMN, NAC) and strongly hydrophobic ingredients (MitoQ, astaxanthin). Long-term storage can easily lead to phase separation, crystallization, and uneven content. After entering the stomach, the strong acid environment directly destroys acid-sensitive ingredients, while the P-glycoprotein efflux pump, which is highly expressed on the apical membrane of intestinal epithelial cells, actively pumps the hard-won drug back into the intestinal lumen, forming an ineffective absorption-efflux cycle. This means that even if existing products have scientifically designed formulations, they cannot exert their due synergistic anti-aging effects due to the inherent deficiencies in their delivery systems.

[0004] Most existing technologies are insufficient to meet the practical requirements of multi-component oral anti-aging products. At the chemical modification level, some approaches attempt to protect active ingredients through prodrug strategies such as borate esters, acetals, or sulfates. However, the former suffers from a pH response direction opposite to the actual gastrointestinal environment, while the latter presents problems such as excessively high polarity after protection, hindering absorption, and immediate metabolism after release. Neither approach simultaneously addresses the dual requirements of stability and absorption efficiency. At the formulation technology level, conventional enteric coating only provides a single function of gastric acid isolation and cannot offer a systematic solution to issues such as enzymatic degradation of NMN, active efflux of flavonoids, and compatibility problems in multiphase systems. While ordinary nanoemulsions or liposomes can improve the absorption of a single component, they struggle to stably co-load more than ten different physicochemically active molecules under oral conditions for extended periods. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide an oral anti-aging composition based on targeted stem cells and a method for preparing the same.

[0006] The first aspect of this invention is to provide an oral anti-aging composition based on targeted stem cells, comprising a first group of active ingredients, a second group of active ingredients, and a third group of active ingredients; By mass fractions, the first active ingredient group includes: 245-255 parts of glycolipid-modified nicotinamide mononucleotide, 48-52 parts of icariin-II, and 48-52 parts of spermidine; The second active ingredient group includes: 8-12 parts of mitochondrial-targeted coenzyme Q10, 18-22 parts of pyrroloquinoline quinone, and 195-205 parts of R-lipoic acid; The third group of active ingredients includes: 95-105 parts of quercetin, 248-252 parts of quercetin, 10-15 parts of astaxanthin, and 390-410 parts of N-acetylcysteine. Among them, glycolipid-modified nicotinamide mononucleotide is formed by attaching a glucose-fatty acid amphiphilic group to the nicotinamide mononucleotide.

[0007] It should be noted that the components added in this invention are divided into the following three groups of active ingredients, which have the following effects: First active ingredient group: Nicotinamide mononucleotide (NMN): It can efficiently increase intracellular NAD+ levels, strongly activate the SIRT1-7 family of longevity proteins, improve telomere maintenance, DNA repair and mitochondrial function, and create a "youthful" epigenetic environment for stem cells. Icariin-II: It can specifically activate telomerase (TERT) and promote the Wnt / β-catenin signaling pathway, directly driving stem cell proliferation and self-renewal; Spermine: A natural autophagy inducer that promotes autophagy to clear damaged proteins and organelles, maintains protein homeostasis, and provides a clean internal environment for stem cell function.

[0008] NMN enhances NAD + Levels of this substance fuel SIRT1 activation, which in turn deacetylates and enhances key proteins in the Wnt signaling pathway, thereby synergistically amplifying the activation effect on stem cells with icariin-II. Spermine, on the other hand, ensures that activated stem cells function efficiently by maintaining cell cleanliness.

[0009] Second active ingredient group: MitoQ: A compound in which coenzyme Q10 is linked to a triphenylphosphine cation, which can autonomously cross the membrane and accumulate in the inner mitochondrial membrane, precisely neutralizing mitochondrial ROS with an efficiency hundreds of times that of ordinary coenzyme Q10. Pyrroloquinoline quinone (PQQ): Not only a powerful antioxidant, but also a direct inducer of mitochondrial biosynthesis, stimulating the generation of new, healthy mitochondria; R-lipoic acid (ALA): is a versatile antioxidant that can regenerate other antioxidants in the body (such as glutathione, vitamin C / E), improve insulin sensitivity, and optimize overall energy metabolism.

[0010] MitoQ provides point-to-point mitochondrial protection, PQQ is responsible for expanding a healthy mitochondrial network, and R-ALA provides antioxidant support and metabolic optimization. Together, these three reverse the cellular energy crisis and provide stem cells with abundant ATP.

[0011] Third active ingredient group: Fisetin and quercetin: can selectively induce apoptosis in senescent cells by activating specific apoptosis pathways (such as the BH3 protein-dependent pathway) without harming normal cells; Astaxanthin: One of the strongest natural antioxidants, it can cross the blood-brain barrier and blood-retinal barrier, comprehensively inhibit inflammatory pathways such as NF-κB, and strongly reduce the toxicity of SASP. N-acetylcysteine ​​(NAC): It is used to enhance the body's reserves of the main antioxidant system, and at the same time, it can break down the pro-inflammatory mucus secreted by aging cells, thus purifying the microenvironment from another pathway.

[0012] The combination of quercetin and astaxanthin can remove senescent cells, astaxanthin can extinguish the inflammatory fire (SASP) released by them, and NAC is used to enhance the body's overall resistance. The three work together to systematically purify the microenvironment of the stem cell nest (Niche).

[0013] In some embodiments, glycolipid-modified nicotinamide mononucleotide is prepared by the following steps: (1) Dissolve D-glucose in solvent I, add octanoyl chloride solution dropwise to react, and after the reaction is completed, extract, wash, purify and dry to obtain 6-O-octanoyl-D-glucose; (2) 6-O-octanoyl-D-glucose was dispersed in solvent II, phosphorus oxychloride was added dropwise and stirred, ice water was added dropwise to carry out hydrolysis reaction, then the pH was adjusted to neutralize, and after purification and drying, 6-O-octanoyl-glucose-1-phosphate was obtained. (3) 6-O-octanoyl-glucose-1-phosphate, nicotinamide mononucleotide, dicyclohexylcarbodiimide, 4-dimethylaminopyridine and solvent III were reacted in the dark, and the mixture was purified and dried to obtain glycolipid-modified nicotinamide mononucleotide.

[0014] It should be noted that this invention innovatively uses nicotinamide mononucleotide (NMN) as the core to construct an amphiphilic prodrug molecule with a glycosylated end and a fatty acyl group as the hydrophobic end. First, D-glucose is selectively acylated with octanoyl chloride to obtain 6-O-octanoylglucose (Glc-C8), which is then phosphorylated by phosphorus oxychloride to generate Glc-C8-1-P. Finally, under the catalysis of dicyclohexylcarbodiimide and 4-dimethylaminopyridine, it is dehydrated and coupled with the riboglycolic hydroxyl group of NMN to form a phosphodiester bond. After oral administration, the resulting product is efficiently taken up by the intestinal epithelium via the glucose-fatty acid dual transport pathway, and then hydrolyzed by phosphodiesterase in cells to release proto-NMN, which is finally converted to NAD via NMNAT. + .

[0015] In some embodiments, the octanoyl chloride solution is prepared by mixing octanoyl chloride and solvent IV at a ratio of 1.05-1.12 g: 4.5-5.5 mL; the mass ratio of D-glucose to octanoyl chloride is 0.95-1.05: 1.05-1.13; and the volume ratio of D-glucose to solvent I is 0.8-1.2 g: 23-27 mL.

[0016] In some embodiments, the mass ratio of 6-O-octanoyl-D-glucose to phosphorus oxychloride is 48-52:35-40; the volume ratio of solvent II to 6-O-octanoyl-D-glucose is 8-12 mL:0.48-0.53 g; and the mass of ice water is the same as that of solvent II.

[0017] In some embodiments, the mass ratio of 6-O-octanoyl-glucose-1-phosphate, nicotinamide mononucleotide, dicyclohexylcarbodiimide, and 4-dimethylaminopyridine is 61-67:23-27:28-33:16-20; and the volume ratio of solvent III to 6-O-octanoyl-glucose-1-phosphate is 8 mL:0.62-0.67 g.

[0018] In some embodiments, solvent I, solvent II, and solvent IV are each selected from at least one of anhydrous pyridine and anhydrous dimethylformamide; solvent III is selected from at least one of anhydrous dimethylformamide, anhydrous dimethyl sulfoxide, and anhydrous acetonitrile.

