An anti-aging composition based on mitochondrial quality control, oral formulations thereof and uses thereof

Abstract

The application discloses an anti-aging composition based on mitochondrial quality control and an oral preparation and use thereof, and comprises, in terms of a single daily dose, 200-300 mg of urolithin A, 2-4 mg of spermidine, 200-300 mg of beta-nicotinamide mononucleotide, 4-6 mg of L-ergothioneine, 4-8 mg of astaxanthin, 80-120 mg of R-alpha-lipoic acid, 120-180 mg of magnesium glycinate, 8-12 mg of pyrroloquinoline quinone and 400-600 mg of acetyl-L-carnitine, wherein the mass ratio of the L-ergothioneine, the astaxanthin and the R-alpha-lipoic acid is 1-1.5:1-2:20-30, and the mass ratio of the urolithin A, the beta-nicotinamide mononucleotide, the R-alpha-lipoic acid and the pyrroloquinoline quinone is 20-30:20-30:8-12:0.8-1.2. Through the synergy of the four units of elimination-repair-remodeling-update and the ternary mitochondrial membrane protection system, and further through the day-night rhythm time delivery, the mitochondrial quality control function is significantly improved, and the purpose of systematic anti-aging is achieved.

Description

Technical Field

[0001] This invention relates to the field of health food technology, specifically to an anti-aging composition based on mitochondrial quality control, its oral formulation, and its uses. Background Technology

[0002] During aging, mitochondria not only exhibit decreased respiratory chain efficiency and reduced ATP synthesis capacity, but also show a series of pathological changes at the ultrastructural level, such as weakened autophagic flux, cristae disintegration, respiratory chain supercomplex dissociation, and selective loss of type II myofibrils.

[0003] Studies have shown that the structure of the mitochondrial cristae directly determines the assembly efficiency and stability of the respiratory chain supercomplex. Optic atrophy protein 1 (OPA1) stabilizes the supercomplex and maximizes electron transport efficiency by forming oligomers at the cristae junctions, maintaining cristae compactness and negative curvature. In aging skeletal muscle, downregulation of OPA1 expression and weakened SIRT3-mediated OPA1 deacetylation lead to widening of cristae junctions, supercomplex dissociation, and a decrease in respiratory control ratio, ultimately causing selective atrophy of type II muscle fibers due to insufficient energy supply.

[0004] To systematically describe the body's multidimensional maintenance processes, existing academic literature summarizes mitochondrial quality control (MQC) into four interrelated units: Removal: Selectively removes damaged and depolarized mitochondria through PINK1 / Parkin-mediated mitophagy; Repair: Repairing mitochondrial protein function through deacetylases (such as SIRT3) and protecting the integrity of mtDNA and membrane lipids through endogenous antioxidants; Remodeling: Reconstructing the physical scaffold of the respiratory supercomplex through OPA1-mediated cristae stability and mitochondrial fusion / fission balance regulation; Renewal: Drives mitochondrial biogenesis through the PGC-1α / TFAM axis, replenishing newly generated healthy mitochondria.

[0005] The core insight of this framework is that the four units do not operate independently, but rather form a closed-loop chain of clearance, repair, remodeling, and renewal. If only clearance and renewal are activated while repair and remodeling are lacking, newly formed mitochondria will still rapidly become inoperable due to cristae structural defects and DNA damage; if clearance and renewal are activated simultaneously, they will share metabolic resources (such as AMPK, NAD) + Competition exists among the four units (acetyl-CoA pool, etc.), which may actually reduce overall efficiency. Therefore, ideal MQC intervention should cover all four units simultaneously and achieve temporal synergy and metabolic resource balance among the units.

[0006] Limitations of existing oral intervention techniques: 1. Incomplete unit coverage, missing Repair and Remodeling dimensions: In the field of oral dietary supplements, there are already many publicly available solutions that attempt to activate mitochondrial function through multi-component compounding.

[0007] Existing technology CN122461327A discloses a five-fold anti-aging composition and its liposome formulation based on the coupling of mitochondrial regeneration and autophagy. It discloses the combination of urolithin A (UA), spermidine, pyrroloquinoline quinone (PQQ), nicotinamide riboside (NR), L-ergothioneine (EGT), and magnesium citrate, and proposes a five-fold coupling logic of autophagy, regeneration, fuel, energy production, and gene repair. However, this scheme essentially only covers two units: Removal (UA + spermidine) and Renewal (PQQ + NR), and its gene repair is limited to NAD. + The precursor generalizes and activates SIRT1.

[0008] CN111479580A discloses the synergistic induction of muscle autophagy by the combination of urolithin A and spermidine for use in people at risk of sarcopenia, but only involves a single clearance unit.

[0009] CN121890744A discloses a multi-component anti-aging hard capsule containing EGT, PQQ, UA, spermidine, etc., which is formulated according to its antioxidant, autophagy, and regeneration functions, without specifying the unit node mapping.

[0010] 2. Unresolved circadian rhythm conflict and metabolic resource competition during simultaneous administration: Mitochondrial quality control exhibits significant circadian rhythm characteristics. The core clock gene Bmal1 / Clock regulates the temporal expression of mitochondrial biosynthesis and autophagy: autophagy peaks are concentrated in deep sleep (mTOR inhibition window), while PGC-1α-driven biosynthesis peaks are concentrated several hours after wakefulness. From a metabolic resource perspective, autophagy initiation (AMPK activation, ULK1 phosphorylation) and biosynthesis activation (PGC-1α transcription, NRF1 / TFAM expression) occur in NAD+. + There is competition between them for the demand for acetyl-CoA and ATP.

[0011] Existing multi-component mitochondrial supplements (such as CN121890744A) physically mix the active ingredients in the same dosage form and release them simultaneously. This causes autophagy-inducing components and biosynthetic activating components to compete for limited metabolic regulatory resources within the same time window. Theoretically, it is difficult to achieve the expected effect of multi-unit synergy, and they may even cancel each other out due to metabolic conflicts.

[0012] In summary, existing technologies for oral mitochondrial systemic anti-aging interventions have at least two major systemic defects: First, multi-component regimens are incomplete in terms of unit coverage, generally lacking the Repair (SIRT3 / OPA1-mediated repair) and Remodeling (cristoirex remodeling) dimensions, making it difficult to stabilize the respiratory supercomplex at the ultrastructural level; Second, even regimens covering more units cannot achieve the expected synergistic effect because the synchronous release formulation causes autophagy and biosynthesis to compete for rhythmic phases and metabolic resources. Summary of the Invention

[0013] The present invention aims to overcome at least one of the defects of the prior art and provide an anti-aging composition based on mitochondrial quality control, its oral formulation and uses, which significantly improves mitochondrial quality control function through the synergistic effect of a four-unit system of clearance-repair-remodeling-renewal and a ternary mitochondrial membrane protection system, and further through circadian rhythm time-shaping and sustained-release delivery of active ingredients.

[0014] Specifically, the present invention provides an anti-aging composition based on mitochondrial quality control, comprising: Scavenging unit: UA 200~300mg, spermidine 2~4mg; Repairing agents: β-nicotinamide mononucleotide (NMN) 200~300mg, EGT 4~6mg, astaxanthin 4~8mg; Remodeling units: R-alpha-Lipoic Acid (RALA) 80~120mg, Magnesium Glycine 120~180mg; Updated unit: PQQ 8~12mg, Acetyl-L-Carnitine (ALCAR) 400~600mg; The mass ratio of EGT, astaxanthin and RALA is 1~1.5:1~2:20~30; The mass ratio of UA, NMN, RALA to PQQ is 20~30 : 20~30 : 8~12 : 0.8~1.2.

