Multi-target-point composition for delaying senescence synergistically and dual application of multi-target-point composition

Abstract

The invention relates to the technical field of biological medicines and cosmetics, and particularly discloses a multi-target-point composition for delaying senescence synergistically and dual application of the multi-target-point composition. The core of the composition is that the composition contains four active components with synergistic effects in safe and effective doses: an aging cell scavenger, an NAD + precursor, a mitochondrial function optimizer and an immunomodulator. The main innovation of the invention lies in that the same core composition can be configured for two different ways of oral administration and external application: during oral administration, senescent cells can be systematically reduced, the overall energy metabolism and immune function can be improved, and the overall aging of the body can be delayed; when externally applied, the skin aging characterization can be improved in a targeted manner, for example, collagen regeneration is promoted, and wrinkles are reduced. In-vitro and in-vivo experiments prove that the composition has an anti-aging effect obviously superior to that of a single component under two application ways through a multi-target synergistic mechanism.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine, nutrition and health care, and cosmetics, specifically to a multi-target synergistic anti-aging composition and its dual applications. Background Technology

[0002] Aging is a complex biological process involving cumulative damage at multiple cellular and molecular levels. Its core mechanisms include the accumulation of senescent cells, a decline in nicotinamide adenine dinucleotide levels, mitochondrial dysfunction, oxidative stress, and immune system dysregulation. These factors collectively drive functional decline and the development of age-related diseases. At the skin level, these systemic and localized changes directly manifest as the loss of collagen and elastin, wrinkle formation, dull skin tone, and impaired skin's physical barrier function.

[0003] Recent studies have further revealed a close "dialogue" mechanism between the skin and other tissues and organs of the body. Senescent cells accumulated in the skin can enter the circulatory system by secreting aging-related phenotypes, such as various pro-inflammatory factors and matrix metalloproteinases, accelerating the systemic aging process. Conversely, the systemic aging internal environment can also have a reciprocal effect on the skin, exacerbating its aging manifestations. Therefore, a synergistic "inside-out" strategy combining oral systemic intervention and local skin intervention has become a cutting-edge direction in the field of anti-aging, especially in skin anti-aging.

[0004] However, existing anti-aging technologies have significant limitations: First, most solutions target only a single or a few pathways of aging, lacking comprehensive solutions that can systematically target multiple core mechanisms of aging; second, the simple physical combination of oral nutritional supplements and topical skincare products on the market is not based on a clear synergistic mechanism design, making it difficult to achieve the synergistic effect of "1+1>2"; finally, most solutions neglect the crucial role of the immune system in recognizing and clearing senescent cells and regulating chronic inflammation.

[0005] Therefore, there is an urgent need in this field for an innovative composition that can simultaneously and synergistically target multiple core pathways such as senescent cell clearance, cellular energy metabolism, mitochondrial function optimization, and immune regulation, and can be flexibly and effectively applied via both oral and topical routes. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a multi-target synergistic anti-aging composition with both oral and topical efficacy, as well as its applications. This composition, through carefully designed ingredients and proportions, achieves a comprehensive anti-aging effect, addressing both internal and external factors.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a composition for delaying aging, comprising the following four active ingredients in safe and effective doses: a) senescent cell scavenger; b) NAD+ precursor; c) mitochondrial function optimizer; d) immunomodulator.

[0008] Preferably, the senescent cell scavenger is fisetin; the NAD+ precursor is nicotinamide mononucleotide or nicotinamide ribose; the mitochondrial function optimizer is pyrroloquinoline quinone, α-ketoglutarate, or urolithin A; and the immunomodulator is β-glucan, Ganoderma lucidum polysaccharide, or Lycium barbarum polysaccharide.

[0009] Preferably, the four active ingredients are fisetin, nicotinamide mononucleotide, pyrroloquinoline quinone, and β-glucan.

[0010] Preferably, the composition comprises, by weight, 1-5 parts of fisetin and 5-20 parts of nicotinamide mononucleotide;

[0011] Pyrroloquinoline quinone 0.1-0.5 parts; β-glucan 1-5 parts;

[0012] The relative proportions of each active ingredient in the core raw material combination (Fisserone:NMN:PQQ:β-glucan≈(1-5):(5-20):(0.1-0.5):(1-5)). This ratio is based on the optimal synergistic range determined by in vitro synergistic effect screening experiments, reflecting the contribution weight and interaction relationship of each component in the biological effect, and is the chemometric basis for the "multi-target synergy" of this invention.