[0019] In some embodiments, in step (2), the stirring is carried out at -8 to -12°C for 50-65 min, and then at 0±2°C for 1.8-2.2 h, and the hydrolysis reaction is carried out at room temperature for 25-35 min; in step (3), the light-protected reaction is carried out at room temperature under nitrogen protection for 23-25 ​​h.

[0020] A second aspect of this invention provides a method for preparing an oral anti-aging composition based on targeted stem cells, comprising the following steps: S1: Mix choline chloride and anhydrous ethanol and react them. Add lactic acid to the mixture to obtain a choline chloride-lactic acid ionic liquid. S2: Mix choline chloride-lactic acid ionic liquid and deionized water I, add surfactant, sodium cholate and vitamin E to obtain a composite matrix; S3: Mix glycolipid-modified nicotinamide mononucleotide and deionized water II, add to the complex matrix and stir, then add mitochondrial-targeted coenzyme Q10, pyrroloquinoline quinone, R-lipoic acid and astaxanthin in sequence, and mix in the dark. S4: Add icariin-II, spermidine, N-acetylcysteine, quercetin, and rosin to the S3 mixture system, and mix to obtain an oral anti-aging composition based on targeted stem cells.

[0021] The surfactant is either Pluronic F127 or Pluronic P12.

[0022] In some embodiments, the ratio of choline chloride, anhydrous ethanol, and lactic acid is 1.3-1.5 g: 1.03-1.1 g: 9-11 mL; the mass ratio of choline chloride-lactic acid ionic liquid, deionized water I, surfactant, sodium cholate, and vitamin E is 6.5-7.2: 0.18-0.22: 1-1.5: 0.3-0.7: 0.28-0.32.

[0023] In some embodiments, the ratio of glycolipid-modified nicotinamide mononucleotide to deionized water II is 5.28-5.34 g: 2.8-3.2 mL; the mass of the composite matrix is ​​125-150% of the sum of the masses of glycolipid-modified nicotinamide mononucleotide, mitochondrial-targeted coenzyme Q10, pyrroloquinoline quinone, R-lipoic acid, astaxanthin, icariin-II, spermidine, N-acetylcysteine, quercetin, and rutin.

[0024] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention prepares an oral anti-aging composition based on targeted stem cells through precise dosage ratios, glycolipid modification of nicotinamide mononucleotide, and complex matrix formulation. This allows the components to enhance each other in absorption, distribution, metabolism, and action, achieving synergistic effects; thus improving the overall efficacy of anti-aging supplements. On the one hand, the core NAD+... + plasma exposure of precursors and intracellular NAD +The efficiency is significantly enhanced, and the first-pass metabolic loss of senolytic flavonoids is greatly reduced, enabling the active ingredients of each functional module to reach effective concentrations in the target tissue. Furthermore, the stable coexistence of multiple components in a homogeneous gel matrix ensures content uniformity during long-term storage, while the gastric protection mechanism reduces degradation loss of active ingredients before absorption. Therefore, this approach enables multi-level synergistic intervention on the stem cell microenvironment, mitochondrial function, and clearance of senescent cells, thereby more effectively delaying age-related functional decline. Through multi-target, multi-level systematic intervention, it delays, prevents, and even partially reverses age-related stem cell functional decline, maintains tissue regeneration capacity, and optimizes physiological age.

[0025] 2. This invention achieves a significant improvement in molecular delivery efficiency by modifying nicotinamide mononucleotide (Glyco-NMN) with glycolipids. Glyco-modified nicotinamide mononucleotide overcomes the shortcomings of free NMN, which is easily degraded by CD38 enzymes and has low passive diffusion absorption efficiency after oral administration. In its molecular structure, the glucose terminus can be recognized by the intestinal epithelial SGLT1 transporter, and the octyl terminus can insert into the cell membrane lipid bilayer and undergo FATP4 / CD36-mediated endocytosis, forming a synergistic absorption mechanism of active transport and membrane fusion diffusion. After entering the cell, the phosphodiester bond is controlled by intracellular phosphodiesterase to release original NMN, which is then converted to NAD through NMNAT. + This avoids the direct attack of N-glycosidic bonds by CD38 in the intestinal lumen and achieves NAD+ activation. + Intracellular sustained release of the precursor fundamentally enhances the conversion of NMN to NAD. + Conversion efficiency.

[0026] 3. This invention also addresses the challenges of homogeneous coexistence of polar and lipid-soluble components in multi-component anti-aging preparations, as well as bioavailability losses due to gastric acid degradation and P-glycoprotein efflux, through a composite matrix. This matrix uses choline chloride-lactic acid ionic liquid as the continuous phase. Through the thermosensitive phase transition properties of surfactants, it transforms from a fluid state to a gel state after oral administration, forming a physical adhesion barrier in the stomach and preventing degradation of acid-sensitive active ingredients by the strong acid environment. Simultaneously, sodium cholate in the matrix constructs a bile-like solubilizing microenvironment, allowing lipid-soluble components such as MitoQ and astaxanthin to be stably dispersed with water-soluble components such as NAC and PQQ in the same homogeneous phase, avoiding phase separation and crystallization during long-term storage. Furthermore, vitamin E TPGS, as a P-glycoprotein inhibitor, can inhibit the active efflux of absorbed drugs back into the intestinal lumen by intestinal epithelial cells, significantly increasing the exposure of active ingredients. Attached Figure Description

[0027] Figure 1 For cellular NAD + Horizontal test results diagram; Figure 2The graph shows the hMSC proliferation rate test results. Figure 3 The graph shows the results of the SA-β-gal positive cell reduction rate test. Figure 4 The graph shows the results of the ATP production test. Figure 5 Image showing the results of mitochondrial ROS level testing; Figure 6 The graph shows the results of the senescent cell clearance rate test. Figure 7 This is a graph showing the results of a normal cell viability test. Figure 8 This is a graph showing the average grip strength test results; Figure 9 The graph shows the results of the exhaustion time test; Figure 10 A diagram showing the results of the water maze escape latency test. Figure 11 The image shows the results of the SA-β-gal positive area test in the liver. Detailed Implementation

[0028] The present invention will now be described in further detail with reference to the accompanying drawings.

[0029] Example 1 An oral anti-aging composition based on targeted stem cells, comprising the following ingredients by weight: First active ingredient group: 250 parts of glycolipid-modified nicotinamide mononucleotide, 50 parts of icariin-II, and 50 parts of spermidine; Second active ingredient group: 10 parts of mitochondrial-targeted coenzyme Q10, 20 parts of pyrroloquinoline quinone, and 200 parts of R-lipoic acid; The third active ingredient group consists of: 100 parts of quercetin, 250 parts of quercetin, 12 parts of astaxanthin, and 400 parts of N-acetylcysteine. The glycolipid-modified nicotinamide mononucleotide was prepared by the following steps: (1) Dissolve D-glucose in anhydrous pyridine, add octanoyl chloride solution dropwise under nitrogen protection and light protection at 0℃, stir at room temperature for 4h, pour the reaction solution into ice-cold ethyl acetate, stir for 5 min, transfer to a separatory funnel, wash 3 times with 1M HCl, then wash 3 times with saturated NaHCO3, transfer to a chromatography column, elute with dichloromethane and methanol (volume ratio 10:1), collect the chromatographic product and dry it under vacuum at 40℃ to constant weight to obtain 6-O-octanoyl-D-glucose; wherein, the octanoyl chloride solution is prepared by mixing octanoyl chloride and anhydrous pyridine at a ratio of 1.08g:5mL; the mass ratio of D-glucose to octanoyl chloride is 1:1.08; the volume ratio of D-glucose to anhydrous pyridine is 1g:25mL; (2) 6-O-octanoyl-D-glucose was dispersed in anhydrous pyridine, phosphorus oxychloride was added dropwise and stirred at -10℃ for 55 min, then stirred at 0±2℃ for 2 h, ice water was added dropwise and stirred at room temperature for 30 min to carry out hydrolysis reaction, then the pH was adjusted for neutralization, the aqueous phase was extracted with ethyl acetate, the aqueous phase was collected, filtered through a 0.22 μm aqueous microporous membrane, and freeze-dried to obtain 6-O-octanoyl-glucose-1-phosphate; wherein, the mass ratio of 6-O-octanoyl-D-glucose to phosphorus oxychloride was 50:38; the mass ratio of anhydrous pyridine to 6-O-octanoyl-D-glucose was 10 mL:0.5 g; the mass of ice water was the same as the mass of anhydrous pyridine; (3) 6-O-octanoyl-glucose-1-phosphate, nicotinamide mononucleotide, dicyclohexylcarbodiimide and 4-dimethylaminopyridine in a mass ratio of 64:25:30:18 were added to anhydrous dimethylformamide and stirred for 24 h under light protection, room temperature and nitrogen protection. The reaction solution was filtered through a 0.45 μm PTFE filter membrane and the filtrate was slowly poured into ice-cold anhydrous diethyl ether with stirring. The flocculent precipitate was collected and washed three times with cold diethyl ether and once with cold methanol. The precipitate was then dissolved in a 1:1 volume ratio of methanol and water. A C18 column was used for elution with water as mobile phase A and methanol as mobile phase B. The elution product was collected and freeze-dried to obtain glycolipid modified nicotinamide mononucleotide. The mass ratio of anhydrous dimethylformamide to 6-O-octanoyl-glucose-1-phosphate was 8 mL:0.65 g.

