Use of falcarindiol and derivatives thereof in the preparation of a medicament for delaying aging or immunomodulation

Abstract

The application discloses application of falcarinol-7-methyl ether and derivatives thereof in preparation of medicines and functional foods for delaying aging or immune regulation. In the application, the falcarinol-7-methyl ether and derivatives thereof are derived from falcarinol-7-methyl ether, and can effectively delay replicative senescence of mesenchymal stem cells, inhibit expression of aging-related markers of mouse liver and lung induced by adriamycin, improve grip strength of the mouse, enhance muscle function, and promote proliferation of T cells separated from human peripheral blood mononuclear cells, and have an immune regulation effect. Therefore, the falcarinol-7-methyl ether and derivatives thereof have good effects of improving telomerase activity, anti-aging and immune regulation, and can be developed into anti-aging or immune regulation products.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of isopyramone-7-methyl ether from ox-bean fruit and its derivatives in the preparation of drugs for delaying aging or regulating immunity. Background Technology

[0002] Aging is a spontaneous and inevitable process that occurs naturally in living organisms over time, manifested as a decline in structure and function, and a weakening of adaptability and resistance. Currently, population aging and the high incidence of age-related diseases are major social problems facing the world. Therefore, the search for novel drugs to delay aging or modulate immunity has significant practical value.

[0003] Telomere shortening is one of the fundamental causes of human cellular senescence. Telomeres are highly ordered, specialized "cap" structures at the ends of eukaryotic chromosomes, composed of highly repetitive DNA sequences (TTAGGG) and their interacting proteins. They play a crucial role in protecting chromosome ends, maintaining genome stability, and participating in biological processes such as cellular senescence regulation. Human cell telomeres are 5-15 kb long. With each cell division in normal humans, telomere length gradually shortens due to end replication. When telomeres shorten to a certain limit, cell division ceases; this is a significant cause of senescence in most somatic cells, known as replicative senescence. In vitro cultured normal cells undergo a limited number of divisions, resulting in gradual telomere shortening, DNA damage accumulation, and alterations in cell morphology and physiological metabolic activities, manifesting as slowed cell growth, increased β-galactosidase activity, and increased secretion of inflammatory factors. Many aging-related genes are considered biomarkers of aging, including cyclins (p16, p21), p53, and aging-associated secretory phenotypes (SASP) such as IL1-α, IL-6, and TNF-α. These genes serve as both "indicators" of aging and targets for intervention.

[0004] Telomerase is a crucial mechanism for telomere elongation, and interventions targeting telomeres, such as small molecule drugs, are essential for delaying the development of age-related diseases. Immune system aging also affects overall health; for example, the immune response after vaccination is often weaker in older adults, potentially leading to less effective protection compared to younger individuals. Immune system aging is primarily manifested in the aging of immune organs and immune cells. Telomerase plays a vital role in the function of immune cells, such as lymphocytes. Lymphocytes, especially T cells, require upregulation of telomerase activity after activation by antigen stimulation to maintain telomere length during rapid cell replication. Telomerase-dependent T cell activation and proliferation help the body fight infection. A lack of telomerase activity can also lead to T cell dysfunction. Therefore, regulating telomerase activity in T cells is a viable immunomodulatory approach.

[0005] Currently, many natural extracts are being researched for their potential to slow down the aging process. Astragalus extract TA-65 is currently the only telomerase activator marketed in the United States, delaying cellular aging by maintaining telomere length. Clinically, it can improve metabolism and immunity in the elderly and has a certain effect on slowing the progression of age-related diseases such as osteoporosis, cardiovascular disease, and Alzheimer's disease. However, its high price and unstable efficacy make the development of safe, effective, and affordable anti-aging or immune-modulating products with independent intellectual property rights in my country of great significance.