[0013] Preferably, the molar ratio of fisetin, nicotinamide mononucleotide, and pyrroloquinoline quinone is (5-15):(25-50):1.

[0014] Preferably, the composition is formulated for oral administration; the dosage range of the active ingredients, based on the recommended daily intake, is: fisetin 100-500 mg; nicotinamide mononucleotide 100-1000 mg; pyrroloquinoline quinone 10-40 mg; β-glucan 100-500 mg;

[0015] Based on the established relative synergistic ratios, and taking into account the standard safe dosage range for adult dietary supplements, the bioavailability of the ingredients, and the required in vivo exposure to achieve systemic anti-aging effects, these ratios are converted into absolute daily intakes per person. For example, the daily dose of fisetin is set at 100-500 mg, a range that covers the doses shown to be effective in animal models and is strictly controlled within recognized safety thresholds. The daily dosage ranges for other ingredients also follow the same principle, ensuring that the entire composition is safe and effective as an oral product.

[0016] Preferably, the composition is formulated for external use; based on the total weight of the external composition, the weight percentages of each active ingredient are: fisetin 0.01%-1%; nicotinamide mononucleotide 0.1%-5%; pyrroloquinoline quinone 0.001%-0.08%; β-glucan 0.1%-2%;

[0017] Based on the same core synergistic ratios mentioned above, the specific characteristics of topical skin applications must be prioritized. Topical formulations must balance transdermal efficiency, skin tolerance (irritation), and the usual requirements of cosmetic regulations regarding raw material concentrations. Therefore, the core ratios are translated into safe effective concentrations in the final dosage form (such as serums and creams). For example, the topical concentration of fisetin is set at 0.01%-1%, which ensures that it reaches an effective concentration at the target site (such as dermal fibroblasts) after transdermal absorption, while remaining well below the threshold that may cause skin irritation.

[0018] Preferably, the oral composition is prepared in the form of capsules, tablets, powders or oral liquids.

[0019] Preferably, the topical composition is prepared in the form of a serum, lotion, cream, gel, mask, ampoule, microneedle patch, or spray.

[0020] The present invention also provides the use of the composition in the preparation of health products, dietary supplements or medicines for oral administration to delay aging and prolong healthy lifespan.

[0021] The present invention also provides the use of the described composition in the preparation of cosmetics, skin care products or medical skin dressings for external use to improve skin aging.

[0022] Preferably, the improvement of skin aging includes: promoting skin collagen regeneration, reducing wrinkles, improving skin elasticity, brightening skin tone, and / or enhancing skin barrier function.

[0023] The beneficial effects of this invention are as follows:

[0024] 1. This invention is the first to propose that the same core composition can be used both orally and topically, achieving a "holistic" anti-aging strategy. When taken orally, it acts on the whole body, systematically reducing senescent cells and enhancing overall cellular energy and immune function; when applied topically, it precisely targets the skin, locally improving signs of aging.

[0025] 2. This composition, through its four core components, acts on multiple key pathways of aging, producing a synergistic effect. Its effects in reducing senescent cells, decreasing the expression of SASP and aging-related factors, and promoting collagen production are significantly superior to any single-component, two-component, or three-component combination. Furthermore, the synergistic value between the components has been demonstrated.

[0026] 3. This composition can be developed into oral forms such as capsules and tablets, or formulated into topical dosage forms such as serums and creams to meet consumers' urgent need for comprehensive and systematic anti-aging solutions.

[0027] Furthermore, the four components of the composition of the present invention are not simply superimposed, but constitute a multi-target synergistic anti-aging network. Its core lies in breaking the vicious cycle of mutual promotion between the accumulation of senescent cells and systemic inflammation, and rebuilding tissue homeostasis.

[0028] Initiating clearance (targeting senescent cells): Fiseridone, as a senescent cell scavenger, can specifically induce apoptosis in senescent cells. Its action is related to the regulation of signaling pathways such as p53 / p21 and PI3K / Akt. Clearing senescent cells is fundamental to reducing the aging-associated secretory phenotype (SASP).

