Application of pseudo-ginsenoside RT5 in preparation of anti-cell and overall aging product

Abstract

The invention discloses an application of pseudo-ginsenoside RT5 in preparation of anti-cell and overall aging products. Pseudoginsenoside RT5 is protocatechol type saponin, and it is proved for the first time that pseudoginsenoside RT5 has remarkable anti-aging activity. In-vitro experiments show that the pseudoginsenoside RT5 relieves cell senescence through a multi-target mechanism (such as improvement of cell viability, reduction of SA-beta-gal and inhibition of SASP), and particularly, the average inhibition rate of the pseudoginsenoside RT5 to reactive oxygen species (ROS) reaches up to 62.9%. An in-vivo zebra fish experiment further verifies the overall anti-aging effect, and the SA-beta-gal staining index score in a D-galactose induced rapid aging model can be remarkably reduced. The pseudo-ginsenoside RT5 provided by the invention is derived from medicinal and edible plants, has the advantages of high safety and suitability for long-term use, and provides an efficient and multi-target acting natural active ingredient for preparing anti-aging products (such as foods, medicines, skin care products, cosmetics and the like).

Description

Technical Field

[0001] This invention belongs to the field of anti-aging technology of small molecule compounds derived from natural products, specifically involving a protocatechuic saponin compound, Pseudoginsenoside Rt5, which has significant anti-cellular and overall aging activity, and its application in the preparation of anti-aging active products. Background Technology

[0002] Aging is a natural process of gradual functional decline in organisms from the molecular to the systemic level, involving the gradual decline of cellular, tissue, and overall physiological functions, and accompanied by an increased risk of age-related diseases such as cardiovascular disease, diabetes, and neurodegenerative diseases. Statistical studies have found that physiological age is the leading risk factor for major diseases and causes of death worldwide, with approximately half of all people over 65 years of age suffering from at least one chronic disease. As the aging of society intensifies, people's healthy lifespan will face even more severe challenges, making aging a major issue in the field of life and health.

[0003] Currently, the main characteristics driving aging are considered to include genomic instability, epigenetic alterations, mitochondrial dysfunction, cellular senescence, stem cell depletion, and chronic inflammation. Among these, cellular senescence is the foundation of organ and individual aging and is considered a key driver of aging and age-related diseases. Senescent cells stably exit the cell cycle and lose their proliferative capacity, entering a terminal state of growth arrest. At the molecular level, senescent cells are characterized by high expression of cell cycle repressor proteins such as p16 and p21, increased activity of senescence-associated β-galactosidase (SA-β-gal), and accumulation of DNA damage markers such as γH2AX. Furthermore, one of the most prominent characteristics of senescent cells is the senescence-associated secretory phenotype (SASP), with most senescent functions involving SASP. SASP mainly includes pro-inflammatory factors such as IL-6, IL-8, and TNF-α, as well as lipids and matrix metalloproteinases. Senescent cells establish a chronic inflammatory microenvironment by secreting SASP. Over time, sustained SASP enables senescent cells to resist elimination and causes more surrounding cells to senescent, leading to fibrosis and functional decline in surrounding tissues, and promoting the development of age-related diseases such as Alzheimer's disease and cardiovascular disease. Currently, therapies targeting senescent cells can be broadly divided into two categories. One category consists of drugs that treat chronic diseases by eliminating senescent cells, but these generally suffer from problems such as insufficient targeting and systemic side effects. The other category comprises drugs that can alleviate cellular senescence phenotypes, which is currently a more feasible research direction.

[0004] Humans have used natural products to treat various diseases for thousands of years. Natural products possess rich structural diversity and exhibit a wide range of biological activities. Compared with synthetic drugs, these natural products often have fewer adverse reactions and higher acceptability among a large number of patients worldwide, and are also less expensive. Therefore, natural products remain a very important source of pharmaceutical formulations. In the nearly 40 years from 1981 to 2019, two-thirds of the 1394 small molecule drugs approved by the FDA were derived directly or indirectly from natural products. In recent years, the anti-aging activities of more and more natural products have been discovered. For example, astragalus extract can increase telomerase activity, and extracts of Ganoderma lucidum and Panax notoginseng have been reported to improve heart function and prolong healthy lifespan. In conclusion, natural product molecules have strong application potential in the field of anti-aging.