[0030] The glycolipid-modified nicotinamide mononucleotide (NMN) prepared in this embodiment had a yield of 38% based on the amount of NMN fed into the reactor. HRMS (ESI) - The measured m / z is 701.1726, which is consistent with the theoretical value [MH]. - (C) 25 H 39 N2O 17 P2 (calculated value 701.1724) is highly consistent with the value, with an error of 0.3 ppm. 11H NMR (400 MHz, D2O) showed complete retention of the heterocyclic protons of nicotinamide. H-2 showed a singlet at δ 9.42, H-6 and H-4 showed characteristic coupling at δ 9.15 (d, J = 6.0 Hz) and δ 8.92 (dd, J = 6.0, 1.5 Hz), respectively, and H-5 showed a singlet at δ 8.32. The ribose terminal proton H-1' showed a characteristic doublet at δ 6.12 (J = 5.5 Hz). Compared with the raw NMN, the proton signal in the 2' / 3' region of the ribose was shifted to a lower field by about 0.3–0.5 ppm, suggesting that this site was modified. The glucose anomeric region showed coexistence of α / β isomers, with α-H-1” at δ 5.55 (d, J = 3.5 Hz) and β-H-1” at δ 5.12 (d, J = 7.8 Hz). (Hz), with an integration ratio of approximately 2:3; the triplet of octanoyl COCH2 at δ 2.32 and the triplet of terminal methyl at δ 0.85 confirm the successful introduction of the aliphatic chain. 13 In C NMR (100 MHz, D2O), δ 175.0 is assigned to the octanoyl ester carbonyl group, δ 166.3 to the nicotinamide amide carbonyl group, δ 98.0 and 93.5 in the sugar ring region correspond to the α- and β-anomeric carbons of glucose, respectively, and δ 88.3 to the C-1' of ribose. The HSQC spectrum clearly assigned the one-to-one correspondence between the protons of the sugar ring and the directly attached carbons, and the HMBC spectrum further confirmed the spin coupling networks of glucose and ribose. 31 P NMR (162 MHz, D2O) revealed two characteristic signals: a single peak at δ 4.05 ppm attributed to the original 5'-phosphate monoester of NMN, and a signal at δ -1.52 ppm corresponding to the newly formed phosphodiester bond. The integrated area ratio of the two peaks was approximately 1:1, and the phosphate monoester signal (δ ~0.5 ppm) of the glycolipid-modified nicotinamide mononucleotide had completely disappeared. HPLC (C18 column, water-methanol gradient elution, 260 nm detection) showed a purity of 95.2% and a retention time of approximately 12.8 min, with no detectable NMN or 6-O-octanoyl-glucose-1-phosphate residues.

[0031] The above-mentioned oral anti-aging composition based on targeted stem cells is prepared by the following steps: S1: Mix choline chloride and anhydrous ethanol, stir at 40°C for 10 min, add lactic acid and stir at 40°C for 1 h to obtain choline chloride-lactic acid ionic liquid; wherein, the ratio of choline chloride, anhydrous ethanol and lactic acid is 1.4 g: 1.07 g: 10 mL. The structure of the choline chloride-lactic acid ionic liquid prepared in this embodiment was confirmed by nuclear magnetic resonance spectroscopy: 1 In ¹H NMR (400 MHz, D₂O), the N₂ of choline cations +The (CH3)3 proton appears at δ 3.18 (s, 9H), N + The two sets of methylene protons in CH2CH2OH are located at δ 3.55 (m, 2H) and δ 3.85 (m, 2H), respectively. The signal at δ 3.85 is slightly shifted to a lower field due to its connection with the hydroxyl group and the influence of the strong hydrogen bonding network of the ionic liquid. The chiral proton of the lactate anion is located at δ 4.05 (q, J = 6.8 Hz, 1H), and the methyl proton is located at δ 1.28 (d, J = 6.8 Hz, 3H), with matching coupling constants. No characteristic signals of ethanol residue at δ 1.10 (t) and δ 3.55 (q) were observed in the full spectrum. 13 In C NMR (100 MHz, D2O), the lactate carboxylate carbon signal is located at δ 177.0, the chiral carbon at δ 69.5, and the methyl carbon at δ 21.0; the N-terminus signal of the choline cation is... + The carbon atom in CH2 is located at δ 66.3, the carbon atom in HOCH2 is located at δ 56.1, and N... + The (CH3)3 carbon is located at δ 54.2; the above chemical shift is in perfect agreement with the standard spectrum of the choline-lactic acid ion pair, and no free Cl- was detected in the choline chloride raw material. - Any corresponding impurity signal indicates that the ion exchange reaction is complete.

[0032] S2: Choline chloride-lactic acid ionic liquid and deionized water I were stirred at 40°C for 5 min, and surfactant Pluronic F127, sodium cholate and vitamin E were added and stirred until completely dissolved to obtain a composite matrix; wherein, the mass ratio of choline chloride-lactic acid ionic liquid, deionized water I, surfactant, sodium cholate and vitamin E was 6.8:0.2:1.3:0.5:0.3; S3: Glycolipid-modified nicotinamide mononucleotide (NMN) and deionized water II were ultrasonically dispersed and mixed at a ratio of 5.3 g: 3 mL. The mixture was then added to the composite matrix and stirred at 40 °C for 10 min. Mitochondrial-targeted coenzyme Q10, pyrrolidone, R-lipoic acid, and astaxanthin were added sequentially and mixed in the dark. The mass of the composite matrix was 135% of the sum of the masses of glycolipid-modified nicotinamide mononucleotide, mitochondrial-targeted coenzyme Q10, pyrrolidone, R-lipoic acid, astaxanthin, icariin-II, spermidine, N-acetylcysteine, quercetin, and quercetin. S4: Add icariin-II, spermidine, N-acetylcysteine, quercetin, and rosin to the S3 mixture system, and mix evenly to obtain an oral anti-aging composition based on targeted stem cells.