[0015] Preferably, the mass ratio of EGT, astaxanthin and RALA is 1:1.2:20.

[0016] Preferably, the mass ratio of UA, NMN, RALA to PQQ is 25:25:10:1.

[0017] Preferably, the dosage, based on a single daily administration, includes: 250 mg of UA and 3 mg of spermidine. NMN 250mg, EGT 5mg, astaxanthin 6mg; RALA 100mg, magnesium glycine 150mg; PQQ 10mg, ALCAR 500mg.

[0018] The present invention also provides an anti-aging oral formulation comprising three types of granules for a daily dose, wherein the three types of granules are physically mixed and then filled into an oral solid dosage unit: Immediate-release granules: containing PQQ 8~12mg, NMN 200~300mg, ALCAR 400~600mg, RALA 80~120mg, and magnesium glycine 120~180mg; cumulative release ≥80% within 30 minutes in vitro; Time-delayed sustained-release granules: containing UA 200–300 mg and spermidine 2–4 mg; coated with a 14–16 h time-delayed controlled-release coating, release begins after a 12 h delay in vitro, with a cumulative release of ≥70% after 12–18 h; Sustained-release liposome particles: Liposomes containing 4-8 mg of astaxanthin and 4-6 mg of EGT, continuously released in vitro for 16 hours, with a cumulative release of ≥80% over 16 hours, and the effective concentration in the release medium is maintained for up to 18 hours.

[0019] Those skilled in the art will understand that, depending on the total daily dose, the above three types of granules can be packaged into 2 to 4 capsules without affecting the time-release effect of the present invention.

[0020] The present invention also provides the application of the aforementioned anti-aging composition or oral anti-aging preparation in oral health foods that regulate abnormal mitochondrial energy metabolism.

[0021] This invention also provides the use of the aforementioned anti-aging composition or oral anti-aging formulation in oral health foods for maintaining muscle and cognitive health-related mitochondrial function.

[0022] Furthermore, maintaining muscle-related mitochondrial function involves reducing selective loss of type II muscle fibers or reducing respiratory chain supercomplex dissociation.

[0023] Compared with the prior art, the present invention has the following beneficial effects: (1) Four-unit full coverage synergy: This invention fully maps the 4R framework (Removal, Repair, Remodeling, Renewal) of mitochondrial quality control (MQC) to the multi-component combination of oral dietary supplements. By matching specific components with specific units, it achieves the synergistic effect of clearing damaged mitochondria, repairing protein function and mtDNA, remodeling cristae structure and respiratory supercomplex, and renewing healthy mitochondrial biogenesis, overcoming the one-sidedness of existing technologies that only cover 2 to 3 units.

[0024] (2) Tripartite mitochondrial membrane protection system: By using a specific ratio of EGT, astaxanthin and RALA to synergistically (4~6:4~8:80~120), a hierarchical protection network from membrane lipid antioxidation (EGT+AST) to cristae remodeling (RALA) was constructed, which significantly stabilized OPA1 oligomerization and respiratory chain supercomplex assembly and improved oxidative phosphorylation coupling efficiency.

[0025] (3) Circadian rhythm decoupling: Through the design of three particle sequences—immediate-release particles (released during the day to activate biosynthesis), time-delayed sustained-release particles (released at night to match the autophagy window), and sustained-release liposome particles (sustaining release throughout the process, membrane protection)—under standardized fasting administration conditions, the peak release of time-delayed sustained-release particles can cover the active period of autophagy at night (12-18 hours after administration), thereby avoiding the decoupling of autophagy clearance and biosynthesis in AMPK and NAD. + Competition for metabolic resources significantly improves ATP synthesis efficiency compared to simultaneous release formulations. Attached Figure Description

[0026] Figure 1 A bar chart showing the muscle mitochondrial respiratory control ratio (RCR) in test example 5.

[0027] Figure 2 This is a bar chart showing the rate of ATP synthesis in liver mitochondria in test example 5.

[0028] Figure 3 This is a bar chart showing the time to exhaustion during weighted swimming in Test Example 5.

[0029] Figure 4 This is a bar chart showing the dwell time of the rotating rod in Test Example 5.

[0030] Figure 5 This is a bar chart showing the escape latency period in the Morris water maze test case 5.

[0031] Figure 6 This is a bar chart showing the percentage of time spent in the target quadrant of the Morris water maze in Test Example 5. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below with reference to specific embodiments, but the scope of protection of this invention is not limited thereto. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or according to the manufacturer's recommendations. Unless otherwise specified, all reagents and materials used are commercially available.

[0033] Example 1 Based on a single daily dose or divided doses, the mitochondrial-based quality control-based anti-aging composition comprises: Scavenging unit: UA 250 mg, spermidine 3 mg; Repairing components: NMN 250 mg, EGT 5 mg, astaxanthin 6 mg; Remodeling unit: RALA 100 mg, magnesium glycine 150 mg; Updated units: PQQ 10 mg, ALCAR 500 mg.

[0034] Example 2 Based on a single daily dose or divided daily dose, the composition comprises: Scavenging unit: UA 300 mg, spermidine 4 mg; Repair components: NMN 300 mg, EGT 6 mg, astaxanthin 8 mg; Remodeling unit: RALA 120 mg, magnesium glycine 180 mg; Updated units: PQQ 12 mg, ALCAR 600 mg.

[0035] Example 3 Based on a single daily dose or divided daily dose, the composition comprises: Scavenging unit: UA 200 mg, spermidine 2 mg; Repairing components: NMN 200 mg, EGT 4 mg, astaxanthin 4 mg; Remodeling unit: RALA 80 mg, magnesium glycine 120 mg; Updated units: PQQ 8 mg, ALCAR 400 mg.

[0036] Example 4 Based on a single daily dose or divided daily dose, the composition comprises: Scavenging unit: UA 300 mg, spermidine 4 mg; Repair components: NMN 300 mg, EGT 6 mg, astaxanthin 7.5 mg; Remodeling unit: RALA 120 mg, magnesium glycine 180 mg; Updated units: PQQ 12 mg, ALCAR 600 mg.

[0037] The mass ratio of EGT:Astaxanthin:RALA is 6:7.5:120, or 1:1.25:20.

[0038] Example 5 Based on a single daily dose or divided daily dose, the composition comprises: Scavenging unit: UA 200 mg, spermidine 2 mg; Repair components: NMN 200 mg, EGT 4 mg, astaxanthin 6 mg; Remodeling unit: RALA 80 mg, magnesium glycine 120 mg; Updated units: PQQ 8 mg, ALCAR 400 mg.

[0039] The mass ratio of EGT:Astaxanthin:RALA is 4:6:80, or 1:1.5:20.

[0040] Example 6 An oral health food based on the composition described in Example 1, comprising three types of granules for a daily dose, wherein the three types of granules are physically mixed and then packaged into four No. 00 gelatin hard capsules for once-daily administration.

[0041] (1) Immediate-release granules Ingredients: PQQ 10 mg, NMN 250 mg, ALCAR 500 mg, RALA 100 mg, magnesium glycine 150 mg.

[0042] Preparation method: Mix the above components with microcrystalline cellulose (MCC) at a mass ratio of 1:1.5, use 5% polyvinylpyrrolidone (PVP K30) ethanol solution as binder, granulate in a fluidized bed, dry at 40°C, and granulate through a 24-mesh sieve to obtain immediate-release granules.