[0005] Ginsenoside RT5 (CAS No.: 98474-78-3) is a ginsenoside originally derived from American ginseng (Panax quinquefolius). Panax quinquefolium The protocatechuic saponins were extracted and purified from [the sample / material]. Existing research indicates that ginsenoside RT5 has renal protective, anti-tumor, and anti-myocardial ischemia effects. However, no published literature reports any anti-cellular aging activities of ginsenoside RT5, such as restoring cell vitality, reducing aging-related β-galactosidase activity, inhibiting SASP component secretion, and possessing strong antioxidant effects. Furthermore, there is no verification of its anti-aging effects in in vivo animal models (overall aging).

[0006] Therefore, based on existing technologies, this invention is the first to demonstrate that ginsenoside RT5 possesses significant dual anti-cellular and systemic aging activities. Ginsenoside RT5 can effectively enhance cell viability through multiple target pathways, significantly reduce aging-related β-galactosidase (SA-β-gal) activity, inhibit the secretion of key factors of the aging-related secretory phenotype (SASP) (such as IL-6 and IL-8), and effectively reduce intracellular reactive oxygen species (ROS) levels. In a D-galactose-induced rapid aging model in zebrafish, RT5 also exhibited significant in vivo systemic anti-aging effects. Compared to existing anti-aging therapies (such as limited drug types and potential toxic side effects), the ginsenoside RT5 provided by this invention is a multi-target, highly effective, and naturally active ingredient derived from a "food and medicine homology" substance (American ginseng). Its raw material, American ginseng, is included in the National Food and Drug Catalogue, ensuring high safety and suitability for long-term use. This characteristic gives it a significant advantage in developing anti-aging related products. It can be used not only in pharmaceuticals, but also in functional foods, dietary supplements, skin care products, and even fragrance products. It provides new core ingredients for developing safe, effective, and easy-to-use anti-aging solutions, with broad application prospects. Summary of the Invention

[0007] This invention discloses the application of ginsenoside RT5 in the preparation of anti-cellular and overall aging products. The purpose of this invention is to address the limitations of existing anti-aging technologies, such as the limited variety of naturally derived active molecules and the common problems of insufficient targeting and systemic side effects in therapies targeting senescent cells. This invention aims to overcome the shortcomings of existing anti-aging drugs.

[0008] To achieve the above objectives, the present invention provides the application of ginsenoside RT5 in the preparation of anti-cellular and overall aging products.

[0009] The ginsenoside RT5 (CAS No.: 98474-78-3) is a protocatechuic acid type saponin, initially extracted and purified from the medicinal and edible plant Panax quinquefolium, with the chemical formula C. 36 H 62 O 10 The structural formula is as follows:

[0010] The three-dimensional configuration of position C-20 is S-type, R-type, or a mixture thereof.

[0011] The anti-cellular and overall aging products have one or more of the following functions: improving cell viability; inhibiting the secretion of aging-associated secretory phenotype (SASP) factors (such as IL-6 and IL-8); reducing intracellular reactive oxygen species (ROS) levels; and reducing the activity of aging-associated β-galactosidase in zebrafish as a whole.

[0012] The enhancement of cell vitality refers to increasing the vitality of senescent cells by at least 45.0% or more. The senescence-related secretory phenotypic factors include interleukin-6 and interleukin-8. The average reduction rate of intracellular reactive oxygen species levels is at least 62.9% or more.

[0013] The reduction of aging-related β-galactosidase activity in zebrafish refers to a significant reduction of β-galactosidase activity by 25% or more in a D-galactose-induced rapid aging model of zebrafish.