[0033] Example 2 An oral anti-aging composition based on targeted stem cells, comprising the following ingredients by weight: First active ingredient group: 255 parts of glycolipid-modified nicotinamide mononucleotide, 52 parts of icariin-II, and 52 parts of spermidine; Second active ingredient group: 12 parts of mitochondrial-targeting coenzyme Q10, 22 parts of pyrroloquinoline quinone, and 205 parts of R-lipoic acid; The third active ingredient group consists of: 105 parts of quercetin, 252 parts of quercetin, 15 parts of astaxanthin, and 410 parts of N-acetylcysteine. The glycolipid-modified nicotinamide mononucleotide was prepared by the following steps: (1) D-glucose was dissolved in anhydrous dimethylformamide. Octanoyl chloride solution was added dropwise under nitrogen protection and light protection at 0℃. The mixture was stirred at room temperature for 4 hours. After the reaction was completed, the reaction solution was poured into ice-cold ethyl acetate and stirred for 5 minutes. The solution was then transferred to a separatory funnel, washed three times with 1M HCl, and then washed three times with saturated NaHCO3. The solution was transferred to a chromatography column and eluted with dichloromethane and methanol (volume ratio 10:1). The chromatographic product was collected and dried under vacuum at 40℃ to constant weight to obtain 6-O-octanoyl-D-glucose. The octanoyl chloride solution was prepared by mixing octanoyl chloride and anhydrous dimethylformamide at a ratio of 1.12 g: 5.5 mL. The mass ratio of D-glucose to octanoyl chloride was 1.05:1.13. The molar ratio of D-glucose to anhydrous dimethylformamide was 1.2 g: 27 mL. (2) 6-O-octanoyl-D-glucose was dispersed in anhydrous dimethylformamide, phosphorus oxychloride was added dropwise and stirred at -12℃ for 50 min, then stirred at 0±2℃ for 2.2 h, ice water was added dropwise and stirred at room temperature for 35 min to carry out hydrolysis reaction, then the pH was adjusted for neutralization, the aqueous phase was extracted with ethyl acetate, the aqueous phase was collected, filtered through a 0.22 μm aqueous microporous membrane, and freeze-dried to obtain 6-O-octanoyl-glucose-1-phosphate; wherein, the mass ratio of 6-O-octanoyl-D-glucose to phosphorus oxychloride was 52:40; the mass ratio of anhydrous dimethylformamide to 6-O-octanoyl-D-glucose was 12 mL:0.53 g; the mass of ice water was the same as the mass of anhydrous dimethylformamide; (3) 6-O-octanoyl-glucose-1-phosphate, nicotinamide mononucleotide, dicyclohexylcarbodiimide and 4-dimethylaminopyridine in a mass ratio of 67:27:33:20 were added to anhydrous dimethyl sulfoxide and stirred for 25 h under light protection, room temperature and nitrogen protection. The reaction solution was filtered through a 0.45 μm PTFE filter membrane and the filtrate was slowly poured into ice-cold anhydrous diethyl ether with stirring. The flocculent precipitate was collected and washed three times with cold diethyl ether and once with cold methanol. The precipitate was then dissolved in a 1:1 volume ratio of methanol and water. A C18 column was used for elution with water as mobile phase A and methanol as mobile phase B. The elution product was collected and freeze-dried to obtain glycolipid modified nicotinamide mononucleotide. The mass ratio of anhydrous dimethyl sulfoxide and 6-O-octanoyl-glucose-1-phosphate was 8 mL:0.67 g.

[0034] The glycolipid-modified nicotinamide mononucleotide prepared in this embodiment had a yield of 41% based on the NMN feed amount. HRMS (ES I- The measured m / z is 701.1730, which is consistent with the theoretical value [MH]. - 701.1724 is highly consistent, with an error of 0.9 ppm. 1 The 1H NMR (400MHz, D2O) spectrum characteristics are consistent with those of Example 1. The proton signals of the nicotinamide heterocycle are distributed at δ 9.43 (s, H-2), 9.16 (d, J = 6.0 Hz, H-6), 8.93 (dd, J = 6.0, 1.5 Hz, H-4), and 8.33 (d, J = 1.5 Hz, H-5). The ribose H-1' exhibits a double peak at δ 6.13 (J = 5.5 Hz), and the protons in the 2' / 3' region of the ribose show a low-field shift relative to the starting material NMN. The glucose anomeric protons α-H-1" are at δ 5.56 (d, J = 3.5 Hz), and β-H-1" are at δ 5.14 (d, J = 7.8 Hz). The proton signals of the octanoyl fatty chain are clearly distinguishable. 13 The main signal assignments of the C NMR (100 MHz, D2O) and HSQC spectra are consistent with those in Example 1, and the assignments of the glucose and ribose spin systems in the HMBC spectrum are clear. 31 P NMR (162 MHz, D2O) showed two signals with an area ratio of approximately 1:1 at δ 4.08 and δ -1.50 ppm, corresponding to the 5'-phosphate monoester and the newly formed phosphate diester, respectively. The phosphorus signal from the starting material disappeared, suggesting that DMSO as a coupling solvent is beneficial to the complete reaction. The HPLC purity was 96.0%, the retention time was approximately 12.7 min, and the impurity content was less than 4%.

[0035] The above-mentioned oral anti-aging composition based on targeted stem cells is prepared by the following steps: S1: Mix choline chloride and anhydrous ethanol, stir at 40°C for 10 min, add lactic acid and stir at 40°C for 1 h to obtain choline chloride-lactic acid ionic liquid; wherein, the ratio of choline chloride, anhydrous ethanol and lactic acid is 1.5 g: 1.1 g: 11 mL. S2: Choline chloride-lactic acid ionic liquid and deionized water I were stirred at 40°C for 5 min, and surfactant Pluronic P12, sodium cholate and vitamin E were added and stirred until completely dissolved to obtain a composite matrix; wherein, the mass ratio of choline chloride-lactic acid ionic liquid, deionized water I, surfactant, sodium cholate and vitamin E was 7.2:0.22:1.5:0.7:0.32; S3: Glycolipid-modified nicotinamide mononucleotide (NMN) and deionized water II were ultrasonically dispersed and mixed at a ratio of 5.34 g: 3.2 mL. The mixture was then added to the composite matrix and stirred at 40 °C for 10 min. Mitochondrial-targeted coenzyme Q10, pyrrolidone, R-lipoic acid, and astaxanthin were added sequentially and mixed in the dark. The mass of the composite matrix was 150% of the sum of the masses of glycolipid-modified nicotinamide mononucleotide, mitochondrial-targeted coenzyme Q10, pyrrolidone, R-lipoic acid, astaxanthin, icariin-II, spermidine, N-acetylcysteine, quercetin, and quercetin. S4: Add icariin-II, spermidine, N-acetylcysteine, quercetin, and rosin to the S3 mixture system, and mix evenly to obtain an oral anti-aging composition based on targeted stem cells.

[0036] Example 3 An oral anti-aging composition based on targeted stem cells, comprising the following ingredients by weight: First active ingredient group: 245 parts of glycolipid-modified nicotinamide mononucleotide, 48 parts of icariin-II, and 48 parts of spermidine; Second active ingredient group: Mitochondrial-targeted coenzyme Q10 8 parts, pyrroloquinoline quinone 18 parts, R-lipoic acid 195 parts; The third group of active ingredients: 95 parts of quercetin, 248 parts of quercetin, 10 parts of astaxanthin, and 390 parts of N-acetylcysteine; The glycolipid-modified nicotinamide mononucleotide was prepared by the following steps: (1) Dissolve D-glucose in anhydrous pyridine, add octanoyl chloride solution dropwise under nitrogen protection and light protection at 0℃, stir at room temperature for 4h, pour the reaction solution into ice-cold ethyl acetate, stir for 5 min, transfer to a separatory funnel, wash 3 times with 1M HCl, then wash 3 times with saturated NaHCO3, transfer to a chromatography column, elute with dichloromethane and methanol (volume ratio 10:1), collect the chromatographic product and dry it under vacuum at 40℃ to constant weight to obtain 6-O-octanoyl-D-glucose; wherein, the octanoyl chloride solution is prepared by mixing octanoyl chloride and anhydrous pyridine at a ratio of 1.05g:4.5mL; the mass ratio of D-glucose to octanoyl chloride is 0.95:1.05; the volume ratio of D-glucose to anhydrous pyridine is 0.8g:23mL; (2) 6-O-octanoyl-D-glucose was dispersed in anhydrous pyridine, phosphorus oxychloride was added dropwise and stirred at -8℃ for 65 min, then stirred at 0±2℃ for 1.8 h, ice water was added dropwise and stirred at room temperature for 25 min to carry out hydrolysis reaction, then the pH was adjusted for neutralization, the aqueous phase was extracted with ethyl acetate, the aqueous phase was collected, filtered through a 0.22 μm aqueous microporous membrane, and freeze-dried to obtain 6-O-octanoyl-glucose-1-phosphate; wherein, the mass ratio of 6-O-octanoyl-D-glucose to phosphorus oxychloride was 48:35; the mass ratio of anhydrous pyridine to 6-O-octanoyl-D-glucose was 8 mL:0.48 g; the mass of ice water and the mass of anhydrous pyridine were the same; (3) 6-O-octanoyl-glucose-1-phosphate, nicotinamide mononucleotide, dicyclohexylcarbodiimide and 4-dimethylaminopyridine in a mass ratio of 61:23:28:16 were added to anhydrous acetonitrile and stirred for 23 h under light protection, room temperature and nitrogen protection. The reaction solution was filtered through a 0.45 μm PTFE filter membrane. The filtrate was slowly poured into ice-cold anhydrous diethyl ether with stirring. The flocculent precipitate was collected and washed three times with cold diethyl ether and once with cold methanol. The precipitate was then dissolved in a 1:1 volume ratio of methanol and water. A C18 column was used for elution with water as mobile phase A and methanol as mobile phase B. The elution product was collected and freeze-dried to obtain glycolipid modified nicotinamide mononucleotide. The mass ratio of anhydrous acetonitrile to 6-O-octanoyl-glucose-1-phosphate was 8 mL:0.62 g.