[0029] Energy Enhancement and Cell Repair (Targeting Cellular Function): As an NAD+ precursor, NMN increases intracellular NAD+ levels and activates deacetylases such as SIRT1. SIRT1 deacetylates and inhibits pro-inflammatory transcription factors such as NF-κB, thereby suppressing SASP production. Simultaneously, it synergizes with pyrroloquinoline quinone (PQQ), which promotes mitochondrial biogenesis by activating the PGC-1α signaling pathway and enhances NMN's activation effect on SIRT1. Together, they optimize mitochondrial function, reduce oxidative stress (ROS), and increase ATP, providing energy for cell repair.

[0030] Immune Regulation and Homeostasis Reconstruction (Targeting the Systemic Environment): Immunomodulators such as β-glucan are the system integrators in this protocol. Their role goes far beyond simply "enhancing immunity," but rather achieving immune reprogramming.

[0031] Promotes clearance: By activating pattern recognition receptors (such as Dectin-1) on the surface of immune cells such as macrophages and dendritic cells, it enhances their ability to recognize and phagocytose senescent cells, forming a dual clearance mechanism of "chemical + immune" with the chemical clearance of fisetin.

[0032] Regulating inflammation: It guides macrophages to polarize from the pro-inflammatory M1 type to the anti-inflammatory and repair-promoting M2 type, thereby precisely regulating the local inflammatory response that may be triggered after SASP clearance, avoiding excessive inflammation that could cause damage, and secreting growth factors to promote tissue repair.

[0033] Improve immune aging: Reverse age-related T cell depletion and decline in immune surveillance function, restore the body's ability to continuously monitor senescent and abnormal cells, and form a long-lasting anti-aging immune environment.

[0034] In summary: fisetin clears senescent cells, reducing the burden of SASP (saturated cellular inflammatory processes); NMN and PQQ empower and repair remaining functional cells, enhancing their health and vitality; β-glucan, by regulating the immune system, assists in clearing senescent cells while stabilizing the internal environment, shifting the tissue after senescent cell clearance to a regenerative repair mode, rather than fibrosis or chronic inflammation. The resulting healthier cells produce fewer SASPs, further reducing the burden on the immune system, thus forming a positive synergistic anti-aging cycle. Together, they break the vicious cycle of "accumulation of senescent cells → chronic SASP inflammation → damage to healthy cells → production of more senescent cells," promoting a virtuous cycle of "clearing senescent cells → inflammation subsiding → restoration of cell function → tissue regeneration and repair." This multi-target, synergistic approach, covering the three dimensions of cells themselves, energy metabolism, and systemic immunity, is the fundamental mechanism by which it exerts a "1+1>2" anti-aging effect. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 A bar chart showing the effects of different treatment groups on the proportion of senescent cells, the secretion of SASP factor in cell supernatant, and the expression of senescence-related genes p16 and p21 in cell lysate in a replicative senescent human embryonic lung fibroblast or human dermal fibroblast model.

[0037] Figure 2 Bar chart showing the effects of different treatment groups on mitochondrial ROS and ATP in replicative senescent MRC-5 or HDF cell models.

[0038] Figure 3 Bar chart showing the effect of each treatment group on the content of type I and III collagen in a replicative senescent HDF cell model.

[0039] Figure 4 : A bar chart showing the effects of different treatment groups on the behavioral status and the proportion of senescent cells in the liver of aging mice in a naturally aging mouse model;

[0040] Figure 5 A bar chart showing the effects of different treatment groups on the content of type I and III collagen in the skin tissue of aging mice in a naturally aging mouse model. Detailed Implementation

[0041] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] Example 1: In vitro validation of the synergistic anti-aging effect of the composition

[0043] Establishment of a cellular senescence model and compound treatment: Human embryonic lung fibroblasts (MRC-5) or human dermal fibroblasts (HDF) were continuously passaged to the population doubling arrest phase to establish a replicative senescent cell model. Young cells (low passage count) served as a negative control, and dasatinib (D, 0.1 μM) + quercetin (Q, 10 μM) served as a positive control. When cells adhered and grew to 70%-80% confluence, the medium was replaced with fresh medium containing the corresponding test compound, and cultured for another 72 hours. The model group was then replaced with an equal volume of solvent medium. Cells were randomly divided into the following groups:

[0044]

[0045] Experimental methods:

[0046] (1) Verify the effect of the composition on senescence markers in senescent MRC-5 and HDF cells: The proportion of senescent cells in each treatment group was detected by SA-β-Gal method; the level of SASP factors (IL-6 and IL-1β) in cell supernatant was detected by ELISA; and the expression of senescence-related genes p16, p21 and MMP3 was detected by PCR.