[0014] The products include, but are not limited to: food, medicine, skin care products, cosmetics or fragrance products.

[0015] The beneficial effects of this invention are as follows: This invention represents a breakthrough based on existing technologies, providing the first clear evidence that ginsenoside RT5 possesses significant dual activities against both cellular and overall aging. Ginsenoside RT5 can effectively enhance cell viability (increasing the viability of senescent cells by 45.0%), significantly reduce aging-related β-galactosidase (SA-β-gal) activity (decreasing by 17.5%), inhibit the secretion of key factors in the aging-related secretory phenotype (SASP) (such as IL-6 and IL-8), and effectively reduce intracellular reactive oxygen species (ROS) levels (with an average inhibition rate of 62.9%) through multiple target pathways. In a D-galactose-induced rapid aging model of zebrafish, RT5 also exhibited a significant in vivo overall anti-aging effect (reducing aging by 25%).

[0016] Compared to the limitations of existing anti-aging molecules, which have single-target effects and potential toxic side effects, the ginsenoside RT5 provided by this invention is a multi-target, highly effective, and naturally active ingredient derived from the "food and medicine homology" plant American ginseng. It boasts advantages such as high safety and suitability for long-term use, greatly expanding its application prospects in the preparation of food, pharmaceuticals, skincare products, cosmetics, and fragrance products. This invention possesses outstanding novelty, inventiveness, and significant application value. Attached Figure Description

[0017] Figure 1 A diagram of an etoposide-induced senescence model of IMR-90 cells, used to detect senescence-associated β-galactosidase. Figure 2 The images show the chemical structure of ginsenoside RT5 and a bar graph of cell viability assays. Figure A shows the chemical structure of ginsenoside RT5, and Figure B shows the bar graph of cell viability assays. Proliferating represents the proliferating group, and Senescent represents the senescent group. Figure 3 The bar graphs show the detection and quantitative results of senescence-related β-galactosidase in cells treated with ginsenoside RT5. Figure A shows the detection of senescence-related β-galactosidase in cells treated with ginsenoside RT5, and Figure B shows the quantitative results. Control represents the proliferating group, and Old represents the senescent group. Figure 4 The images show the ELISA standard curve and bar graph of the inhibitory effect of ginsenoside RT5 on SASP active factors IL6 and IL8. Figure A shows the ELISA standard curve for SASP active factor IL6, Figure B shows the ELISA standard curve for SASP active factor IL8, Figure C shows the quantitative results of SASP active factor IL6, and Figure D shows the quantitative results of SASP active factor IL8. Figure 5Figure A shows the peak shape and bar graph of fluorescence signal intensity detected by flow cytometry to show the inhibitory effect of ginsenoside RT5 on ROS. Figure B shows the peak shape of fluorescence signal intensity detected by flow cytometry and the bar graph of the inhibitory effect of ginsenoside RT5 on ROS. Figure 6 The images show the grouped anthropometric images of a D-galactose-induced rapid aging model in zebrafish. Group A is the proliferating group, group B is the aging group, normal is the blank control group, D+ is the D-galactose monotherapy group, and RT5 is D-galactose plus ginsenoside RT5.

[0018] Figure 7 Quantitative analysis of β-galactosidase activity of ginsenoside RT5 in a D-galactose-induced rapid aging model of zebrafish. Detailed Implementation

[0019] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0020] Example 1. Construction of a chemotherapy-induced IMR-90 senescent cell model (1) Culture of IMR90 cells The IMR-90 human female fetal lung fibroblast cell line was purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number GNHu32. IMR-90 cells were cultured normally in DMEM (Dulbecco's Modified Eagle Medium) complete medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution. The culture environment was 37°C and 5% carbon dioxide, with the medium replaced fresh every 48 hours.