[0037] The glycolipid-modified nicotinamide mononucleotide prepared in this embodiment had a yield of 33% based on the NMN feed amount. HRMS (ESI) - The measured m / z is 701.1721, which is consistent with the theoretical value [MH]. - 701.1724 is consistent, with an error of 0.4 ppm. 1¹H NMR (400 MHz, D₂O) showed proton signals of the nicotinamide heterocycle at δ 9.41 (s, H⁻²), 9.14 (d, J = 6.0 Hz, H⁻⁶), 8.91 (dd, J = 6.0, 1.5 Hz, H⁻⁴), and 8.31 (d, J = 1.5 Hz, H⁻⁵). The ribose H⁻¹' showed a double peak at δ 6.11 (J = 5.5 Hz), the glucose anomeric region α-H⁻¹” at δ 5.53 (d, J = 3.5 Hz), and β-H⁻¹” at δ 5.10 (d, J = 7.8 Hz). The octanoyl chain proton signal was intact. However, due to the slightly poor solubility of acetonitrile in the intermediate, a trace amount of unreacted 6-O-octanoyl-glucose-1-phosphate anomeric proton signal (around δ 5.35) was visible in the spectrum. 13 The main signal attributions of the C NMR (100 MHz, D2O) and HSQC spectra are consistent with those in Example 1. 31 The main P NMR (162 MHz, D2O) signals were located at δ 4.02 and δ -1.55 ppm, corresponding to the two phosphorus environments of the target product. However, a trace amount of residual phosphate monoester signal (area ratio <3%) was observed at δ 0.6 ppm, indicating that the coupling reaction was incomplete. The HPLC purity was 92.5%, and the retention time was approximately 12.9 min. A small amount of residual peaks from the starting material were detected, consistent with the low yield.

[0038] The above-mentioned oral anti-aging composition based on targeted stem cells is prepared by the following steps: S1: Mix choline chloride and anhydrous ethanol, stir at 40°C for 10 min, add lactic acid and stir at 40°C for 1 h to obtain choline chloride-lactic acid ionic liquid; wherein, the ratio of choline chloride, anhydrous ethanol and lactic acid is 1.3 g: 1.03 g: 9 mL. S2: Choline chloride-lactic acid ionic liquid and deionized water I were stirred at 40°C for 5 min, and surfactant Pluronic F127, sodium cholate and vitamin E were added and stirred until completely dissolved to obtain a composite matrix; wherein, the mass ratio of choline chloride-lactic acid ionic liquid, deionized water I, surfactant, sodium cholate and vitamin E was 6.5:0.18:10.3:0.28; S3: Glycolipid-modified nicotinamide mononucleotide (NMN) and deionized water II were ultrasonically dispersed and mixed at a ratio of 5.28 g: 2.8 mL. The mixture was then added to the composite matrix and stirred at 40 °C for 10 min. Mitochondrial-targeted coenzyme Q10, pyrrolidone, R-lipoic acid, and astaxanthin were added sequentially and mixed in the dark. The mass of the composite matrix was 125% of the sum of the masses of glycolipid-modified nicotinamide mononucleotide, mitochondrial-targeted coenzyme Q10, pyrrolidone, R-lipoic acid, astaxanthin, icariin-II, spermidine, N-acetylcysteine, quercetin, and quercetin. S4: Add icariin-II, spermidine, N-acetylcysteine, quercetin, and rosin to the S3 mixture system, and mix evenly to obtain an oral anti-aging composition based on targeted stem cells.

[0039] Example 4 An oral anti-aging composition based on targeted stem cells, comprising the following ingredients by weight: First active ingredient group: 248 parts of glycolipid-modified nicotinamide mononucleotide, 49 parts of icariin-II, and 49 parts of spermidine; Second active ingredient group: 9 parts of mitochondrial-targeted coenzyme Q10, 19 parts of pyrroloquinoline quinone, and 198 parts of R-lipoic acid; The third group of active ingredients: 98 parts of quercetin, 249 parts of quercetin, 11 parts of astaxanthin, and 393 parts of N-acetylcysteine; The glycolipid-modified nicotinamide mononucleotide was prepared by the following steps: (1) D-glucose was dissolved in anhydrous dimethylformamide. Octanoyl chloride solution was added dropwise under nitrogen protection and light protection at 0℃. The mixture was stirred at room temperature for 4 hours. After the reaction was completed, the reaction solution was poured into ice-cold ethyl acetate and stirred for 5 minutes. The solution was then transferred to a separatory funnel, washed three times with 1M HCl, and then washed three times with saturated NaHCO3. The solution was transferred to a chromatography column and eluted with dichloromethane and methanol (volume ratio 10:1). The chromatographic product was collected and dried under vacuum at 40℃ to constant weight to obtain 6-O-octanoyl-D-glucose. The octanoyl chloride solution was prepared by mixing octanoyl chloride and anhydrous dimethylformamide at a ratio of 1.07 g: 4.8 mL. The mass ratio of D-glucose to octanoyl chloride was 0.98:1.08. The molar ratio of D-glucose to anhydrous dimethylformamide was 0.9 g: 24 mL. (2) 6-O-octanoyl-D-glucose was dispersed in anhydrous dimethylformamide, phosphorus oxychloride was added dropwise and stirred at -9℃ for 55 min, then stirred at 0±2℃ for 1.9 h, ice water was added dropwise and stirred at room temperature for 30 min to carry out hydrolysis reaction, then the pH was adjusted for neutralization, the aqueous phase was extracted with ethyl acetate, the aqueous phase was collected, filtered through a 0.22 μm aqueous microporous membrane, and freeze-dried to obtain 6-O-octanoyl-glucose-1-phosphate; wherein, the mass ratio of 6-O-octanoyl-D-glucose to phosphorus oxychloride was 49:36; the mass ratio of anhydrous dimethylformamide to 6-O-octanoyl-D-glucose was 9 mL:0.49 g; the mass of ice water was the same as the mass of anhydrous dimethylformamide; (3) 6-O-octanoyl-glucose-1-phosphate, nicotinamide mononucleotide, dicyclohexylcarbodiimide and 4-dimethylaminopyridine in a mass ratio of 62:24:29:17 were added to anhydrous dimethylformamide and stirred for 24 h under light protection, room temperature and nitrogen protection. The reaction solution was filtered through a 0.45 μm PTFE filter membrane and the filtrate was slowly poured into ice-cold anhydrous diethyl ether with stirring. The flocculent precipitate was collected and washed three times with cold diethyl ether and once with cold methanol. The precipitate was then dissolved in a 1:1 volume ratio of methanol and water. A C18 column was used for elution with water as mobile phase A and methanol as mobile phase B. The elution product was collected and freeze-dried to obtain glycolipid modified nicotinamide mononucleotide. The mass ratio of anhydrous dimethylformamide to 6-O-octanoyl-glucose-1-phosphate was 8 mL:0.63 g.

[0040] The glycolipid-modified nicotinamide mononucleotide prepared in this embodiment had a yield of 39% based on the NMN feed amount. HRMS (ESI) - The measured m / z is 701.1728, which is consistent with the theoretical value [MH]. - The error for 701.1724 is 0.6 ppm, and the molecular weight has been confirmed to be correct. 1 In 1H NMR (400 MHz, D2O), the proton signals of the nicotinamide heterocycle were located at δ 9.42 (s, H-2), 9.15 (d, J = 6.0 Hz, H-6), 8.92 (dd, J = 6.0, 1.5 Hz, H-4), and 8.32 (d, J = 1.5 Hz, H-5), respectively. The ribose H-1' region showed a doublet at δ 6.12 (J = 5.5 Hz), with the ribose 2' / 3' region protons exhibiting a low-field shift relative to the starting material. The α / β isomer signals in the glucose anomeric region were clear, with α-H-1” at δ 5.54 (d, J = 3.5 Hz) and β-H-1” at δ 5.13 (d, J = 7.8 Hz), with an integration ratio of approximately 2:3. The octanoyl COCH2 group showed a triplet at δ 2.33, and the terminal methyl group showed a doublet at δ 1.5 Hz. There is a triple peak at 0.86. 13 C NMR (100 MHz, D2O) and HSQC / HMBC two-dimensional spectra showed clear assignment of carbon signals in the sugar rings, and clear spin networks of glucose and ribose in HMBC. 31 PNMR (162 MHz, D2O) showed two signals with an area ratio of approximately 1:1 at δ 4.06 and δ -1.51 ppm. No signals from the starting material or pyrophosphate byproduct were detected. The HPLC purity was 95.8%, the retention time was approximately 12.8 min, and the sum of the peak areas of all impurities was less than 4.2%.