[0006] Heteropeucenin 7-methyl ether is a chromogen derived from the root of *Harrisonia perforata* (Bl.) Merr., a plant belonging to the genus *Harrisonia* in the Simararubaceae family. It is mainly distributed in Fujian, Guangdong, and Hainan provinces of my country, and also found in the Malay Peninsula, Indochina Peninsula, the Philippines, and Indonesia, commonly found in low-altitude shrublands and sparse forests. Simararubaceae plants are abundant in my country; clinically, *Brucea javanica* oil and other plants are mainly used to treat cancer, peptic ulcers, and skin diseases. *Harrisonia perforata* has the effect of clearing heat and treating malaria. The root is used medicinally; it is bitter and cold in nature. In folk medicine, the root, stem, and leaves are used to treat malaria, skin ulcers, fever, cough, and sore throat. Scholars have isolated 32 chromogenin compounds from *Eleusine indica* fruit, with basic skeletons based on benzene and pyran rings. Among them, Wei Xincheng et al. isolated *Niujing I* (i.e., isopeucedanum chromogenin-7-methyl ether) from *Eleusine indica* fruit in 1986 and reported its antitumor activities. Other chromogenin compounds, such as pyrano-2-benzopyrans (*Eleusine indica* chromogenin A, *Niujing III*), have antihypertensive, stimulant, and excitatory activities. However, to date, there are no reports in the existing technology on the activities of *Eleusine indica* isopeucedanum chromogenin-7-methyl ether and its derivatives in anti-aging, immune regulation, and muscle function.

[0007] The structural formulas of compounds isopyramidal chromogenone-7-methyl ether, compound 9 (8-dimethylallyl)-7-hydroxy-6-methoxycoumarin, and compound 44 (5,7-dimethoxy-2-methylchromogenone) are as follows:

[0008]

[0009] The compound isopyramone-7-methyl ether is disclosed in the literature: Molecular and crystal structure of the active ingredient of ox-ear fruit, Niujing 1, Wei Xincheng et al., Journal of Peking University (Natural Science Edition), No. 4, 1986.

[0010] The compound 8-dimethylallyl)-7-hydroxy-6-methoxycoumarin is disclosed in the literature: Cytotoxicconstituents of Oldenlandia umbellata and isolation of a new symmetrical coumarin dimer, Senthi Mahibalan etl. Med Chem Res (2016) 25:466–472.

[0011] Compound 5,7-dimethoxy-2-methylchromone is disclosed in the literature: Chemical constituents of Penicillium ferraniaense GE-7 and their cytotoxicities, Yan Wang etl. Natural Product Research 2025, VOL. 39, NO. 14, 3915–3922. Summary of the Invention

[0012] The purpose of this invention is to provide the application of isopyramidal chromogenone-7-methyl ether and its derivatives in the preparation of drugs for delaying aging or immune regulation, especially targeting telomerase activity.

[0013] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0014] This invention provides the application of isopyramidal chromogenone-7-methyl ether or its derivatives in the preparation of drugs for delaying aging or regulating immunity;

[0015] The structural formula of the aforementioned Heteropeucenin 7-methyl ether is shown below:

[0016] .

[0017] Preferably, it is used in the preparation of drugs that enhance telomerase activity in cells.

[0018] Preferably, it is used in the preparation of drugs that delay cell aging and improve the function of immune cells.

[0019] Preferably, the cells are mesenchymal stem cells, immune cells, muscle cells, lung cells, hepatocytes, kidney cells, germ cells, nerve cells, epithelial cells, or endothelial cells.

[0020] Preferably, the derivative is (8-(dimethylallyl)-7-hydroxy-6-methoxycoumarin, cedrelopsin) or (5,7-dimethoxy-2-methylchromone), with the following structural formula:

[0021]

[0022] Preferably, the drug is used directly or in combination with isopyramone-7-methyl ether or its derivatives, or pharmaceutically acceptable salts, esters, acids or stereoisomers thereof as active ingredients, with the addition of acceptable excipients or auxiliary ingredients.

[0023] Preferably, the dosage form of the drug includes oral preparations, injectable preparations, topical preparations, and microcrystalline microneedle preparations.

[0024] More preferably, the oral preparation includes hard capsules, tablets, oral liquids, granules, soft capsules, and drop pills.