[0021] Cell passage: Aspirate the culture medium, wash the cells three times with PBS, then digest with 0.05% trypsin at 37°C for 1.5 minutes. Terminate the reaction by adding complete culture medium, centrifuge at 1000 rpm for 3 minutes, aspirate the supernatant, resuspend the cells in fresh complete culture medium, and passage at a 1:2 ratio. All cells used in the experiment were passaged no more than 18 times.

[0022] Cell cryopreservation and thawing: The cryopreservation solution for IMR-90 cells was prepared using 60% DMEM complete medium supplemented with 30% fetal bovine serum and 10% DMSO. After trypsin digestion and centrifugation, the cells were resuspended in the cryopreservation solution, then aliquoted into cryovials (1 ml each), placed in a cryovial box for slow freezing, and stored at -80°C or in liquid nitrogen. For IMR-90 cell thawing, the cells were removed from the freezer or liquid nitrogen tank and rapidly thawed in a 37°C water bath. The cell suspension was then centrifuged at 1000 rpm for 3 minutes, the supernatant was collected, and the cells were resuspended in fresh DMEM complete medium. The cells were then seeded into cell culture dishes and cultured in a cell culture incubator.

[0023] (2) IMR90 cell-induced senescence model Etoposide acts on DNA topoisomerase II, inducing apoptosis in tumor cells by blocking the cell cycle. Currently, etoposide is mainly used clinically for chemotherapy treatment of cancers such as small cell lung cancer. In basic research, etoposide has been found to induce cellular senescence. Using etoposide to induce cellular senescence not only allows for the study of cellular senescence but also simulates the scenario of cellular senescence induced by the side effects of chemotherapy drugs in real-world tumor treatment.

[0024] Etoposide was dissolved in DMSO solution to prepare a 50 mM stock solution, which was then diluted and aliquoted into a 10 mM concentrate for use. After cell passage, when the cell density reached 30%-50% confluence, the 10 mM etoposide solution was added to the culture medium at a volume ratio of 1:200, resulting in a working concentration of 50 μM etoposide. Etoposide induced cell senescence for 48 hours, followed by replacement with fresh DMEM complete medium to allow the cells to recover for 72 hours. These cells were designated as senescent-induced cells. The control group cells were treated with 0.5% DMSO for 48 hours, with the remaining procedures identical to the senescent-induced cells. These cells served as the normal proliferation control group for subsequent experiments. After induction treatment, the senescence marker β-galactosidase was detected, and IMR-90 cells exhibited significant cellular senescence (…). Figure 1 ).

[0025] Example 2. Determination of the anti-cellular aging activity of ginsenoside RT5 Seed cells into 96-well plates, approximately 10 cells per well. 4Cells were cultured. After 24 hours of normal cell adhesion and growth, the test drug was added for the experiment. The ginsenoside RT5 used was purchased from MedChemExpress and prepared as a 10mM stock solution using DMSO. During the experiment, the compound was diluted in the cell culture medium to working concentrations of 10μM and 1μM. A negative control group was also set up, with 0.1% DMSO added to the culture medium volume as a substitute. Each different drug treatment group was set up with 3 wells as replicates. After adding the drug, the cells were incubated in a cell culture incubator for 72 hours. Cell viability was detected using CCK8. CCK8 was diluted 1:10 by volume in the cell culture medium, and 100μl of CCK8 working solution was added to each well. The cells were incubated in a cell culture incubator for 40 min. Then, the signal intensity was detected at a wavelength of 450nm using a microplate reader. 620nm was used as the background reference wavelength. The signal intensity was subtracted from the signal at the reference wavelength to obtain the true cell viability value. A bar graph was plotted based on the experimental results. Figure 2 B).

[0026] The results are as follows Figure 2 As shown in Figure B, ginsenoside RT5 at a concentration of 10 μM effectively increased the cell viability of IMR-90 cells, increasing cell viability by 26.4% and 45.0% in the normal proliferation group and the senescent group, respectively. In particular, ginsenoside RT5 showed a significantly stronger cell viability-enhancing effect in the senescent group, reflecting its good anti-cellular senescence activity.