[0041] The preparation steps of the oral anti-aging composition based on targeted stem cells described above are the same as those in Example 1.

[0042] Example 5 An oral anti-aging composition based on targeted stem cells, comprising the following ingredients by weight: First active ingredient group: 252 parts of glycolipid-modified nicotinamide mononucleotide, 51 parts of icariin-II, and 51 parts of spermidine; Second active ingredient group: 11 parts of mitochondrial-targeting coenzyme Q10, 21 parts of pyrroloquinoline quinone, and 203 parts of R-lipoic acid; The third group of active ingredients: 103 parts of quercetin, 251 parts of astaxanthin, 14 parts of astaxanthin, and 408 parts of N-acetylcysteine; The glycolipid-modified nicotinamide mononucleotide was prepared by the following steps: (1) Dissolve D-glucose in anhydrous pyridine, add octanoyl chloride solution dropwise under nitrogen protection and light protection at 0℃, stir at room temperature for 4h, pour the reaction solution into ice-cold ethyl acetate, stir for 5 min, transfer to a separatory funnel, wash 3 times with 1M HCl, then wash 3 times with saturated NaHCO3, transfer to a chromatography column, elute with dichloromethane and methanol (volume ratio 10:1), collect the chromatographic product and dry it under vacuum at 40℃ to constant weight to obtain 6-O-octanoyl-D-glucose; wherein, the octanoyl chloride solution is prepared by mixing octanoyl chloride and anhydrous pyridine at a ratio of 1.11g:4.9mL; the mass ratio of D-glucose to octanoyl chloride is 1.03:1.12; the volume ratio of D-glucose to anhydrous pyridine is 1.1g:26mL; (2) 6-O-octanoyl-D-glucose was dispersed in anhydrous dimethylformamide, phosphorus oxychloride was added dropwise and stirred at -11℃ for 62 min, then stirred at 0±2℃ for 2.1 h, ice water was added dropwise and stirred at room temperature for 32 min to carry out hydrolysis reaction, then the pH was adjusted for neutralization, the aqueous phase was extracted with ethyl acetate, the aqueous phase was collected, filtered through a 0.22 μm aqueous microporous membrane, and freeze-dried to obtain 6-O-octanoyl-glucose-1-phosphate; wherein, the mass ratio of 6-O-octanoyl-D-glucose to phosphorus oxychloride was 51:38; the mass ratio of anhydrous dimethylformamide to 6-O-octanoyl-D-glucose was 11 mL:0.52 g; the mass of ice water was the same as the mass of anhydrous dimethylformamide; (3) 6-O-octanoyl-glucose-1-phosphate, nicotinamide mononucleotide, dicyclohexylcarbodiimide and 4-dimethylaminopyridine in a mass ratio of 66:25:32:19 were added to anhydrous dimethyl sulfoxide and stirred for 25 h under light protection, room temperature and nitrogen protection. The reaction solution was filtered through a 0.45 μm PTFE filter membrane and the filtrate was slowly poured into ice-cold anhydrous diethyl ether with stirring. The flocculent precipitate was collected and washed three times with cold diethyl ether and once with cold methanol. The precipitate was then dissolved in a 1:1 volume ratio of methanol and water. A C18 column was used for elution with mobile phase A being water and mobile phase B being methanol. The elution product was collected and freeze-dried to obtain glycolipid modified nicotinamide mononucleotide. The mass ratio of anhydrous dimethyl sulfoxide and 6-O-octanoyl-glucose-1-phosphate was 8 mL:0.66 g.

[0043] The glycolipid-modified nicotinamide mononucleotide prepared in this embodiment had a yield of 42% based on the NMN feed amount. HRMS (ESI) - The measured m / z is 701.1731, which is consistent with the theoretical value [MH]. - 701.1724 is highly consistent, with an error of 1.0 ppm. 1 1H NMR (400MHz, D2O) spectra showed that the proton signals of the nicotinamide heterocycle were distributed at δ 9.44 (s, H-2), 9.17 (d, J = 6.0 Hz, H-6), 8.94 (dd, J = 6.0, 1.5 Hz, H-4), and 8.34 (d, J = 1.5 Hz, H-5). The ribose H-1' showed a double peak at δ 6.14 (J = 5.5 Hz), and the low-field shift of the protons in the 2' / 3' region of the ribose was obvious. The glucose anomeric protons α-H-1” were at δ 5.57 (d, J = 3.5 Hz), and β-H-1” were at δ 5.15 (d, J = 7.8 Hz). The octanoyl chain proton signal was intact and the signal-to-noise ratio was good. 13 CNMR (100 MHz, D2O) and HSQC / HMBC two-dimensional spectroscopy further confirmed the molecular skeleton, and the assignment of the glucose and ribose spin system in the HMBC spectrum was sharp and clear. 31 P NMR (162 MHz, D2O) showed two signals with an area ratio of approximately 1:1 at δ 4.10 and δ -1.49 ppm, corresponding to 5'-phosphate monoester and newly formed phosphate diester, respectively. No signals from the starting material or byproducts were detected, suggesting high coupling efficiency in the DMSO solvent system. The HPLC purity was 96.5%, and the retention time was approximately 12.6 min.

[0044] The preparation steps of the oral anti-aging composition based on targeted stem cells described above are the same as those in Example 1.

[0045] Experiment 1 NAD+ Level enhancement and stem cell vitality assessment 1. NAD + Content detection Experimental objective: To verify the effect of the complete formula of the first active ingredient group on NAD3 in stem cells. + The effect of improving skill level.

[0046] Groups: blank control group, NMN single group (Glyco-NMN only), icariin-II / spermidine group (icariin-II and spermidine only), first active ingredient group full formula group (Glyco-NMN, icariin-II and spermidine only), the dosage is the same as in Example 1; Cell model: Human mesenchymal stem cells (hMSCs), young generation hMSCs (P5) for NAD + Content determination experiment; Detection indicator: NAD + / NADH Detection Kit (Beyotime, #S0175) for NAD Measurement + Content. Experimental results are shown in Table 1 and... Figure 1 .

[0047] Table 1

[0048] Statistical Notes: Data are expressed as mean ± standard deviation, with n=6 for each group. One-way ANOVA was used in conjunction with... Tukey's multiple comparison test.

[0049] Compared with the blank control group, p<0.05, p<0.01, p<0.001.

[0050] #: Compared with all single-component intervention groups (NMN single group, icariin-II / spermidine group), p<0.05.

[0051] From Table 1 and Figure 1 As can be seen, the NMN single-component group, the icariin-II / spermine group, and the full formulation of the first active ingredient group can all enhance NAD in hMSCs (P5) cells. + The content of NMN was most significantly increased in the full-formula group (P<0.001), demonstrating that NMN can effectively enhance NAD+ levels. + At the level, the three active ingredients work synergistically to enhance NAD+ levels. + The level has been further improved.

[0052] 2. Functional assessment of human mesenchymal stem cells (hMSCs) Experimental objective: To verify the promoting effect of the complete formula of the first active ingredient group on stem cell proliferation. Groups: blank control group, NMN single group (only Glyco-NMN added), icariin-II / spermidine group (only icariin-II and spermidine added), and first active ingredient group full formula group (only Glyco-NMN, icariin-II and spermidine added), with the same dosage as in Example 1.

[0053] Cell model: human mesenchymal stem cells (hMSCs), young generation hMSCs (P5) were used for proliferation experiments.