[0043] Release rate: The release rate of the three types of particles was determined using the first method (basket method or paddle method) of General Chapter 0931, Part IV of the 2025 edition of the Chinese Pharmacopoeia, with 900 mL of pH 6.8 phosphate buffer (containing 0.5% Tween 80) as the medium, at 37±0.5℃ and 50 r / min. Among them, the sustained-release liposome particles, due to their particle size of approximately 120 nm, were measured using dynamic dialysis (MWCO 100 kDa, medium as above) as an equivalent alternative. The equivalence between the two methods was confirmed by a preliminary experiment (n=3, correlation coefficient r>0.95).

[0044] (2) Time-delayed slow-release granules Ingredients: UA 250 mg, spermidine 3 mg.

[0045] Preparation method: UA and spermidine are mixed with hydroxypropyl methylcellulose (HPMC E5) as the backbone material (UA and spermidine:HPMC = 1:0.3) to form a soft material, which is then extruded and spheroidized to obtain pellet cores. A coating pan coating technology is used to coat the pellet core surface with three layers of coating film from the inside out, the total weight of the three coating films accounting for 18% of the pellet core weight (i.e., a total coating weight gain of 18%): (i) Inner porous layer: composed of hydroxypropyl methylcellulose (HPMC E5) and polyethylene glycol 6000 at a mass ratio of 1:0.5, used to control the release slope after initial hysteresis; (ii) Middle release-inhibiting layer: composed of ethyl cellulose (viscosity 50 cps) and polyacrylic acid resin Eudragit RS PO at a mass ratio of 4:1, accounting for 87% of the total coating film weight; (iii) Outer anti-adhesion layer: magnesium stearate, accounting for 5% of the total coating film weight. Adjusting the total coating weight gain to 15%, 18%, and 21% (based on the weight of the pellet core) yields time delays of approximately 12h, 14h, and 16h, respectively. The time delay is defined as the time elapsed from 0h until the cumulative release of active material in the release medium reaches 10% (n=6, RSD<10%).

[0046] Release rate: As measured by the same method above, the results showed that release began after a 12-hour delay in vitro, with a cumulative release of ≥70% from 12 to 18 hours. In vivo release behavior suggests that the 14-16 hour time-delay controlled-release garment's release time is the in vitro release rate measurement value; actual in vivo release behavior is affected by gastric emptying time, intestinal peristalsis speed, and food intake. It is recommended to take it on an empty stomach or at fixed mealtimes to ensure the time-series decoupling effect.

[0047] (3) Sustained-release liposome particles Ingredients: Astaxanthin 6 mg, EGT 5 mg.

[0048] Preparation method: Liposomes were prepared using a thin-film dispersion-extrusion method. The liposome membrane material consisted of soybean phosphatidylcholine (SPC), cholesterol, and distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) in a mass ratio of 55:40:5. Astaxanthin and EGT were dissolved in ethanol and rotary evaporated together with the membrane material to form a membrane. The membrane was then hydrated with pH 7.4 phosphate buffer and extruded 10 times through a 0.1 μm polycarbonate membrane to obtain a liposome suspension with a particle size of approximately 120 nm. The suspension was then freeze-dried to obtain liposome powder.

[0049] Release rate: Using dynamic dialysis with pH 6.8 phosphate buffer containing 0.5% Tween 80 as the release medium, the results showed that the release was continuous within 16 h in vitro, with a cumulative release of ≥82% over 16 h, and the effective concentration in the release medium was maintained for up to 18 h.

[0050] The three types of granules were physically mixed evenly according to the prescription amount and then packaged into four No. 00 gelatin hard capsules using an equal-volume incremental method. Each capsule contains approximately 1 / 3 immediate-release granules, 1 / 3 time-delayed sustained-release microspheres, and 1 / 3 lyophilized liposome powder, ensuring that the number of time-delayed sustained-release microspheres in each capsule is consistent to guarantee uniform release behavior.

[0051] Comparative Example The following comparative examples are used to further illustrate the advantages of the technical solution of the present invention compared with the prior art.

[0052] Comparative Example 1 Referring to the formulation and preparation method of Example 1 in CN122461327A, the composition comprises, based on the daily dosage: PQQ 5 mg, NR 50 mg, L-ergothioneine 3 mg, urolithin A 100 mg, spermidine 5 mg, riboflavin 1 mg, magnesium citrate 55 mg, γ-aminobutyric acid (GABA) 25 mg, piperine 1.5 mg, vitamin C 25 mg, and vitamin E 5 mg.

[0053] Comparative Example 2 The difference between this comparative example and Example 1 is that RALA and magnesium glycine in the remodeling unit are removed, while the remaining components and dosages remain unchanged.

[0054] Comparative Example 3 The difference between this comparative example and Example 1 is that L-ergothioneine and astaxanthin were removed from the repair unit, while the remaining components and dosages remained unchanged.

[0055] Comparative Example 4 The difference between this comparative example and Example 1 is that PQQ and ALCAR are removed, while the remaining components and dosages remain unchanged.

[0056] Comparative Example 5 The difference between this comparative example and Example 1 is that spermidine is removed, while the other components and dosages remain unchanged.

[0057] Comparative Example 6 In this comparative example, all nine components described in Example 1 were physically mixed and then prepared into ordinary immediate-release granules using a conventional wet granulation process. These granules were then packaged into four 00-size hard capsules. In vitro release studies showed that ≥80% of all components were released within 30 minutes, with no time-lag release characteristics.

[0058] Comparative Example 7 The difference between this comparative example and Example 1 is that NMN in the formula is replaced with NR in an equimolar amount, while the other ingredients and dosages remain unchanged.

[0059] Comparative Example 8 The difference between this comparative example and Example 1 is that magnesium glycine in the formulation is replaced with magnesium citrate in equal molar amounts, while the other components and dosages remain unchanged.

[0060] Comparative examples 9-12 were used to investigate the effect of the proportion of the membrane-protected ternary system on the experimental results.

[0061] Comparative Example 9 Based on Example 1, the mass ratio of L-ergothioneine, astaxanthin and R-α-lipoic acid was adjusted from 1:1.2:20 to 1:2.5:20 (EGT 5mg, astaxanthin 12.5mg, RALA 100mg), while the other components and dosages remained unchanged.

[0062] Comparative Example 10 Based on Example 1, the mass ratio of L-ergothioneine, astaxanthin and R-α-lipoic acid was adjusted from 1:1.2:20 to 2:1.2:20 (EGT 10mg, astaxanthin 6mg, RALA 100mg), while the other components and dosages remained unchanged.

[0063] Comparative Example 11 Based on Example 1, the mass ratio of L-ergothioneine, astaxanthin and R-α-lipoic acid was adjusted from 1:1.2:20 to 1:1.2:35 (EGT 5mg, astaxanthin 6mg, RALA 175mg), while the other components and dosages remained unchanged.

[0064] Comparative Example 12 Based on Example 1, the mass ratio of L-ergothioneine, astaxanthin and R-α-lipoic acid was adjusted from 1:1.2:20 to 2:0.5:15 (EGT 10mg, astaxanthin 2.5mg, RALA 75mg), while the other components and dosages remained unchanged.

[0065] Comparative Examples 13-17 were used to investigate the effect of the proportion of the four core components on the experimental results.

[0066] Comparative Example 13 Based on Example 1, the mass ratio of UA, NMN, RALA and PQQ was adjusted from 25:25:10:1 to 15:25:10:1 (UA 150mg, NMN 250mg, RALA 100mg, PQQ 10mg), while the other components and dosages remained unchanged.