[0027] Example 3. Effects of ginsenoside RT5 on cellular senescence markers (β-galactosidase) Cells were seeded in 6-well plates and cultured, with separate groups for normal proliferation and senescence. The experimental groups were treated with 10 μM ginsenoside RT5 for 72 h before detection. The negative control group was treated with 0.1% DMSO (by volume of culture medium). The experiment was performed using a β-galactosidase staining kit (Solarbio). After aspirating the culture medium, the cells were washed once with PBS, and 1 ml of β-Gal fixative was added for fixation at room temperature for 15 min. The fixative was then aspirated, and the cells were washed three times with PBS (3 min each time). A staining working solution was prepared fresh at a reagent volume ratio of B:C:D:E = 5:1:1:93. After aspirating the PBS, 1 ml of the staining working solution was added to each well, and the cells were incubated overnight at 37°C for 12 h. The staining solution was then aspirated, and 2 ml of PBS was added to each well. The cells were immediately observed and photographed under an optical microscope (5× objective lens), or stored at 4°C and photographed within 48 hours. The results are shown below. Figure 3A. At least 15 images were taken per well for statistical quantitative analysis. Quantitative analysis of β-galactosidase staining images was performed using the OpenCV (v4.8.1) library in Python (v3.8) to calculate the β-galactosidase signal intensity for each image. The rank-sum test was used to calculate the significance of differences between groups using the mannwhitneyu function of the stats module in the scipy (v1.8.0) library.

[0028] Quantitative analysis results of β-galactosidase staining images are as follows: Figure 3 As shown in Figure B, after treatment with ginsenoside RT5, the positive signal of β-galactosidase in the cells of the normal proliferation group decreased significantly. In the senescence group, the average positive signal of β-galactosidase in the drug-treated group decreased by 17.5% compared with the control group, demonstrating anti-cellular senescence activity.

[0029] Example 4. Inhibitory activity of ginsenoside RT5 against SASP inflammatory factors (IL-6 and IL-8) SASP (Saponins Acid-Reducing Acids) is one of the most prominent characteristics of senescent cells and a key driver of the development of age-related diseases due to its continuous influence on the tissue microenvironment of senescent cells. SASP has a complex composition, with IL-6 and IL-8 being important members. Disordered synthesis of IL-6 plays a pathological role in chronic inflammation and autoimmune diseases. IL-8 is an important chemokine; its continuous release leads to chronic inflammation, affects the function of normal cells, and accelerates tissue aging. This study used ELISA to detect the important active components of SASP, IL-6 and IL-8, in IMR-90 cells. Cells were seeded in 6-well plates, with separate groups for normal proliferation and senescent cells. The experimental groups were treated with 10 μM of ginsenoside RT5, while the negative control group was treated with 0.1% DMSO (by volume of the culture medium). After 72 hours of treatment, 1 ml of culture medium was aspirated from each well and centrifuged at 500 rpm for 5 minutes to remove cell debris from the culture medium. The supernatant was aspirated and transferred to a new centrifuge tube. The culture medium supernatant was stored at -80°C and used for subsequent experiments within one week.

[0030] The assay was performed using the Xinbosheng Human IL-6 and IL-8 ELISA kit. A standard curve was calculated using IL-6 standard dilution gradients (0–200 pg / ml) and IL-8 gradients (0–500 pg / ml). After equilibrating the pre-coated plate to room temperature, add 100 μl of sample / standard to each well and incubate at 37°C in the dark for 90 min → wash 5 times → add 100 μl of biotinylated antibody and incubate at 37°C in the dark for 60 min → wash 5 times → add 100 μl of enzyme conjugate and incubate at 37°C in the dark for 30 min → wash 5 times → add 100 μl of TMP substrate and develop color for 15 min in the dark → add 100 μl of stop solution and immediately measure the absorbance at 450 nm using a microplate reader. For result processing, the absorbance of each standard and experimental sample was subtracted from the absorbance of the blank well to obtain the actual measured value. A standard curve was plotted using Boster Biotech's ELISA calculation software (http: / / boster.com / index / index / show / id / 114.html), and the results are as follows. Figure 4 As shown. Using a quadratic polynomial equation for fitting, the standard curve of IL-6 is calculated as: y = 0.6599 + 0.01712x - 0.00003x², R0 2 The value is 0.998, such as Figure 4 A. The standard curve for IL-8 is calculated as: y = 0.16865 + 0.01268x - 0.00001x², R 2 The value is 0.995, such as Figure 4 B.