[0054] Detection indicators: Cell viability (proliferation) was measured using the CCK-8 assay, and the proportion of senescent cells was measured using SA-β-gal staining. Results are shown in Table 2 and... Figure 2 , 3 .

[0055] Table 2

[0056] Statistical Notes: Data are expressed as mean ± standard deviation, with n=6 for each group. One-way ANOVA was used in conjunction with... Tukey's multiple comparison test.

[0057] Compared with the blank control group, p<0.05, p<0.01, p<0.001.

[0058] #: Compared with all single-component intervention groups (NMN single group, icariin-II / spermidine group), p<0.05.

[0059] From Table 2 and Figure 2 , 3 As can be seen, the data of the first active ingredient group of the whole formula group met or exceeded the standards set by the protocol: "the cell proliferation rate should be increased by ≥30%" and "the proportion of SA-β-gal positive cells should be reduced by ≥40%".

[0060] Experiment 2: Evaluation of Mitochondrial Function Optimization Experimental objective: To verify the product's effect on improving mitochondrial energy metabolism and oxidative stress.

[0061] Groups: blank control group, oxidative stress model group (hydrogen peroxide treatment), MitoQ single group (only mitochondrial-targeted coenzyme Q10 added), second active ingredient group full formula group (only mitochondrial-targeted coenzyme Q10, pyrroloquinoline quinone and R-lipoic acid added), the dosage of each component is the same as in Example 1.

[0062] Cell model: human mesenchymal stem cells (hMSCs).

[0063] Detection indicators: ATP content detection, MitoSOX TM Fluorescent probes were used to detect mitochondrial superoxide; the results are shown in Table 3 and... Figure 4 , 5 .

[0064] Table 3

[0065] Statistical Notes: Data are expressed as mean ± standard deviation, with n=6 for each group. One-way ANOVA was used in conjunction with... Tukey's multiple comparison test.

[0066] Compared with the oxidative stress model group, p<0.05, p<0.01, p<0.001.

[0067] #: Compared with MitoQ single-group comparison, p<0.05.

[0068] From Table 3 and Figure 4 , 5 As can be seen, the full formulation of the second active ingredient group significantly reversed the decrease in ATP caused by oxidative stress, increasing it by 28.7% (target ≥25%), and effectively reduced mitochondrial ROS production by 38.4% (target ≥35%). These data are consistent with the expected goals for mitochondrial optimization and energy metabolism. The full formulation is superior to the single MitoQ component, demonstrating the synergistic effect of PQQ and R-ALA.

[0069] Experiment 3 Senolytic activity verification Experimental objective: To verify the ability of the third active ingredient group formula to eliminate senescent cells. Groups: blank control group, quercetin / quercetin group (only quercetin and quercetin added), and third active ingredient group full formula group (only quercetin, quercetin, astaxanthin and N-acetylcysteine ​​added). The dosage of each component is consistent with the example.

[0070] Cell model: Human mesenchymal stem cells (hMSCs). Young generation hMSCs (P5) were senescent-induced by irradiation (10 Gy) for clearance experiments.

[0071] Detection indicators: Flow cytometry was used to measure apoptosis (to assess selectivity for senescent cells). Experimental results are shown in Table 4 and... Figure 6 , 7 .

[0072] Table 4

[0073] Statistical Notes: Data are expressed as mean ± standard deviation, with n=6 for each group. One-way ANOVA was used in conjunction with... Tukey's multiple comparison test.

[0074] Compared with the blank control group, p<0.05, p<0.01, p<0.001.

[0075] #: Compared with the quercetin / quercetin group, p<0.05.

[0076] From Table 4 and Figure 6 , 7The third active ingredient group demonstrates highly efficient selective senescent cell scavenging (Senolytic activity) with minimal impact on normal cells, validating the synergistic effect of senescent cell scavenging and microenvironment purification.

[0077] Experiment 4: Validation of the efficacy of the natural aging mouse model Experimental objective: To verify the anti-aging physiological function of the product at the whole animal level.

[0078] Experimental Design Animals and grouping: 24-month-old naturally aging C57BL / 6J male mice were divided into an aging control group, a low-dose product group, a medium-dose product group, and a high-dose product group (3 times the low dose). A 6-month-old young control group was also included. The product was the anti-aging composition prepared in Example 1. Each group had n=15 mice, and the intervention lasted for 3 months.

[0079] Test indicators: muscle grip strength, treadmill endurance, Morris water maze cognitive test, and tissue SA-β-gal staining. Results are shown in Table 5. Figure 8 , 9 10, 11.

[0080] Table 5

[0081] Statistical Notes: Data are expressed as mean ± standard deviation, with n=15 individuals per group. One-way ANOVA was used. Combined with Tukey's multiple comparison test.

[0082] Compared with the aging control group, p<0.05, p<0.01, p<0.001.

[0083] From Table 5 and Figure 8-11 As can be seen, medium-dose intervention significantly improved the muscle grip strength of aging mice by 32.4% and endurance by 41.4%, and improved their spatial learning and memory abilities, bringing their water maze performance close to that of the young control group (anti-aging). Histological analysis showed that the accumulation of senescent cells in the liver was reduced by 52% in the medium-dose group, which was significantly better than that in the aging control group (P<0.001). This is consistent with the in vitro senolytic activity data and achieved the target of reducing the accumulation of senescent cells in the preclinical efficacy criteria. Moreover, the high-dose group was more effective than the medium-dose group.

[0084] Experiment 5 Oral Absorption of Glycolipid-Modified NMN (Glyco-NMN) and NAD + Improvement effect verification Experimental objective: To verify, at the whole animal level, the oral absorption efficiency, plasma exposure, and tissue NAD ratio of Glyco-NMN compared to free NMN. + The efficacy was enhanced, and its resistance to CD38 degradation was verified in vitro.

[0085] In vitro component (CD38 resistance): Reaction system: Recombinant human CD38 (100 ng / mL) + NAD + Detection substrate system Groups: Blank control, free NMN group (Glyco-NMN in the oral anti-aging composition of Example 1 was replaced with the same amount of unmodified NMN), and Glyco-NMN group (oral anti-aging composition of Example 1). Conditions: 37℃, pH 7.4, incubation times: 0, 30, 60, 120 min Detection indicators: The concentration of residual NMN equivalent was determined by HPLC, and the results are shown in Table 6. In vivo components (pharmacokinetics and tissue NAD) + ): Animals and grouping: C57BL / 6J male mice, 8 weeks old, weighing 20-22 g; n=10 per group, single gavage administration; blood collection time points: 0, 0.5, 1, 2, 4, 6, 8 h (orbital blood collection, heparin anticoagulation); endpoint tissue collection: 5 mice were sacrificed 4 h after administration (near Cmax) for liver, skeletal muscle, and brain tissue; the remaining 5 mice were sacrificed 24 h later for tissue collection.

[0086] Detection indicators: Plasma NMN concentration: LC-MS / MS (MRM mode); Tissue NAD5 concentration. + / NADH level: NAD + / NADH assay kit (Beyotime, #S0175), test results are shown in Table 7; key pharmacokinetic parameters: Cmax, Tmax, AUC 0-8 h (calculated using the trapezoidal method), and the test results are shown in Table 6.

[0087] Table 6

[0088] Table 7

[0089] Statistical explanation: Data are expressed as mean ± standard deviation. In vitro experiments n=6; in vivo pharmacokinetics n=10; tissue biochemistry n =5. One-way ANOVA combined with Tukey's multiple comparison test was used. Compared with the blank control group, p < 0.05， p<0.01， p<0.001. #: Compared with the free NMN group, p<0.05.

[0090] As shown in Tables 6 and 7, Glyco-NMN exhibits significant resistance to CD38 degradation in vitro (88.5% retention rate at 120 min vs. 42.0% for free NMN); in vivo pharmacokinetics show a 3.4-fold increase in Cmax and AUC. 0-8 The h-day rate increased 4.4 times, and the Tmax was delayed to 1 hour, indicating sustained-release characteristics; NAD3 levels in liver, skeletal muscle, and brain tissue were also observed. + The levels were increased by 68%, 55%, and 42%, respectively, significantly better than the same dose of free NMN. This verifies the substantial enhancement of oral NMN delivery efficiency by the glycolipid modification strategy.

[0091] Experiment 6: Verification of the delivery efficiency and gastric protective effect of the composite matrix Experimental objective: To verify the advantages of composite matrix compared to ordinary oral preparations in terms of gastric acid protection, synergistic absorption of multiple components, and bioavailability of active ingredients.