[0047] SA-β-gal staining: The proportion of senescent cells in a cell range was determined using a cellular senescence β-galactosidase staining kit. The kit uses X-Gal as a substrate, which generates a deep blue product under the catalysis of senescence-specific β-Gal. Therefore, the degree of cellular senescence is measured by observing the cells that turn blue. The assay was performed according to the kit instructions. Images were captured under a fluorescence microscope, and the number of blue-positive cells was counted as senescent cells.

[0048] ELISA assay: Collect the culture supernatant of cells from each group, centrifuge at 4°C and 1000×g for 10 minutes, and collect the supernatant. IL-6 and IL-1β levels were detected using an ELISA kit, strictly following the instructions.

[0049] Detection of aging-related gene expression levels: Total RNA was extracted from cells in each group using TRIzol reagent; then, 1 μg of total RNA was reverse transcribed into cDNA using a reverse transcription kit, following the kit instructions; using cDNA as a template, amplification was performed on a real-time quantitative PCR instrument using SYBR Green qPCR premixed reagent. GAPDH was used as an internal control, and the relative expression levels of p16, p21, and MMP3 genes were calculated using the 2-ΔΔCt method. The primer sequences for the above genes are as follows:

[0050]

[0051] (2) Verify the effect of the composition on mitochondrial function in senescent MRC-5 and HDF cells: Characterize changes in mitochondrial function by measuring mitochondrial ROS and ATP levels in cells.

[0052] Mitochondrial ROS level detection: After cell treatment in each group, the culture medium was discarded, and the cells were washed once with pre-warmed serum-free medium. MitoSOX™ Red mitochondrial superoxide indicator diluted with serum-free medium was added, and the cells were incubated in a 37°C cell culture incubator in the dark for 30 minutes. After incubation, the cells were washed three times with pre-warmed PBS to thoroughly remove any probes that had not entered the cells. The cells were digested and resuspended with trypsin, and immediately detected by flow cytometry (excitation / emission wavelengths: 510nm / 580nm), or observed and images were acquired using a fluorescence microscope, and the average fluorescence intensity was calculated using image analysis software.

[0053] ATP level detection: After cell treatment in each group, the culture medium was discarded, and the cells were gently washed once with pre-chilled PBS. An appropriate amount of ATP lysis buffer was immediately added, and the cells were lysed on ice. The cell lysis buffer was collected and centrifuged at 4°C and 12000×g for 5 minutes, and the supernatant was collected. The ATP detection kit was used for detection. The sample was mixed with the ATP detection working solution in a white, opaque 96-well plate, and the relative photon value was read using a chemiluminescence analyzer. The ATP concentration of each sample was calculated based on the simultaneously plotted ATP standard curve and standardized using the protein concentration determined by the BCA method.

[0054] (3) Verify the effect of the composition on the content of type I and III collagen in senescent HDF cells: by detection using an ELISA kit.

[0055] Experimental results:

[0056] (1) Verification of the effect of the composition on senescence markers in senescent MRC-5 and HDF cells: Since fisetin has been reported to have a clear ability to clear senescent cells, and its combination with the immune enhancer β-glucan can exert a synergistic effect in clearing senescent cells, in verifying the effect of the composition on senescence markers in senescent MRC-5 and HDF cells, in addition to the four single-component groups, fisetin + β-glucan was used as a two-component comparative group, and nicotinamide mononucleotide and / or pyrroloquinoline quinone were added on this basis. The specific cell groups were as follows: Figure 1 The cells are grouped as shown. Figure 1 As shown in the figure. The results showed that compared with the control group, the proportion of senescent cells, the levels of SASP factors such as IL-6 and IL-1β, and the relative expression levels of aging markers such as p16, p21, and MMP3 genes were significantly increased in the model group. After administration of four single components—fisetin, nicotinamide mononucleotide, pyrroloquinoline quinone, and β-glucan—only fisetin could significantly reduce the proportion of senescent cells and decrease the levels of SASP factors and aging-related genes. When fisetin was used in combination with β-glucan (Comparative Example 5, F+G), the anti-aging activity was further significantly enhanced, while continued use in combination with nicotinamide mononucleotide (Comparative Example 7, F+N+G) or pyrroloquinoline quinone (Comparative Example 8, F+N+G) showed a significantly enhanced anti-aging activity. In Example 8, when the four components were used in combination (Examples 1 and 2, F+N+P+G), the anti-aging activity was not significantly enhanced. However, when the four components were used in combination (Examples 1 and 2, F+N+P+G), the proportion of senescent cells, the level of SASP factor, and the level of aging-related genes were significantly reduced. This effect was superior to the F+G, F+N+G, and F+P+G groups, and significantly superior to the four single-component groups (Comparative Examples 1-4) and the positive control group. However, when the ratio of the four components was not within the synergistic effect range protected by this invention (Example 3, F+N+P+G), the anti-aging activity of the composition was significantly weakened compared to Examples 1 and 2, and comparable to the anti-aging activity of the F+G, F+N+G, and F+P+G groups. The above demonstrates that the anti-aging efficacy of the composition of this invention is superior to that of any single component, bimolecular combination, or trimolecular combination, and can synergistically reduce senescent cells and lower the levels of SASP factor and aging-related genes.