[0025] The beneficial effects of this invention are as follows:

[0026] This invention utilizes isoimperatorin-7-methyl ether monomer and its derivatives derived from *Eleusine indica* fruit. By increasing telomerase activity and reducing the expression of aging-related genes, it can effectively delay the replicative aging of mesenchymal stem cells, inhibit the expression of doxorubicin-induced aging-related markers in mouse lungs and liver, improve grip strength and muscle function in mice, and promote the proliferation of T cells isolated from human peripheral blood mononuclear cells, thus exhibiting immunomodulatory effects. Therefore, isoimperatorin-7-methyl ether from *Eleusine indica* fruit and its derivatives possess good effects in increasing telomerase activity and anti-aging or immunomodulatory effects, and are expected to be developed into anti-aging or immunomodulatory products. Attached Figure Description

[0027] Figure 1 The figures show the results of telomerase activation in mesenchymal stem cells by isoperitone-7-methyl ether from *Polygonum cuspidatum* in Example 1. Figure A shows the change in telomerase activity in cells 24 hours after the addition of isoperitone-7-methyl ether to the culture medium; Figure B shows the change in telomerase mRNA levels in cells 24 hours after the addition of isoperitone-7-methyl ether to the culture medium; and Figure C shows the change in telomerase protein expression levels in cells 24 hours after the addition of isoperitone-7-methyl ether to the culture medium.

[0028] Figure 2 This is a graph showing the effect of 7-methyl ether from *Polygonum cuspidatum* on the proliferation of mesenchymal stem cells under long-term cell culture conditions in Example 2.

[0029] Figure 3 This is a diagram showing the staining results of senescence-related galactosidase in mesenchymal stem cells by isopian tuber chromogen-7-methyl ether under long-term cell culture conditions in Example 3.

[0030] Figure 4 The figure shows the effect of 7-methyl ether of *Polygonum cuspidatum* on some aging-related biomarkers of mesenchymal stem cells under long-term cell culture conditions in Example 4; where A represents the expression level of aging-related genes in mesenchymal stem cells after long-term drug treatment; and B represents the expression level of aging-related genes in mesenchymal stem cells after long-term drug treatment.

[0031] Figure 5 The figure shows the effect of isopyramone-7-methyl ether and its derivatives on telomerase in mesenchymal stem cells in Example 5.

[0032] Figure 6 The figure shows the effects of isopian tuber chromogenone-7-methyl ether from *Eucalyptus chinensis* on aging indicators and muscle function in doxorubicin-induced aging mice; where A represents the grip strength level of mice; B represents the expression levels of aging-related genes p16, p21, p53, IL1-α, IL-6, and TNF-α in mouse lung tissue; and C represents the expression levels of aging-related genes p16 and p21 in mouse lung tissue.

[0033] Figure 7 The figure shows the effect of isopeucedanum chinense chromogenone-7-methyl ether on T cells in Example 7; where A is the effect of isopeucedanum chinense chromogenone-7-methyl ether on T cell telomerase activity; and B is the effect of isopeucedanum chinense chromogenone-7-methyl ether on T cell telomerase activity. Detailed Implementation

[0034] Those skilled in the art will understand that the techniques disclosed in the following embodiments represent those discovered by the inventors that work well in the practice of this invention. However, many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the invention, still yielding the same or similar results. Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field.

[0035] Example 1: Effects of isopyramidal chromogenone-7-methyl ether on telomerase activity and expression in mesenchymal stem cells.

[0036] I. Experimental Methods

[0037] Cell culture: Human umbilical cord mesenchymal stem cells were cultured in DMEM / F12 medium with 10% fetal bovine serum at 37°C in a cell culture incubator with 5% CO2 concentration.

[0038] Drug treatment: Weigh an appropriate amount of isopyram-7-methyl ether powder and dissolve it in DMSO to a final concentration of 100 mM. Serially dilute with DMSO according to the required drug concentration, then add to the culture medium containing human umbilical cord mesenchymal stem cells to achieve a final DMSO concentration of 0.05%, for example, adding 0.5 μL of 600 μM isopyram-7-methyl ether DMSO solution to each 1 mL of culture medium. After cell adhesion, replace the medium with fresh drug-containing medium and culture for 24 hours. Collect adherent cells by trypsin digestion. The positive control is to add a total concentration of 10 μg / mL of vitamin C (stock solution dissolved in PBS, aliquoted and stored at -20°C protected from light) to the culture medium for 24 hours.