[0067] Comparative Example 14 Based on Example 1, the mass ratio of UA, NMN, RALA and PQQ was adjusted from 25:25:10:1 to 25:15:10:1 (UA 250mg, NMN 150mg, RALA 100mg, PQQ 10mg), while the other components and dosages remained unchanged.

[0068] Comparative Example 15 Based on Example 1, the mass ratio of UA, NMN, RALA and PQQ was adjusted from 25:25:10:1 to 25:25:5:1 (UA 250mg, NMN 250mg, RALA 50mg, PQQ 10mg), while the other components and dosages remained unchanged.

[0069] Comparative Example 16 Based on Example 1, the mass ratio of UA, NMN, RALA and PQQ was adjusted from 25:25:10:1 to 25:25:10:0.5 (UA 250mg, NMN 250mg, RALA 100mg, PQQ 5mg), while the other components and dosages remained unchanged.

[0070] Comparative Example 17 Based on Example 1, the mass ratio of UA, NMN, RALA and PQQ was adjusted from 25:25:10:1 to 10:10:20:2 (UA 100mg, NMN 100mg, RALA 200mg, PQQ 20mg), while the other components and dosages remained unchanged.

[0071] Comparative Example 18 This comparative example is based on the capsule formulation described in Example 6, except that the total coating weight gain of the time-delayed sustained-release granules is reduced from 18% to 9% (the proportion of each layer to the total weight of the coating remains unchanged), which shortens the in vitro time delay from 14-16 h to 4-6 h. The rest of the granule formulation and process remain unchanged.

[0072] Effect verification test The following experiments were conducted to verify the technical effectiveness of the compositions and formulations described in the above examples in improving mitochondrial quality control.

[0073] Experiment 1: Network Pharmacology and Molecular Docking Simulation Validation 1.1 Experimental Methods The targets of each component were predicted using the PharmMapper and SwissTargetPrediction databases. Targets related to mitochondrial functional decline and aging were collected from the GeneCards and OMIM databases. The intersection of these targets yielded the potential targets of the composition. A "component-target-pathway" network was constructed using Cytoscape. Molecular docking between the core components and key targets was performed using AutoDock Vina, and the binding energy (ΔG, kcal / mol) was calculated to verify the binding stability.

[0074] 1.2 Selection of core targets Key proteins from four major units of mitochondrial quality control were selected: PINK1 and Parkin (Removal unit), targets of mitophagy; PGC-1α and TFAM (Renewal unit), targets of biosynthesis; SIRT1, a target of energy metabolism; and OPA1 (Remodeling unit), a target related to mitochondrial dynamics / crisp remodeling. The core target protein structures were derived from: PINK1 (PDB: 6G9X), Parkin (PDB: 5C1Z), PGC-1α (PDB: 6M3D), TFAM (PDB: 3TMM), SIRT1 (PDB: 4I5I), and OPA1 (PDB: 7MGX). Molecular docking was performed using AutoDock Vina 1.2.0, with the grid box centered on the active site, side length 30 Å × 30 Å × 30 Å, exhaustiveness=32, and other parameters set to default values. A binding energy ≤ -7.0 kcal / mol was considered significant binding.

[0075] 1.3 Experimental Results Table 1. Binding energies (ΔG, kcal / mol) of core components to key target molecules for mitochondrial quality control

[0076] 1.4 Conclusion Molecular docking results showed that each core component exhibited good binding stability with its corresponding target site: urolithin A had low binding energies with PINK1 and Parkin, validating its potential to strongly induce mitophagy; PQQ bound tightly with PGC-1α and TFAM, supporting its core role in promoting mitochondrial biosynthesis; NMN had the lowest binding energy with SIRT1, confirming its role in promoting mitochondrial biosynthesis through NAD+. + The SIRT1 axis regulates mitochondrial function; RALA and OPA1 also exhibit good binding stability, suggesting they may be involved in the regulation of mitochondrial cristae remodeling. The extensive binding of multiple components and targets validates, at the molecular level, the design logic of the composition for multi-pathway synergistic regulation of mitochondrial quality control.

[0077] Experiment 2: In vitro cell experiments: Effects on improving mitochondrial function in senescent cells 2.1 Experimental Design Cell model: Mouse C2C12 myoblasts were used to establish a premature aging model of oxidative damage induced by H2O2.

[0078] Experimental groups (n=6): Normal control group (normal culture medium); Aging model group (treated with H2O2); Positive control group (H2O2 + rapamycin 100 nM); and Examples 1-5 and Comparative Examples 1-5 and 7-17.

[0079] 2.2 Detection Indicators Cell viability (CCK-8 assay); mitochondrial membrane potential (JC-1 fluorescence assay); intracellular ATP content (luciferase assay); mitochondrial ROS level (MitoSOX fluorescent probe assay); mitophagy and expression of biosynthetic proteins (Western Blot: PINK1, LC3-II / LC3-I, PGC-1α, TFAM).

[0080] 2.3 Experimental Results Table 2. Effects of the composition on mitochondrial function-related indicators in H2O2-induced senescent cells.

[0081] Note: ### p<0.001 vs normal control group; p<0.05, p<0.01 vs aging model group, Δ p<0.05 vs Example 1 group, n=6.

[0082] Table 3. Effects of the composition on the expression of mitochondrial quality control proteins in senescent cells (relative expression levels)

[0083] Note: ### p<0.001 vs normal control group; p<0.05, p<0.01 vs aging model group, Δ p<0.05 vs Example 1 group, n=6.

[0084] 2.4 Conclusion The composition can dose-dependently enhance the viability, mitochondrial membrane potential, and ATP content of senescent cells, reduce ROS levels, and effectively reverse mitochondrial dysfunction caused by oxidative damage. Example 1 significantly upregulated the expression of PINK1, LC3-II / LC3-I, PGC-1α, and TFAM, confirming the synergistic activation of the four units.

[0085] The comparative examples of unit deletions exhibit characteristic defect spectra, confirming that each of the four units performs its own function: (1) In Comparative Example 2, the membrane potential decreased, but PINK1 was close to that in Example 1, suggesting that crest remodeling deficiency impairs energy conversion efficiency rather than autophagy initiation; (2) In Comparative Example 4, ATP and PGC-1α both decreased significantly, but PINK1 increased compensatorily, suggesting that autophagy pathways are activated compensatorily when biosynthesis is insufficient, forming a consumption imbalance of only output and no input; (3) In Comparative Example 5, the levels of PINK1 and LC3-II / LC3-I both decreased, which were the lowest autophagy markers among all unit deletion groups, confirming the irreplaceable role of spermidine in the Removal unit. The above characteristic defect spectra confirm that the four units form a functional closed loop of clearance-repair-remodeling-renewal, and the deletion of any unit leads to the breakage of a specific functional axis.

[0086] Although Comparative Example 1 showed acceptable autophagy metrics, its PGC-1α, TFAM, and membrane potential were significantly lower than those of Example 1, confirming its design flaw of only covering the Removal and Renewal units and lacking Repair and Remodeling. It should be noted that the total active ingredient dose in Comparative Example 1 was approximately 21.6% of that in Example 1, and its NR of 50 mg was far below the SIRT1 activation saturation dose, suggesting a potential structural defect in the unit node dose design.

[0087] The synergistic effect of Comparative Example 9 and Comparative Examples 13-17 decreased gradually with increasing deviation. When Comparative Examples 12 and 17 deviated completely, they approached an additive effect, confirming that a specific range of proportions is a necessary condition for the synergistic effect.

[0088] Experiment 3: Verification Experiment of Four-Unit Collaboration and Cross-Unit Coupling 3.1 Experimental Design Cell model: Same as experiment 2, H2O2-induced premature aging model of C2C12 cells.