[0057] (2) Validation of the effect of the composition on mitochondrial function in senescent MRC-5 and HDF cells: Since nicotinamide mononucleotide and pyrroloquinoline quinone have been reported to optimize mitochondrial function, in validating the effect of the composition on senescence markers in senescent MRC-5 and HDF cells, in addition to the four single-component groups, nicotinamide mononucleotide + pyrroloquinoline quinone was used as a two-component control group, and F and / or G were added in combination. The specific cell groups were as follows: Figure 2As shown. The results showed that compared with the control group, the ROS level of the model group cells was significantly increased and the ATP content was significantly decreased. After administration of the four single components, namely fisetin, nicotinamide mononucleotide, pyrroloquinoline quinone and β-glucan, the ROS level and ATP content did not change significantly compared with the model group. When nicotinamide mononucleotide was used in combination with pyrroloquinoline quinone (Comparative Example 6, N+Q), and continued to be used in combination with fisetin and / or β-glucan (Comparative Example 9, N+P+F; Comparative Example 10, N+P+G; Examples 1 and 2, F+N+P+G), the ROS level and ATP content increased and decreased significantly compared with the model group, respectively, and the effect was better than that of each single component. However, there was no significant difference between the groups in Comparative Examples 6, 9, 10 and Examples 1 and 2. However, when the ratio of the four components used in combination was not within the synergistic effect range protected by this invention (Example 3, F+N+P+G), the composition failed to significantly reduce the ROS level and increase the intracellular ATP content. The above demonstrates that the composition of the present invention can synergistically improve mitochondrial function, and the effect is significantly better than that of any single component.

[0058] (3) Verification of the effect of the composition on the content of type I and III collagen in senescent HDF cells: In order to verify the efficacy of the composition in anti-skin aging, the content of type I and III collagen in HDF cells was used as the detection index. Based on the above experimental results, F+G and N+P were set as the control groups of the two-component composition, and other components were used in combination on this basis. The specific cell groups were as follows: Figure 3 As shown in the figure. The results showed that compared with the control group, the content of type I and III collagen in the model group cells was significantly reduced; among the different treatment groups, only the collagen content in the group with the composition of the present invention (Examples 1 and 2, F+N+P+G) was significantly increased, and significantly better than the F+G and N+P groups; however, when the ratio of the four components used in combination was not within the synergistic effect range protected by the present invention (Example 3, F+N+P+G), the composition failed to significantly increase the collagen content. The above demonstrates that the anti-skin aging effect of the composition of the present invention is superior to that of the bimolecular or trimolecular combination, and can synergistically promote the regeneration of type I and III collagen, thereby reducing wrinkles.

[0059] Example 2: In vivo verification of the oral composition's ability to delay aging, or verification of the combined oral and topical application of the composition to combat skin aging.

[0060] Experimental animals: 18-month-old naturally aging male C57BL / 6J mice were selected. They were housed in an animal room with constant temperature (24±2℃), constant humidity (50-60%), natural light, and good ventilation, with free access to water and food. All experimental procedures complied with the regulations of the Animal Use and Management Committee of Air Force Medical University.