[0039] Telomerase activity assay: Telomere repeat sequence amplification experiment based on real-time PCR

[0040] 1) Digest the cells, wash once with 1 mL PBS, and place the cells on ice. Resuspend the cells in 100 μL of pre-chilled NP-40 lysis buffer and lyse on ice for half an hour.

[0041] 2) Centrifuge at 14000 g, 4℃, for 15 min, and collect 80 μL of supernatant into a new EP tube.

[0042] 3) Use Thermo Scientific™ Pierce™ BCA Protein Assay Kits to quantify protein concentration. Control the total amount of sample loaded subsequently to not exceed 1 μg.

[0043] 4) Prepare 1× 10 5 A standard curve was prepared from 293T cells.

[0044] 5) Calculate and prepare the required mixing system for the number of samples according to Table 1.

[0045] Table 1 Q-trap reaction system

[0046]

[0047] 7) Vortex the cell supernatant and the mixture together, then incubate in an enzyme-linked immunosorbent assay (ELISA) reader at 30°C in the dark for 30 min.

[0048] 8) The TRAP mixture was divided into 3 replicates. 10 μL of the mixture was added to each well of the 96-well plate, and each sample was replicated 3 times.

[0049] 9) The plate-spinning machine spins the plate for 1 minute.

[0050] 10) On-machine testing.

[0051] Total RNA extraction, reverse transcription, and real-time quantitative PCR:

[0052] 1) RNA was extracted from cells using the Trizol method.

[0053] 2) Determine the concentration of extracted RNA. Take 1 μg of RNA and reverse transcriptase M-MLV reagent according to the instructions.

[0054] 3) Quantitative real-time PCR was performed using Novizan ChamQ Universal SYBR qPCR Master Mix, with GAPDH used as an internal reference gene. Primer sequences are shown in Table 2:

[0055]

[0056] Western blot analysis of proteins:

[0057] Add 100 μL of RIPA lysis buffer (RIPA pre-mixed with protease inhibitor) to each sample, lyse on ice for 30 min, sonicate at 4°C for 20 min, then centrifuge at 14000g at 4°C for 10 min in a pre-chilled centrifuge. Aspirate 80 μL of supernatant and add 20 μL of 5× loading buffer (containing β-mercaptoethanol). Boil in water for 10 min, cool, and then load the sample. Select an appropriate protein gel concentration based on the size of the target protein.

[0058] Based on the BCA protein quantification results, the total protein loaded into each sample was controlled to be 80 μg. After loading, the power was turned on and the stacking gel was run at 80V for 30 min, and the separating gel was run at 120V for 1 hour for electrophoresis.

[0059] Transfer membrane (wet transfer method)

[0060] 1) Soak the rectangular filter paper, sponge, PVDF membrane (pre-treated with methanol), and gel in transfer buffer for 3 min.

[0061] 2) Remove the transfer clamp and, from the positive electrode (red side) to the negative electrode (black side), lay out one sponge, filter paper, PVDF membrane, gel, filter paper, and sponge in that order, removing any air bubbles between each layer. Tighten the clamp and place the membrane into the wet transfer tank. Select the appropriate current and transfer time, and connect the power supply. During the transfer process, keep the electrophoresis tank in an ice bath to control the system temperature.

[0062] Blocking and antibody incubation

[0063] 1) Add 1× TBST to 5% skim milk powder to prepare a sealing solution, soak the membrane in a shaker and seal for one hour.

[0064] 2) Primary antibody incubation: Dilute the antibody with 1× TBST according to the ratio and incubate overnight at 4°C.

[0065] 3) Secondary antibody incubation: After recovering the primary antibody, wash the membrane three times with TBST for 10 min each time, add the secondary antibody, incubate at room temperature for 1 h, and wash the membrane three times with TBST for 10 min each time.

[0066] development

[0067] It is divided into chemiluminescent development and fluorescent secondary antibody development.

[0068] Development was performed using the MIKX ultrasensitive chemiluminescence assay kit. After mixing solution A and solution B at a 1:1 ratio, the membrane was immersed in the developer for 30 seconds, and then developed using the BioRad Chemi-Doc XRS assay. + The instrument scans and uses continuous exposure mode to collect the target protein signal.

[0069] Fluorescent secondary antibody was used for imaging with an Odyssey LI-COR dual-fluorescence infrared imager to obtain the target protein band.