[0089] Dosage principle: In all administration groups, the final concentration of a single component is completely consistent with the concentration of the corresponding component in the whole formula, eliminating the interference of dose superposition.

[0090] Experimental groups (16 groups in total, n=6): Group A: Normal control group Group B: Aging Model Group Group C: Removal Unit (Single Component) - Urolithin A Group D: Clearance unit core combined with urolithin A + spermidine Group E: Scavenging unit superimposed with repair unit antioxidant subcomponent - urolithin A + spermidine + astaxanthin (verifying the initial coupling of scavenging and repair) Group F: Renewal unit, single component - pyrroloquinoline quinone Group G: Remodeling unit combined with remodeling unit core - pyrroloquinoline quinone + R-α-lipoic acid Group H: Complete group of renewal and remodeling units - pyrroloquinoline quinone + R-α-lipoic acid + magnesium glycine (to verify the coupling between renewal and remodeling) Group I: Repair Unit (Single Component) - β-Nicotinamide Mononucleotide Group J: Repair unit combined with renewal unit - β-nicotinamide mononucleotide + L-ergothioneine + acetyl-L-carnitine (to verify the coupling of repair and renewal) Group K: Tri-unit core combination - Urolithin A + Pyrroloquinolinequinone + β-Nicotinamide mononucleotide (clearance + renewal + repair) Group L: Full Formulation Group - Example 1 (9 ingredients, complete combination of four units) Group M: Comparative Example 1 - Formulation D1 (Existing Technology Comparison) Group N: Tripartite Mitochondrial Membrane Protection System Validation Group - L-ergothionein + astaxanthin + R-α-lipoic acid, to verify the synergistic effects of EGT, astaxanthin and RALA on hierarchical membrane protection and cristae remodeling.

[0091] Group O: Quadruple core combination group - urolithin A + β-nicotinamide mononucleotide + R-α-lipoic acid + pyrroloquinoline quinone (containing only the representative components of the quadruple core, without auxiliary components), to verify whether the synergy of the quadruple core is superior to that of the tri-core combination.

[0092] Group P: Full formulation with NR as an equimolar substitute for NMN: Based on the full formulation of Comparative Example 7, the molar concentrations of NR and NMN are equal to verify the difference in efficacy between NR and NMN in the complete four-unit combination.

[0093] 3.2 Detection Indicators Select core functional indicators for each unit and cross-unit coupling indicators: Mitochondrial autophagy activity (PINK1 protein expression, Removal unit marker); Mitochondrial biosynthetic activity (PGC-1α protein expression, Renewal unit marker); Mitochondrial cristae remodeling activity (OPA1 protein expression, a marker of the Remodeling unit); Mitochondrial deacetylation repair activity (SIRT3 protein expression, a marker of the coupling between the Repair and Remodeling units). Energy metabolism level (ATP content); Comprehensive effect index (cell viability).

[0094] 3.3 Experimental Results Table 4. Gradient effects of multi-unit components on mitochondrial function in senescent cells

[0095] Note: ### p<0.001 vs normal control group; p<0.05, p<0.01 vs aging model group, n=6.

[0096] 3.4 Results Analysis (1) Four-unit closed-loop is better than three-unit open-loop. Although the three-unit core combination group (Group K, clearing + repair + renewal) significantly improved cell viability and ATP compared to the single-unit group, OPA1 and SIRT3 were still significantly lower than the normal control (p<0.05), suggesting that insufficient crest remodeling led to limited energy conversion efficiency. In the four-unit core combination group (Group O) after introducing the remodeling core RALA, OPA1 and SIRT3 were significantly increased, confirming that the fourth unit is a necessary closed loop in the energy recovery chain, rather than an optional optimization item.

[0097] (2) Network synergistic amplification effect of auxiliary components The four-unit core combination group (Group O, with only 4 components) covers the four-unit framework, but the full formulation group (Group L, with 9 components) is superior to Group O in terms of OPA1, SIRT3, and cell viability. Notably, Group O's PINK1 is close to that of Group L, indicating that autophagy clearance function has been basically achieved in the four-unit core. The significant difference between OPA1 and SIRT3 indicates that auxiliary components such as spermidine, EGT, astaxanthin, ALCAR, and magnesium glycine have generated an indispensable network synergistic amplification effect on cristae remodeling by constructing membrane protection microenvironment, providing acetylation substrates, and stabilizing OPA1 oligomerization.

[0098] (3) Independent cristae remodeling and energy-limited paradox of ternary membrane protection system Group N showed similar OPA1 levels to the full-formula group, but PINK1 levels were not significantly different from the aging model group. SIRT3 and ATP levels were significantly lower in group N than in group L (p<0.01). These results confirm that the ternary membrane protective system can function even in the absence of NAD+. +Under precursor supply conditions, cristae remodeling is achieved independently through EGT-targeted membrane enrichment, astaxanthin lipid peroxidation quenching, and RALA-mediated OPA1 oligomerization stabilization; however, NAD lacks repair units. + The synergy between the SIRT3 axis and the Removal / Update unit means that crest remodeling cannot be converted into energy metabolism recovery, exhibiting a "structure-function decoupling" characteristic. This paradox illustrates that crest structural integrity is a necessary but not sufficient condition for energy metabolism.

[0099] (4) The advantage of NMN over NR in target selectivity in the four-unit system Group P showed lower levels of OPA1 and SIRT3 than Group L (p<0.05), but the difference was smaller in PINK1 and PGC-1α. This difference in target selectivity suggests that although both NMN and NR are NAD+ targets... + Precursor, but NMN may be used in the four-unit synergistic network of this invention via more efficient mitochondrial NAD. + Compartmentalization supply or SIRT3 allosteric activation specifically enhances deacetylation-crispionage remodeling axis function; this selective advantage is evident in single NAD+. + The effect was not significant during prodrug administration and was only amplified under multi-unit synergistic conditions.

[0100] Experiment 4: Validation of Specific Proportional Synergistic Effect (Chou-Talalay CI Method) Using the Chou-Talalay Combination Index (CI) method, we quantitatively demonstrated that two specific mass ratios (L-ergothionein:astaxanthin:R-α-lipoic acid = 1~1.5:1~2:20~30; urolithin A:β-nicotinamide mononucleotide:R-α-lipoic acid:pyrroloquinoline quinone = 20~30:20~30:8~12:0.8~1.2) are necessary conditions for generating strong synergy (CI<0.3): the proportion points within the interval are all strongly synergistic, the synergy of single-factor deviation from the proportion is significantly weakened, the proportions of all deviations are additive, and the proportions without proportion constraints are antagonistic.

[0101] 4.1 Experimental Design Cell model: Isolated mitochondria from naturally aging mice (H2O2 or aging model).

[0102] Experimental groups (n=6): (1) Single-component effect curve group: The effect curves of nine single components, namely urolithin A, spermidine, NMN, L-ergothioneine, astaxanthin, R-α-lipoic acid, glycine magnesium, PQQ and acetyl L-carnitine, were measured at five concentration gradients (0.1×, 0.5×, 1×, 2× and 5× the concentrations corresponding to Example 1), and the median effective concentration (EC50) and m value (Hill coefficient) of each single component were calculated.

[0103] (2) The proportion group of the present invention (the proportion of Example 1): 9 ingredients were taken together according to the proportion of Example 1, with 5 total concentration gradients (0.1×~5×), and the CI value was measured.

[0104] (3) Groups with deviations in the proportion of ternary membrane protection system (4 groups, comparative examples 9~12): Comparative examples 9 to 12.