[0061] Experimental methods:

[0062] (1) To verify the effect of oral composition on the aging phenotype and immune aging, mice were randomly divided into the following groups. After 12 weeks of intervention, the physical and cognitive functions of mice were tested by open field, Y-maze and rotarod tests. In addition, regulatory T cells (Tregs) are the main effector cells of the immune system that regulate disease and body state. Treg dysregulation is one of the important characteristics of immune aging. The spleen is the largest peripheral immune organ in the human body. When the number of Tregs in the spleen increases, it will cause immune decline. Therefore, SA-β-Gal staining and flow cytometry were used to detect the proportion of senescent cells and CD4 in spleen tissue, respectively. + FoxP3 + The proportion of Treg cells was used to verify the efficacy of the oral composition in improving immune aging. Simultaneously, the levels of pro-inflammatory cytokine IL-1β and anti-inflammatory cytokine IL-10 in mouse serum were detected using ELISA.

[0063]

[0064] Open field experiment: The size of the open field reaction chamber is 40×40×40cm. 3 The central area of ​​the test chamber measured 24cm x 24cm, with a digital camera positioned 2m directly above to ensure full coverage of the open field. At the start of the experiment, mice were placed in the center of the chamber, and their behavior was recorded over 15 minutes. After each experiment, the inner walls and bottom of the chamber were cleaned with alcohol to prevent residual odors from the previous test from affecting the next result. Finally, software was used to calculate the total distance the mice traveled in the open field, thus evaluating changes in their spontaneous activity.

[0065] Y-maze experiment: The Y-maze apparatus has three identical arms, each 40×3×15cm in length, arranged at a 120° angle. A spontaneous alternation test is conducted within the Y-maze to assess the learning and memory functions of mice. Mice are randomly placed in one arm of the maze and allowed to explore all three arms undisturbed for 8 minutes. One spontaneous alternation is defined as when a mouse enters all three arms in sequence. The percentage of spontaneous alternations (%) is calculated as [(number of alternations) / (total number of arm entries - 2)] × 100%.

[0066] Spinning bar experiment: An SA102 rotating bar apparatus was used for the experiment. Mice were first pre-trained for three consecutive days: during each training session, the rotating bar speed was gradually increased from 4 revolutions per minute to 40 revolutions per minute over 300 seconds. On the day of the actual test, each mouse was tested, and the time from the start of the spin to the fall was recorded. This test was repeated every 30 minutes for a total of three times. The average of the three fall latency tests was used to assess the mouse's motor coordination and endurance.

[0067] SA-β-gal staining: The proportion of senescent cells in liver and spleen tissues was detected using a cellular senescence β-galactosidase staining kit. The assay was performed according to the kit instructions: frozen sections of mouse tissue were prepared, thawed, and then washed three times with PBS for at least 5 min each time. An appropriate volume of β-Gal staining fixative was added, and the samples were fixed at room temperature for at least 15 min. The samples were then washed three times with PBS for at least 5 min each time and incubated overnight at 37°C. Images were captured under a fluorescence microscope, and the number of blue-positive cells was counted as senescent cells.

[0068] Flow cytometry detection of CD4 in mouse spleen + FoxP3 + Treg: After mouse sacrifice, the spleen was quickly removed and placed in PBS. The spleen was ground into a single-cell suspension using a grinding rod, filtered through a 70μm cell filter, and the filtrate was collected into a 15mL centrifuge tube. The cell suspension was then collected and incubated with the appropriate antibody for staining. The stained cell samples were placed in a flow cytometer, and data were acquired according to the instrument operating procedures. CD4 counts were calculated. + FoxP3 + Treg cells account for a significant portion of total CD4+ cells. + The proportion of T cells.

[0069] ELISA assay: After the behavioral test, blood was collected from the orbital sinus of mice, and serum was collected by centrifugation at 3000-4000 rpm for 20 min at 4℃. The levels of IL-1β and IL-10 in the serum of each group were detected using an ELISA kit according to the manufacturer's instructions.

[0070] (2) To verify the combined oral and topical application of the composition for anti-skin aging, mice were randomly divided into the following groups. After 12 weeks of intervention, skin tissue from the back of the mice was taken, and the content of type I and III collagen was detected by ELISA.