[0092] In vitro component (gastric protection simulation): Grouping: Free mixed powder group (active ingredients physically mixed, administered by gavage, basically the same as in Example 1, except that no composite matrix is ​​used); Ordinary aqueous solution / suspension group (basically the same as in Example 1, except that the active ingredients are dispersed in 0.5% CMC-Na aqueous solution); Composite matrix group (complete formulation of Example 1).

[0093] Model: Simulated gastric juice (SGF, pH 1.5, containing pepsin) was incubated at 37°C for 2 h.

[0094] Detection: The residual contents of quercetin, MitoQ, PQQ and NAC before and after incubation were determined by HPLC. The results are shown in Table 8.

[0095] In vivo components (pharmacokinetics and tissue distribution of multiple components): Animals and grouping: C57BL / 6J male mice, 8 weeks old, weighing 20-22 g.

[0096] Each group had n=15, and the drug was administered by gavage once. The detection time points were: 2 hours after administration (end of gastric emptying), 4 hours (peak of small intestinal absorption), and 8 hours after administration. Blood collection and tissue sampling: blood was collected from the orbital cavity; 5 animals were sacrificed at 2 hours to collect stomach contents and gastric mucosa, 5 animals were sacrificed at 4 hours to collect plasma and liver, and 5 animals were sacrificed at 8 hours to collect plasma.

[0097] Detection indicators: residual rate of active ingredients in gastric contents (HPLC); plasma concentrations of quercetin, MitoQ, PQQ, and NAC (LC-MS / MS), the results are shown in Table 9; liver concentrations of quercetin and MitoQ (LC-MS / MS after tissue homogenization), the results are shown in Table 10.

[0098] Table 8

[0099] Table 9

[0100] Table 10

[0101] Statistical explanation: Data are expressed as mean ± standard deviation. In vitro experiments n=6; in vivo pharmacokinetics n=15; tissue concentration n =5. One-way ANOVA combined with Tukey's multiple comparison test was used.

[0102] Compared with the blank control group, p<0.05, p<0.01， p<0.001。

[0103] #: Compared with the free mixed powder group, p<0.05.

[0104] As shown in Tables 8-10, the retention rates of all active ingredients in the composite matrix group in simulated gastric fluid were >88%, which was significantly better than that of the free powder group (35-52%). The relative bioavailability of plasma was increased by 2.2-2.9 times, and the concentration in liver tissue was increased by 3.5-4.3 times, which verified the isolation effect of the composite matrix on gastric acid and the inhibitory effect of TPGS on P-gp efflux.

[0105] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. An oral anti-aging composition based on targeted stem cells, characterized in that, It includes a first group of active ingredients, a second group of active ingredients, and a third group of active ingredients; By mass fractions, the first active ingredient group includes: 245-255 parts of glycolipid-modified nicotinamide mononucleotide, 48-52 parts of icariin-II, and 48-52 parts of spermidine; The second active ingredient group includes: 8-12 parts of mitochondrial-targeted coenzyme Q10, 18-22 parts of pyrroloquinoline quinone, and 195-205 parts of R-lipoic acid; The third group of active ingredients includes: 95-105 parts of quercetin, 248-252 parts of quercetin, 10-15 parts of astaxanthin, and 390-410 parts of N-acetylcysteine. The glycolipid-modified nicotinamide mononucleotide is a nicotinamide mononucleotide with a glucose-fatty acid amphiphilic group attached to it.

2. The oral anti-aging composition based on targeted stem cells according to claim 1, characterized in that, The glycolipid-modified nicotinamide mononucleotide was prepared by the following steps: (1) Dissolve D-glucose in solvent I, add octanoyl chloride solution dropwise to react, and after the reaction is completed, extract, wash, purify and dry to obtain 6-O-octanoyl-D-glucose; (2) The 6-O-octanoyl-D-glucose was dispersed in solvent II, phosphorus oxychloride was added dropwise and stirred, ice water was added dropwise to carry out hydrolysis reaction, then the pH was adjusted to neutralize, and after purification and drying, 6-O-octanoyl-glucose-1-phosphate was obtained; (3) The 6-O-octanoyl-glucose-1-phosphate, nicotinamide mononucleotide, dicyclohexylcarbodiimide, 4-dimethylaminopyridine and solvent III were reacted in the dark, and the mixture was purified and dried to obtain the glycolipid-modified nicotinamide mononucleotide.

3. The oral anti-aging composition based on targeted stem cells according to claim 2, characterized in that, The octanoyl chloride solution is prepared by mixing octanoyl chloride and solvent IV at a ratio of 1.05-1.12 g: 4.5-5.5 mL; the mass ratio of D-glucose to octanoyl chloride is 0.95-1.05: 1.05-1.13; and the volume ratio of D-glucose to solvent I is 0.8-1.2 g: 23-27 mL.

4. The oral anti-aging composition based on targeted stem cells according to claim 2, characterized in that, The mass ratio of 6-O-octanoyl-D-glucose to phosphorus oxychloride is 48-52:35-40; the volume ratio of solvent II to 6-O-octanoyl-D-glucose is 8-12 mL:0.48-0.53 g; the mass of ice water is the same as the mass of solvent II.

5. The oral anti-aging composition based on targeted stem cells according to claim 2, characterized in that, The mass ratio of 6-O-octanoyl-glucose-1-phosphate, nicotinamide mononucleotide, dicyclohexylcarbodiimide, and 4-dimethylaminopyridine is 61-67:23-27:28-33:16-20; the volume ratio of solvent III to 6-O-octanoyl-glucose-1-phosphate is 8 mL:0.62-0.67 g.

6. The oral anti-aging composition based on targeted stem cells according to claim 3, characterized in that, Solvent I, solvent II and solvent IV are each selected from at least one of anhydrous pyridine and anhydrous dimethylformamide; solvent III is selected from at least one of anhydrous dimethylformamide, anhydrous dimethyl sulfoxide and anhydrous acetonitrile.

7. The oral anti-aging composition based on targeted stem cells according to claim 2, characterized in that, In step (2), the stirring is carried out at -8 to -12℃ for 50-65 min, and then at 0±2℃ for 1.8-2.2 h. The hydrolysis reaction is carried out at room temperature for 25-35 min. In step (3), the light-protected reaction is carried out at room temperature under nitrogen protection for 23-25 ​​h.

8. A method for preparing an oral anti-aging composition based on targeted stem cells according to any one of claims 1-7, characterized in that, Includes the following steps: S1: Mix choline chloride and anhydrous ethanol and react them. Add lactic acid to the mixture to obtain a choline chloride-lactic acid ionic liquid. S2: Mix the choline chloride-lactic acid ionic liquid and deionized water I, and add surfactant, sodium cholate and vitamin E to obtain a composite matrix; S3: Mix glycolipid-modified nicotinamide mononucleotide and deionized water II, add to the composite matrix and stir, then add mitochondrial-targeted coenzyme Q10, pyrroloquinoline quinone, R-lipoic acid and astaxanthin in sequence, and mix in the dark; S4: Add icariin-II, spermidine, N-acetylcysteine, quercetin, and rosin to the S3 mixture system, and mix to obtain the oral anti-aging composition based on targeted stem cells.

9. The method for preparing the oral anti-aging composition based on targeted stem cells according to claim 8, characterized in that, The ratio of choline chloride, anhydrous ethanol, and lactic acid is 1.3-1.5 g: 1.03-1.1 g: 9-11 mL; the mass ratio of choline chloride-lactic acid ionic liquid, deionized water I, surfactant, sodium cholate, and vitamin E is 6.5-7.2: 0.18-0.22: 1-1.5: 0.3-0.7: 0.28-0.

32.

10. The method for preparing the oral anti-aging composition based on targeted stem cells according to claim 8, characterized in that, The ratio of glycolipid-modified nicotinamide mononucleotide to deionized water II is 5.28-5.34 g: 2.8-3.2 mL; the mass of the composite matrix is ​​125-150% of the sum of the masses of glycolipid-modified nicotinamide mononucleotide, mitochondrial-targeted coenzyme Q10, pyrroloquinoline quinone, R-lipoic acid, astaxanthin, icariin-II, spermidine, N-acetylcysteine, quercetin, and rutin.

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