[0105] (4) Four-unit core proportion deviation groups (5 groups, comparative examples 13~17): Comparative examples 13 to 17.

[0106] Effect parameters: Mitochondrial respiratory control ratio (RCR) was measured using a Seahorse XF24 analyzer. Based on OCR, oligomycin (1 μmol / L) and 4-trifluoromethoxyphenylhydrazone (FCCP, 0.5 μmol / L) were injected sequentially, and the RCR was calculated based on the state 3 / state 4 respiratory control ratio. ATP content was measured using the luciferase method. OPA1 oligomerization level was quantified by Western blotting after Blue Native-PAGE. Each combination group was prepared according to its respective formulation ratio and measured at five total concentration gradients. Each group was independently repeated three times, and CI values ​​were calculated.

[0107] 4.2 CI Calculation Method The Combination Index (CI) was calculated using CompuSyn software (Chou-Talalay method) based on the effect-dose data of each group. CI < 1 indicates synergy, CI = 1 indicates additive, CI > 1 indicates antagonism, and CI < 0.3 indicates strong synergy.

[0108] 4.3 Experimental Results Table 5. Synergy Index (CI) and Effects of Different Proportion Combinations

[0109] Table 6. Core Proportion Dimensions of the Four Units: Synergy Index (CI) and Effect of Each Proportion Combination

[0110] Note: ### p<0.001, # p<0.05, ## p<0.01 vs normal control group; p<0.05 p<0.01 vs aging model group; Δ p<0.05 vs Example 1 group. CI<0.3 indicates strong synergy, 0.3≤CI<1 indicates synergy, CI=1 indicates additive, and CI>1 indicates antagonism. RCR is the state 3 / state 4 respiratory control ratio (absolute value); ATP is the mitochondrial ATP production rate (absolute value, nmol / min / mg protein); OPA1 oligomerization was normalized to 1.00 (relative value) with the normal control group as the reference. The normal control group and aging model group used the same batches as Tables 5 and 6 (mitochondrial preparation from the same animal batch); Example 1 is represented in both tables as the corresponding subsystem formulations, and is an independent parallel experiment. Comparative Examples 9-12 and 13-17 are independent experiments. CI values ​​were calculated from three independent replicate experiments, and the dose-response curves (Dx) of each component were pre-determined.

[0111] 4.4 Experimental Conclusions (1) Strong synergistic interval specificity: In Example 1, in the ternary system (1:1.2:20) and the four-unit core system (25:25:10:1), the CI of the three effect indicators of RCR, ATP and OPA1 oligomerization were all <0.3, showing a strong synergistic effect (p<0.01 vs aging model group), which confirmed that a hierarchical membrane protection network can be effectively constructed and the assembly of the respiratory chain supercomplex can be stabilized within this ratio range.

[0112] (2) Single-factor deviation led to a significant reduction in synergy: The CI of comparative examples 9-11 (ternary single-factor deviation) and comparative examples 13-16 (quadrivalent single-factor deviation) were both increased (p<0.05 vs Example 1), and the synergistic effect was significantly weakened. Among them, the synergistic reduction in RALA exceeding the upper limit (comparative example 11) was greater than that in astaxanthin or EGT exceeding the upper limit, suggesting that excessive RALA may overactivate the interfering autophagic flux through the SIRT3-OPA1 axis.

[0113] (3) Overall deviation from the additive effect of regression: The CI of comparative example 12 and comparative example 17 is close to 1, and the effect value falls back to the level close to that of the aging model group, confirming that when the proportions deviate from a specific range, the synergistic effect basically disappears, and the sum of the effects of each individual component is regressed.

[0114] (4) Antagonistic risk of RALA overdose: In Comparative Example 17, the RALA ratio doubled from 10 to 20, and OPA1 oligomerization was significantly reduced, approaching that of the aging model group, with a CI close to 1.0. This result confirms that RALA overdose, without sufficient NAD+ precursor synergy, cannot effectively activate the SIRT3-OPA1 axis, and may instead inhibit autophagic flux by altering mitochondrial redox homeostasis, resulting in mild functional antagonism.

[0115] In summary, the specific ratio ranges of EGT:Astaxanthin:RALA = 1~1.5:1~2:20~30 and UA:NMN:RALA:PQQ = 20~30:20~30:8~12 are necessary conditions for generating a strong synergistic effect (CI<0.3). Deviation from the ratio leads to a weakening or even disappearance of the synergistic effect gradient.

[0116] Experiment 5: Study on anti-aging and mitochondrial function improvement in naturally aging mice 5.1 Experimental Design Experimental animals: SPF-grade C57BL / 6J male mice, with a 3-month-old young control group and 20-month-old naturally aged mice in each senescent group.

[0117] Experimental groups (12 animals per group): Group A: Control group (3 months old, administered via gavage with normal saline) Group B: Aging model group (20 months old, administered via gavage with physiological saline) Group C: Low-dose group of Example 1 (20 months old, 150 mg / kg·d of the composition of Example 1 by gavage) Group D: High-dose group of Example 1 (20 months old, 450 mg / kg·d of the composition of Example 1 by gavage) Group E: Positive control group (20 months old, rapamycin 1 mg / kg·d by gavage) Intervention period: continuous gavage for 8 weeks.

[0118] Groups C and D were prepared by gavage using powders of the physically mixed active ingredients described in Example 1, which were then dissolved in a 0.5% sodium carboxymethyl cellulose solution to form a suspension. Group E was prepared with rapamycin using the same method. The gavage volume for all groups was 10 mL / kg·d.

[0119] 5.2 Detection Indicators and Methods (1) Mitochondrial function detection: Mitochondria were isolated from the gastrocnemius muscle and liver of mice, and the respiratory control ratio (RCR = respiratory rate in state 3 / respiratory rate in state 4) and ATP synthesis rate were measured using a high-resolution respirometer.

[0120] (2) Expression of mitochondrial quality control proteins: Western blot was used to detect the expression of mitochondrial autophagy markers (PINK1, Parkin, LC3-II / LC3-I) and biosynthetic markers (PGC-1α, TFAM) in muscle tissue; the expression of mitochondrial dynamics / cristoire remodeling markers (OPA1, MFN1, DRP1) was also detected.

[0121] (3) Detection of respiratory supercomplex assembly: Blue native-PAGE (BN-PAGE) was used to separate the gastrocnemius mitochondrial respiratory chain complex, and Western blotting was used to detect the assembly level of the supercomplex (I+III2+IV).

[0122] (4) Detection of muscle fiber type composition: Frozen sections of mouse gastrocnemius muscle were stained with ATPase histochemical staining (pre-incubation at pH 4.3) to distinguish between type I (slow-twitch, oxidative) and type II (fast-twitch, glycolytic) muscle fibers. The proportion of each type of muscle fiber was quantified using image analysis software.

[0123] (5) Detection of aging phenotypes: muscle endurance (weighted swimming test, tail load of 5% body weight lead weight, record time to exhaustion); motor coordination (rotary bar fatigue test, record time of rotary bar stay).

[0124] (6) Cognitive function: Morris water maze experiment to detect the escape latency period and the percentage of time spent in the target quadrant.

[0125] (7) Quantitative detection of respiratory chain supercomplex by BN-PAGE: Mitochondria of mouse gastrocnemius muscle were collected, and mitochondrial membrane proteins were solubilized with 1% digitalis saponin. The respiratory chain complex and supercomplex were separated by blue non-denaturing polyacrylamide gel electrophoresis (Blue Native-PAGE, 3~13% gradient gel). Western blotting was performed using anti-NDUFA9 (CI), anti-UQCRC2 (CIII), and anti-COX5A (CIV) antibodies, respectively. ImageJ was used to quantify the percentage of the supercomplex (I+III2+IV, molecular weight about 1500 kDa) in the total CI signal.