[0031] Inhibitory effects on IL-6 and IL-8, such as Figure 4 As shown in C and 4D, ginsenoside RT5 significantly reduced the concentrations of IL-6 and IL-8 in both normal proliferating cells and senescent cells. IL-6 was reduced by 31.2% and 20.6% in the normal proliferating group and the senescent group, respectively, while IL-8 was reduced by 54.2% and 17.1%, respectively. These results indicate that ginsenoside RT5 has a good inhibitory effect on the important components of SASP, IL-6 and IL-8, and also support the anti-aging activity of ginsenoside RT5.

[0032] Example 5. Antioxidant activity of ginsenoside RT5 (detection of reactive oxygen species (ROS)) Reactive oxygen species (ROS) are important drivers of cellular senescence. High doses of ROS can induce cellular senescence by promoting DNA damage, mitochondrial dysfunction, and inhibition of autophagy. Cells were stained using the ROS probe H2DCFDA (2',7'-Dichlorodihydrofluorescein diacetate, MedChemExpress), and the fluorescence signal of ROS was detected by flow cytometry. Specifically, H2DCFDA was dissolved in DMSO to prepare a 10 mM stock solution. For the experiment, H2DCFDA was diluted to 10 μM in serum-free medium as the working solution. Experimental cells were seeded in 6-well plates, with separate groups for normal proliferation and senescence. The experimental groups were treated with a working concentration of 10 μM ginsenoside RT5, while the negative control group was treated with 0.1% DMSO (by volume of the culture medium). After 72 hours of treatment, staining was performed. Cells were then digested with trypsin, centrifuged, and resuspended in PBS and washed twice. Then, 1 ml of H2DCFDA staining working solution was added to each tube, gently mixed, and incubated at 37°C for 30 min. Cells were then resuspended in PBS and washed twice to remove unbound dye. The processed cell samples were placed on ice, and fluorescence intensity was detected by flow cytometry within 45 min. Results were analyzed using BDFACSDiva 9.0 software. Peak plots were generated using Flowjo 10.9, and the mean and standard deviation of fluorescence signals for each group were calculated. Based on the number of cells detected in each group, the mean and standard deviation of fluorescence signals, bar graphs were generated using the pyplot module in the matplotlib (v3.7.5) package, and independent samples t-tests were performed using the stats module in the scipy (v1.8.0) library.

[0033] The results are as follows Figure 5 As shown, in the normal proliferating group cells, ginsenoside RT5 exhibited a certain ROS inhibitory effect, while in the senescent group, ginsenoside RT5 could significantly reduce the ROS level in senescent cells, with an average inhibition rate of 62.9%, demonstrating very good antioxidant activity in senescent cells, and also supporting the anti-cellular senescence activity of ginsenoside RT5.

[0034] Example 6: Construction of a D-galactose-induced rapid aging model in zebrafish (1) Selection of zebrafish materials Juvenile selection: AB wild-type zebrafish, 72 hours after hatching, with a body length of 3.0–3.5 mm. Selection criteria: no deformities, normal heart rate (100–120 beats / minute), and free swimming. Pre-acclimatization environment: 12 hours before the experiment, the juveniles were transferred to fresh E3 medium (pH 7.2) and kept at a constant temperature of 28.5℃ in the dark. Half of the culture medium was replaced every 6 hours to ensure sufficient nutrients in the culture environment.