[0070] II. Experimental Results

[0071] The determination of telomerase activity in human umbilical cord mesenchymal stem cells showed that the addition of isopyram-7-methyl ether for 24 hours significantly increased telomerase activity in cells. Figure 1 A). Detection of telomerase gene (hTERT) mRNA levels showed that the expression of the hTERT gene in cells was significantly increased 24 hours after the addition of isopyramidal chromogenone-7-methyl ether. Figure 1 B). Western blotting analysis of telomerase protein (hTERT) expression levels showed that treatment with isopyramidal chromogenone-7-methyl ether significantly increased hTERT protein expression. Figure 1 C). The above results indicate that isopyramidal chromogenone-7-methyl ether can effectively increase telomerase activity and telomerase expression in mesenchymal stem cells.

[0072] Example 2: Effect of 7-methyl ester glycoside from *Polygonum cuspidatum* on the proliferation of human umbilical cord mesenchymal stem cells under long-term cell culture conditions.

[0073] I. Experimental Methods

[0074] Human umbilical cord mesenchymal stem cells were cultured in DMEM / F12 medium with 10% fetal bovine serum in a cell culture incubator at 37°C and 5% CO2 concentration.

[0075] Adherent cell passage:

[0076] Once human umbilical cord mesenchymal stem cells have filled the entire culture dish, they need to be passaged. Discard the cell culture supernatant. Slowly add PBS along the wall of the culture dish, gently agitate the dish to rinse away any remaining culture medium and dead cells. Add 0.25% trypsin, evenly covering the cell surface, and digest in an incubator for 30 seconds. Under a microscope, the cells will appear rounded. Add culture medium to stop the reaction. Gently pipette the digested cells and transfer them to EP tubes. Centrifuge at 1200 rpm, 25°C for 3 min, then add 1 mL of fresh culture medium to resuspend the cells. Transfer the resuspended cells to a new culture dish, add fresh culture medium, mix thoroughly, and place the dish in a cell culture incubator to continue cell culture.

[0077] Cell growth curve analysis:

[0078] Population doubling (PD) was analyzed using a counting method. Cells were resuspended after trypsin digestion and then counted using a counting chamber. The counting chamber was wiped with alcohol, covered with a coverslip, and 10 μL of cell suspension was slowly added into each well. After adding resuspended suspension to both the upper and lower wells, the number of cells in each of the four diagonal squares was observed and recorded under a microscope. The number of cells in all eight squares (both upper and lower wells) was recorded as N, and the cell concentration was (N / 8) × 10⁻⁶. 4 Cells / mL. Each group was counted three times, and the average value was taken as the total cell count (Nf). Calculations were performed for each group of 10-1. 5 Each cell (Ni) corresponds to a volume, and after each count, it is incremented by 10. 5 Cells were seeded into new six-well plates. After cell adhesion, the culture medium containing the drug was replaced, and drug treatment was performed. The drug-containing culture medium was prepared as follows: an appropriate amount of isopyram-7-methyl ether powder was dissolved in DMSO to a final concentration of 100 mM. The medium was then serially diluted with DMSO according to the required drug concentration and added to the culture medium containing human umbilical cord mesenchymal stem cells to achieve a final DMSO concentration of 0.05%. For example, 0.5 μL of 600 μM isopyram-7-methyl ether DMSO solution was added to each 1 mL of culture medium to obtain the drug-containing culture medium. A control consisting entirely of DMSO and 10 μg / mL vitamin C was used.

[0079] The growth curve is fitted using the formula: PD = log2 (Nf - Ni).

[0080] II. Experimental Results

[0081] During long-term culture, the proliferation of human umbilical cord mesenchymal stem cells slowed down. Under long-term drug-induced culture conditions supplemented with DMSO, vitamin C (positive control), and isopyramidal chromogen-7-methyl ether (100 nM, 300 nM, 1 μM, 3 μM), the cell proliferation rate was as follows: Figure 2 The cell proliferation rates in the isopyram-7-methyl ether treatment groups were significantly higher than those in the DMSO control group. Specifically, the cell proliferation rates in the isopyram-7-methyl ether (300 nM, 1 μM, 3 μM) treatment groups were significantly higher than those in the control group (P < 0.001). These results indicate that isopyram-7-methyl ether from *Eriocaulon buergerianum* can promote the proliferation of mesenchymal stem cells.