[0126] 5.3 Experimental Results Table 7. Effects of the composition on mitochondrial function and aging phenotype in naturally aging mice.

[0127] Note: ### p<0.001 vs. young control group; p<0.05, p<0.01 vs aging model group, n=12.

[0128] Table 8. Effects of the composition on the expression of mitochondrial quality control proteins in the muscle of naturally aging mice.

[0129] Note: ### p<0.001 vs. young control group; p<0.05, p<0.01 vs aging model group, n=12.

[0130] Table 9. Effects of the composition on the composition of myofiber types in naturally aging mice.

[0131] Note: ### p<0.001 vs. young control group; p<0.05, p<0.01 vs aging model group, n=12.

[0132] Table 10 Effects of the composition on cognitive function in naturally aging mice

[0133] Note: ### p<0.001 vs. young control group; p<0.05, p<0.01 vs aging model group Table 11. Respiratory chain supercomplex assembly level (BN-PAGE quantification)

[0134] Note: ### p<0.001 vs. young control group; p<0.05, p<0.01 vs aging model group, n=12.

[0135] 5.4 Experimental Conclusions Combine Tables 7-11 and Figures 1-6 As shown: Significantly improved mitochondrial function: The composition dose-dependently increased the rate of mitochondrial RCR and liver ATP synthesis in the muscles of naturally aging mice, effectively reversing the decline in oxidative phosphorylation coupling efficiency and energy synthesis capacity caused by aging.

[0136] Activation of mitochondrial quality control network: The composition simultaneously upregulates the PINK1 / Parkin-mediated autophagy pathway and the PGC-1α / TFAM-mediated biosynthesis pathway, achieving bidirectional regulation of "clearing damaged mitochondria and replenishing new mitochondria"; at the same time, it significantly upregulates the expression of OPA1 and MFN1, confirming that it has a remodeling effect of stabilizing cristae structure and promoting mitochondrial fusion.

[0137] Myofiber type protection: The aging model group showed significant selective loss of type II myofibers (II / I ratio decreased to 1.37), while the high-dose composition group (Group D) recovered the II / I ratio to 2.47, close to the level of the youth group (2.51), indicating that the composition has a targeted reversal effect on the abnormal conversion of myofiber types characteristic of sarcopenia.

[0138] Validation of the synergistic advantages of multiple targets: The high-dose composition showed better results than the rapamycin positive control (Group E) in multiple core indicators such as RCR, ATP synthesis, myofiber type protection and cognitive function. It overcame the inhibitory effect of a single mTOR inhibitor on biosynthesis and demonstrated the unique advantage of full-chain regulation of "clearance-repair-remodeling-renewal".

[0139] The respiratory chain supercomplex assembly level test results showed that the supercomplex assembly level in the aging model group decreased by 58.7% compared with the young group, confirming that age-related sarcopenia is accompanied by significant dissociation of the respiratory chain supercomplex. The composition of Example 1 (Group C) restored the supercomplex proportion to 55.8%, significantly higher than the aging model group (p<0.01), and the high-dose group (Group D) recovered to near the young level, confirming the efficacy of the remodeling unit of this invention, which maintains OPA1 oligomerization, stabilizes cristae connections, and promotes supercomplex assembly through RALA. The rapamycin-positive control group (Group E) showed limited supercomplex recovery because it only inhibits mTOR / activates autophagy and lacks cristae remodeling targets, further confirming the unique advantages of the four-unit synergy in ultrastructural repair of this invention.

[0140] Experiment 6: Validation of in vivo sequential release of the formulation (PK-PD linkage model) 6.1 Experimental Design Experimental animals: SPF grade C57BL / 6J male mice, 20-month-old naturally aged mice.

[0141] Experimental Groups: Group A: Synchronous administration group (the components of the composition in Example 1 were physically mixed and administered by single gavage, 450 mg / kg·d). Group B: Time-segmented capsule group (capsule formulation as described in Example 6, total dose 450 mg / kg·d, administered by gavage at the same time every day on an empty stomach). Three animals were sampled in each group at each time point, and a parallel group design was used to ensure that the data at each time point were independent.

[0142] Sampling time points: 0, 2, 4, 6, 8, 12, 14, 16, 18, 20, and 24 hours after administration.

[0143] 6.2 Detection Indicators and Methods (1) Concentration of effective plasma components: Blood was collected from the orbital cavity at each time point, plasma was separated, and the plasma concentrations (ng / mL) of urolithiasis A (UA) and β-nicotinamide mononucleotide (NMN) were determined by UPLC-MS / MS.

[0144] (2) Muscle protein expression phases: Gastrocnemius muscle tissue was collected after sacrifice at each time point, and the expression levels of PINK1 and PGC-1α proteins were detected by Western Blot. The relative expression levels were calculated with the 0 h aging baseline as 1.00.

[0145] 6.3 Experimental Results Table 12. Phase changes in plasma effective component concentration and muscle protein expression in the time-lapse capsule group (n=3)

[0146] Note: p<0.05, p<0.01 vs 0 h baseline.

[0147] Table 13. Phase changes in plasma effective component concentration and muscle protein expression in the concurrent administration group (n=3)

[0148] Note: p<0.05, p<0.01 vs 0 h baseline.

[0149] 6.4 Results Analysis (1) Pharmacokinetic characteristics of the time-sharing capsule group (Group B): Plasma UA concentration remained low from 0 to 12 h, began to rise sharply at 14 h, peaked at 16 h, and gradually decreased after 18 h. This release curve was consistent with the in vitro 14–16 h time lag characteristics, confirming that the time-delayed release microspheres achieved the expected delayed release in vivo.

[0150] Plasma NMN concentration increased rapidly from 0 to 4 hours, began to decrease after 6 hours, and returned to a low level after 12 hours, which is consistent with the rapid release characteristics of immediate-release granules.

[0151] (2) Pharmacokinetic characteristics of the concurrent administration group (Group A): Plasma UA and NMN concentrations reached their peak simultaneously between 2 and 4 hours, and then rapidly declined after 8 hours. The absorption phases of the two components completely overlapped.

[0152] (3) Differences in protein expression phases: In the timed capsule group, the peak value of PGC-1α was 6 h after administration, and the peak value of PINK1 was 16 h after administration, with a time difference of 10 h between the two peak values.

[0153] In the concurrent administration group, the peak values ​​of PINK1 and PGC-1α were both located 8 hours after administration, and their peak values ​​completely overlapped. This resulted in autophagic flux and biosynthetic pathways competing for AMPK and NAD within the same time window. + Metabolic resources.

[0154] 6.5 Conclusion The PK-PD linkage model confirmed that the time-segmented capsule formulation described in Example 6 successfully achieved delayed release of UA and rapid release of NMN in vivo, resulting in a 10-hour staggered peak between PINK1-mediated mitophagy and PGC-1α-mediated biosynthesis, effectively avoiding competition for metabolic resources. In the simultaneous administration group, the simultaneous absorption of both components led to overlap between autophagy and biosynthesis signals, resulting in lower overall efficacy compared to the time-segmented formulation group. In the mouse model, 16 hours after administration fell within its active period for autophagy, corresponding to the nocturnal autophagy window mechanism in humans.

[0155] Experiment 7: Comparison of Formulation Release Timing 7.1 Experimental Design Experimental animals: Same as Experiment 5, 20-month-old naturally aged C57BL / 6J male mice.