[0035] (2) Construction of D-galactose-induced rapid aging model in zebrafish and SA-β-Gal staining The inducer used was D-galactose (Sigma-Aldrich, G0750), and a stock solution with a concentration of 25.6 mg / mL was prepared using E3 culture medium. The working solution was prepared by taking 10 μM (1 μL of stock solution + 999 μL of E3 culture medium). 72hpf zebrafish fry were randomly divided into 4 groups (60 fish per group). The model group (D+) was continuously exposed to 25.6mg / mL D-galactose solution for 5 days (with fresh solution changed daily). The zebrafish fry after drug immersion were regarded as rapid aging model individuals. After staining with β-galactosidase staining agent, the blue intensity of the stained images was used as an aging indicator to determine the degree of aging.

[0036] (3) Results Analysis like Figure 6 As shown, after staining treatment, a clear difference in color intensity was observed in the heads and abdomens of zebrafish larvae. The D-galactose model group showed distinct deep blue staining on the heads and abdomens, indicating successful induction of the aging phenotype. The Rt5 treatment group showed a greater reduction in body staining intensity compared to the D-galactose model group, with lighter staining, indicating lower β-galactosidase levels and a significant anti-aging effect in zebrafish larvae.

[0037] Example 7: Anti-aging activity experiment of ginsenoside RT5 (1) Experimental grouping and treatment are shown in Table 1 Table 1 Experimental Grouping Table

[0038] (2) Quantitative analysis of aging phenotypes After the intervention, SA-β-Gal staining was performed according to the protocol. Images of the dorsal somatic segments of zebrafish were acquired using a stereomicroscope. Fifteen zebrafish were randomly selected from each group, and three non-overlapping field-of-view images were acquired for each fish (45 images per group in total). Quantitative analysis of the β-galactosidase staining images was performed using the OpenCV (v4.8.1) library in Python (v3.8) to calculate the β-galactosidase signal intensity of each image. The rank-sum test was performed using the mannwhitneyu function of the stats module in the scipy (v1.8.0) library to calculate the significance of differences between groups.

[0039] (3) Results Analysis like Figure 7 As shown, the β-galactosidase activity in the RT5-treated group was 31.35, while that in the D-galactose model group was 41.84. The RT5-treated group significantly reduced β-galactosidase activity by more than 25% (P < 0.001). This indicates that the ginsenoside RT5 has a significant anti-aging effect on zebrafish larvae. The β-galactosidase activity in the RT5-treated group is closer to that of normally developing zebrafish larvae, further validating the effectiveness and reliability of its anti-aging effect.

Claims

1. Use of ginsenoside RT5 in the preparation of an anti-cellular and whole-body aging product.

2. Use according to claim 1, characterized in that, The anti-cellular and whole-body aging product has one or more of the following functions: improving cell viability; inhibiting secretion of senescence-associated secretory phenotype factors; reducing intracellular reactive oxygen species levels; and reducing whole-body senescence-associated β-galactosidase activity in zebrafish.

3. Use according to claim 2, characterized in that, The improvement in cell viability refers to an increase of 45.0% or more in the viability of senescent cells.

4. Use according to claim 2, characterized in that, The senescence-associated secretory phenotype factors include interleukin-6 and interleukin-8.

5. Use according to claim 2, characterized in that, The average reduction in intracellular reactive oxygen species levels reaches 62.9% or more.

6. Use according to claim 2, characterized in that, The reduction in whole-body senescence-associated β-galactosidase activity in zebrafish refers to a reduction of 25% or more in the activity of β-galactosidase in a D-galactose-induced zebrafish rapid aging model.

7. Use according to claim 1, characterized in that, The anti-cellular and whole-body aging product comprises a pharmaceutical, health food, functional food, cosmetic, or fragrance product containing ginsenoside RT5.

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