[0082] Example 3: Effect of 7-methyl ether from *Polygonum cuspidatum* fruit on senescence-related galactosidase staining in mesenchymal stem cells under long-term cell culture conditions.

[0083] I. Experimental Methods

[0084] Cellular β-galactosidase staining:

[0085] The cells were stained using the Beyotime β-galactosidase staining kit (C0602). After culturing the cells for a certain number of days (30 days for long-term culture, see Example 2 for specific culture method), the experiment was performed (using a six-well plate as an example).

[0086] Discard the culture medium, wash once with 1 mL PBS, add 1 mL β-galactosidase staining and fixation solution, and fix at room temperature for 15 min.

[0087] Remove the fixative and wash the cells three times with 1 mL PBS for 3 min each time.

[0088] After washing, add 1 mL of staining working solution to each well and incubate overnight at 37°C. (The preparation method of the staining working solution is shown in Table 3 below.)

[0089] Table 3. Formulation of staining working solution

[0090]

[0091] 4) Observe and photograph under a regular optical microscope.

[0092] 5) Quantitative analysis of the results was performed using ImageJ. The specific steps were as follows: convert the photographs of the same field of view to 8-bit format and set the OD; adjust the threshold and select an appropriate region (Image-adjust-threshold) to fit the β-galactosidase positive region; select an appropriate threshold algorithm (Image-Adjust-Auto Threshold); set the measurement and export the total gray level (IntDen: Integrated Density); use IntDen to represent the total signal intensity of the β-galactosidase positive region, quantify multiple photographs within a set, and test the significance using one-way ANOVA.

[0093] II. Experimental Results

[0094] During cellular senescence, in addition to changes in cell morphology and slowed cell growth, senescence markers gradually accumulate. These markers reflect the senescence status of cells, with β-galactosidase being an important marker. In senescent cells, SA-β-gal continuously accumulates, thus reflecting the cellular senescence state. SA-β-gal staining was performed on cells treated with a drug for 30 days, and the results are as follows... Figure 3 As shown. Quantitative analysis of SA-β-gal-positive cell regions in the same field of view using ImageJ revealed no significant difference between the 100 nM isopyram-7-methyl ether treatment and the DMSO group, while the 300 nM isopyram-7-methyl ether treatment significantly reduced SA-β-gal accumulation compared to the control group.

[0095] The above results indicate that isopyram chromogenone-7-methyl ether from ox-tendon fruit can delay the replicative senescence of human umbilical cord mesenchymal stem cells.

[0096] Example 4: Effect of 7-methyl ester glycoside from *Polygonum cuspidatum* on the expression levels of aging-related markers in human umbilical cord mesenchymal stem cells under long-term cell culture conditions.

[0097] I. Experimental Methods

[0098] The drug treatment, total RNA extraction, reverse transcription, and real-time quantitative PCR methods for human umbilical cord mesenchymal stem cells were the same as in Example 2, with GAPDH used as an internal reference gene. Primers used are listed in Table 4 below.

[0099] Table 4

[0100]

[0101] II. Experimental Results

[0102] The expression of senescence-related genes in mesenchymal stem cells after long-term treatment (30 days) with 300 nM isopyram-7-methyl ether was as follows: Figure 4 As shown in Figure A. Compared to the control group, the expression levels of p16, p53, and IL-1β in the isopyram-7-methyl ether treatment group were downregulated, and it showed a better effect than the vitamin C (positive control) group. The expression of senescence-related genes in mesenchymal stem cells after short-term (48-hour) treatment with 300 nM isopyram-7-methyl ether is shown in Figure A. Figure 4 As shown in Figure B, compared with the control group, the expression levels of IL-1β, IL-6, and IL-8 were significantly downregulated in the isopyram-7-methyl ether treatment group, indicating that isopyram-7-methyl ether inhibits the expression of aging-related pro-inflammatory factors in mesenchymal stem cells.

[0103] The results above indicate that treatment with isopyramone-7-methyl ether can inhibit the expression of senescence-related pro-inflammatory factors in mesenchymal stem cells.