[0156] Experimental groups (12 animals per group): Group A: Synchronous administration group (After physical mixing of the components of the composition in Example 1, a suspension was prepared with 0.5% sodium carboxymethyl cellulose solution, 450 mg / kg·d, administered by gavage at the same time every day) Group B: Time-segmented capsule group (capsule formulation as described in Example 6, total dose 450 mg / kg·d, administered by gavage at the same time every day). Group C: Simultaneous release of conventional formulation (capsule formulation as described in Comparative Example 6, total dose 450 mg / kg·d, administered by gavage at the same time daily). Group D: The capsule formulation prepared in Comparative Example 18, with a total dose of 450 mg / kg·d, was administered by gavage at the same time every day. Group E: Young control group (3 months old, administered via gavage with normal saline) Group F: Aging model control group (20 months old, administered via gavage with physiological saline). Intervention period: continuous gavage for 8 weeks.

[0157] 7.2 Detection Indicators Muscle mitochondrial RCR and ATP synthesis rate; temporal changes in the expression of PINK1, LC3-II / LC3-I, PGC-1α, and TFAM proteins in muscle tissue (samples were collected at 4 h, 8 h, 12 h, 16 h, and 20 h after administration).

[0158] 7.3 Experimental Results Table 14 Effects of formulation timing design on mitochondrial function and protein expression phases

[0159] Note: ### p<0.001 vs. young control group; p<0.05, p<0.01 vs aging model group, n=12.

[0160] 7.4 Conclusion In group B, the PINK1 peak (16h, corresponding to the nighttime autophagy window) and the PGC-1α peak (6h, corresponding to the daytime biosynthesis window) formed a 10h peak shift, effectively preventing the autophagy flux and biosynthetic pathway from affecting AMPK and NAD. + Competition for metabolic resources enabled temporal synergistic optimization of clearance and renewal units. Group B showed a significantly improved RCR (Recovery Rate) of 4.82 ± 0.41 and an ATP synthesis rate of 17.5 ± 3.5 nmol / min·mg prot compared to the synchronous administration group (Group A) (p < 0.01), recovering to levels close to the young control group (Group E). In both Group A and Group C (the synchronous release of the conventional formulation), the peak values ​​of PINK1 and PGC-1α were at 8 h, with no peak shift at 0 h. There were no statistically significant differences in any indicators between the two groups (p > 0.05), confirming that synchronous release cannot achieve temporal decoupling. In the mouse model, 16 h after administration fell within its active period of activity / autophagy, corresponding to the nocturnal autophagy window mechanism in humans.

[0161] Although Group A (powder mixture) and Group C (conventional wet granulation capsules) of this invention differ in preparation process and physical morphology, both lack the three-layered sequential design of immediate-release granules, time-delayed sustained-release granules, and sustained-release liposome granules of this invention. Therefore, their release behavior is synchronous, and there is no substantial difference in in vivo effects. This result strongly demonstrates that conventional formulation operations in the field, such as conventional granulation and capsule filling, cannot replace the innovative sequential delivery system of this invention based on the biological principles of circadian rhythms. The sequential release characteristics of the formulation process are key to achieving multi-unit synergistic effects, and cannot be expected or replaced by simple dosage form changes.

[0162] In summary, the oral health food of the present invention achieves the circadian rhythm synergy of the 4R unit through a three-layer temporal design of immediate-release particles (released during the day to activate biosynthesis), time-delayed sustained-release particles (released at night to match the autophagy window), and sustained-release liposome particles (sustaining release throughout the process and membrane protection). Its technical effect is significantly better than any form of synchronous release formulation.

[0163] The above embodiments are merely illustrative of the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to the preferred embodiments above, those skilled in the art should understand that modifications or equivalent substitutions to the technical solutions of the present invention should not depart from the spirit and scope of the present invention. Those skilled in the art can also make other changes within the spirit of the present invention and use them in the design of the present invention, as long as they do not deviate from the technical effects of the present invention. These changes made according to the spirit of the present invention should all be included within the scope of protection claimed by the present invention.

Claims

1. An anti-aging composition based on mitochondrial quality control, characterized in that, Based on a single daily dose, it includes: Urolithin A 200~300mg, spermidine 2~4mg; β-Nicotinamide mononucleotide 200~300mg, L-ergothioneine 4~6mg, astaxanthin 4~8mg; R-α-lipoic acid 80~120mg, glycine magnesium 120~180mg; Pyrroloquinoline quinone 8-12 mg, acetyl-L-carnitine 400-600 mg; The mass ratio of L-ergothioneine, astaxanthin, and R-α-lipoic acid is 1~1.5:1~2:20~30; The mass ratio of urolithin A, β-nicotinamide mononucleotide, R-α-lipoic acid and pyrroloquinoline quinone is 20~30 : 20~30 : 8~12 : 0.8~1.

2.

2. The anti-aging composition according to claim 1, characterized in that, The mass ratio of L-ergothioneine, astaxanthin, and R-α-lipoic acid is 1:1.2:

20.

3. The anti-aging composition according to claim 1, characterized in that, The mass ratio of urolithin A, β-nicotinamide mononucleotide, R-α-lipoic acid and pyrroloquinoline quinone is 25:25:10:

1.

4. The anti-aging composition according to claim 1, characterized in that, Based on a single daily dose, it includes: 250 mg of urolithiasis A and 3 mg of spermidine. β-Nicotinamide mononucleotide 250mg, L-ergothioneine 5mg, astaxanthin 6mg; R-α-lipoic acid 100mg, glycine magnesium 150mg; Pyrroloquinoline quinone 10mg, acetyl-L-carnitine 500mg.

5. An anti-aging oral preparation, characterized in that, This includes the anti-aging composition according to any one of claims 1 to 4, as well as excipients acceptable in the food industry.

6. The anti-aging oral preparation according to claim 5, characterized in that, The anti-aging oral preparation is one of the following: tablets, capsules, or granules.

7. The anti-aging oral preparation according to claim 5, characterized in that, The product contains three types of granules for a daily dose, which are physically mixed and then dispensed into oral solid dosage units. A single dose is provided by 1 to 4 of these oral solid dosage units. Immediate-release granules: containing pyrrolizinib 8-12 mg, β-nicotinamide mononucleotide 200-300 mg, acetyl-L-carnitine 400-600 mg, R-α-lipoic acid 80-120 mg, and glycine magnesium 120-180 mg; cumulative release ≥80% in vitro over 30 minutes; Time-delayed sustained-release granules: containing 200-300 mg of urolithin A and 2-4 mg of spermidine; coated with a 12-16 h time-delayed controlled-release coating, releasing ≥70% cumulatively after 12-18 h in vitro; Sustained-release liposome particles: Liposomes containing 4-8 mg of astaxanthin and 4-6 mg of L-ergothioneine, continuously released in vitro for 16 hours, with a cumulative release of ≥80% over 16 hours, and the effective concentration in the release medium is maintained for up to 18 hours.

8. The use of the anti-aging composition according to any one of claims 1 to 4 or the anti-aging oral preparation according to any one of claims 6 to 7 in oral health foods that regulate abnormal mitochondrial energy metabolism.

9. The use of the anti-aging composition according to any one of claims 1 to 4 or the anti-aging oral formulation according to any one of claims 6 to 7 in oral health foods for maintaining muscle and cognitive health-related mitochondrial function.

10. The application according to claim 9, characterized in that, Maintaining muscle-related mitochondrial function involves reducing selective loss of type II muscle fibers or reducing respiratory chain supercomplex dissociation.

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