[0071]

[0072] Experimental results:

[0073] (1) Verify that the oral composition improves the aging phenotype and immune aging: such as Figure 4 As shown: Compared with the young control group, aged mice exhibited significant impairments in autonomous movement and cognitive function, as well as motor dysfunction; and the proportion of senescent cells in spleen tissue was significantly increased, along with CD4+. + FoxP3 +The proportion of Treg cells also increased; the level of pro-inflammatory factor IL-1β in mouse serum was significantly increased, while the level of anti-inflammatory factor IL-10 was significantly decreased. After oral administration of fisetin, nicotinamide mononucleotide, pyrroloquinoline quinone, and β-glucan, the behavioral disorders and the above-mentioned immunosenescence indicators in mice were improved, but not significantly; while the behavioral disorders and immunosenescence characteristics of mice in the F+G, F+N+G, and the four-component composition group of the present invention (F+N+P+G) were significantly improved, among which the anti-aging efficacy of the composition group of the present invention was the best and superior to the positive control drug.

[0074] (2) Verify the combined oral and topical application of the composition to combat skin aging: such as Figure 5 As shown, compared with the young control group, the content of type I and III collagen in the skin of aging mice was significantly reduced. The combined oral and topical application of F+N+G, the oral and topical application of F+N+P+G, and the combined oral and topical application of F+N+P+G significantly increased the content of type I collagen. The combined oral and topical application of F+N+P+G showed the best effect, significantly superior to the oral and topical application of F+G alone, or the oral and topical application of F+N+G alone. Furthermore, the topical and oral application of F+N+P+G, as well as the combined oral and topical application of F+N+P+G, significantly increased the content of type III collagen. The combined oral and topical application of F+N+P+G showed the best effect, significantly superior to the oral, topical, and combined oral and topical application of F+N+G alone. This demonstrates that the composition of the present invention has definite anti-skin aging effects whether taken orally or applied topically, and that the combined use of both achieves the best results.

[0075] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A composition for delaying aging, characterized in that, It contains the following four active ingredients in safe and effective doses: a) Cell scavenger; b) NAD+ precursor; c) Mitochondrial function optimizers; d) Immunomodulators.

2. The composition according to claim 1, characterized in that: The senescent cell scavenger is fisetin; The NAD+ precursor is nicotinamide mononucleotide or nicotinamide ribose; The mitochondrial function optimizer is pyrroloquinoline quinone, α-ketoglutarate, or urolithin A; The immunomodulator is β-glucan, Ganoderma lucidum polysaccharide, or Lycium barbarum polysaccharide.

3. The composition according to claim 2, characterized in that, The four active ingredients are fisetin, nicotinamide mononucleotide, pyrroloquinoline quinone, and β-glucan.

4. The composition according to claim 3, characterized in that, By weight, it comprises: 1-5 parts of fisetin; Nicotinamide mononucleotide 5-20 parts; 0.1-0.5 parts of pyrroloquinoline quinone; 1-5 parts of β-glucan.

5. The composition according to claim 3 or 4, characterized in that, The molar ratio of fisetin, nicotinamide mononucleotide, and pyrroloquinoline quinone is (5-15):(25-50):

1.

6. The composition according to any one of claims 1-5, characterized in that, The composition is formulated for oral administration; the dosage range of the active ingredient, based on the recommended daily intake, is as follows: fisetin 100-500mg; Nicotinamide mononucleotide 100-1000mg; Pyrroloquinoline quinone 10-40 mg; β-glucan 100-500mg.

7. The composition according to any one of claims 1-5, characterized in that, The composition is formulated for external use; the weight percentage of each active ingredient, based on the total weight of the external composition, is as follows: Fesetone 0.01%-1%; Nicotinamide mononucleotide 0.1%-5%; Pyrroloquinoline quinone 0.001%-0.08%; β-glucan 0.1%-2%.

8. The composition according to claim 6, characterized in that, The oral composition is prepared in the form of capsules, tablets, powders or oral liquids.

9. The composition according to claim 7, characterized in that, The topical composition is prepared in the form of serum, lotion, cream, gel, mask, ampoule, microneedle patch or spray.

10. Use of a composition according to any one of claims 1-5 and 8 in the preparation of a health product, dietary supplement or medicine for oral administration to delay aging and prolong healthy life.

11. Use of a composition according to any one of claims 1-5, 7 and 9 in the preparation of a cosmetic, skin care product or medical skin dressing for external use to improve skin aging.

12. The use according to claim 11, characterized in that, The improvements in skin aging include: promoting collagen regeneration, reducing wrinkles, improving skin elasticity, brightening skin tone, and / or enhancing skin barrier function.

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