[0104] Example 5: Effects of isopyramidal chromogenone-7-methyl ether and its derivatives on telomerase in mesenchymal stem cells (Figure 5)

[0105] I. Experimental Methods

[0106] The method for measuring telomerase activity in cells is the same as in Example 1.

[0107] II. Experimental Results

[0108] The results of the telomerase activity assay of human umbilical cord mesenchymal stem cells are as follows: Figure 5 Compounds 44 and 9, derivatives of isopyramidalis chromogenone-7-methyl ether from *Eucalyptus chinensis*, at 300 nM concentrations, both significantly enhanced telomerase activity in mesenchymal stem cells at the same concentration. These results indicate that compounds 44 and 9 also possess the potential to delay cellular senescence through telomerase activity.

[0109] Example 6: Effects of 7-methyl ether of *Polygonum cuspidatum* on lung, liver aging indicators and muscle function in doxorubicin-induced aging mice (Figure 6).

[0110] I. Experimental Methods

[0111] Eighteen 6-week-old male C57BL6 / J mice were selected and divided into three groups: NS group (control group), Doxo group (model group), and isopantegeneronin-7-methyl ether group (drug administration group). Modeling was achieved via intraperitoneal injection. The NS group received 100 μL / 30g saline solution daily, while the Doxo and isopantegeneronin-7-methyl ether groups received 2 mg / kg doxorubicin hydrochloride solution, with an injection volume of 100 μL / 30g, twice weekly for 30 days. Drug administration was administered twice weekly via intraperitoneal injection. The NS and Doxo groups received 100 μL of saline solution each time, while the isopantegeneronin-7-methyl ether group received 100 μL of 1.5 mM / kg isopantegeneronin-7-methyl ether each time, continuing until the end of modeling. After modeling, the mice were weighed, and their forelimb grip strength was measured using a grip strength meter. After the measurement, the mice were anesthetized by injection of 2.5% aphthol solution, and the lung and liver tissues were separated.

[0112] Take a portion of the tissue, add 1 mL of Trizol, and homogenize using a homogenizer at 4°C.

[0113] The subsequent total RNA extraction, reverse transcription, and real-time quantitative PCR methods were the same as in Example 2, with GAPDH used as an internal reference gene.

[0114] II. Experimental Results

[0115] Doxorubicin is a commonly used chemotherapy drug that can intercalate into the DNA double helix, directly causing DNA double-strand breaks and irreversibly leading to cell aging. Furthermore, doxorubicin's intracellular metabolism produces large amounts of reactive oxygen species (ROS), resulting in increased oxidative stress levels, which also contributes to aging. Doxorubicin injection may lead to deterioration of myocardial contractility and fibrosis; hepatocellular aging accompanied by lipofuscin accumulation and hepatotoxicity; proteinuria and glomerulosclerosis; and hematopoietic system stress. This study induced aging in mice through multiple low-dose intraperitoneal injections of doxorubicin. The effect of isopyram chromogenone-7-methyl ether on the grip strength of doxorubicin-induced aging mice is as follows: Figure 6 As shown in Figure A, the treatment group treated with isopyram-7-methyl ether significantly increased the grip strength of mice compared to the doxorubicin treatment group. p16 and p21 are important cell cycle regulatory proteins; p53 induces cell cycle arrest, DNA repair, or apoptosis by transcribedly activating genes such as p21. IL1-α, IL-6, and TNF-α, as components of SASP, accelerate aging and promote related diseases through chronic inflammation. Intervention in the expression of these genes (such as inhibiting p16, p21, or inflammatory factors) may delay lung aging and related diseases (COPD, IPF, and senile lung cancer, etc.). The results of the effect of isopyram-7-methyl ether on doxorubicin-induced lung aging markers in mice are shown below. Figure 6 As shown in Figure B, the treatment group with isopyram-7-methyl ether significantly inhibited the increase in gene expression levels of p16, p21, p53, IL1-α, IL-6, and TNF-α in mouse lung tissue induced by doxorubicin injection. The effects of isopyram-7-methyl ether on doxorubicin-induced liver aging markers in mice are shown in Figure B. Figure 6 As shown in Figure C, the isopyram-7-methyl ether treatment group also showed a trend of inhibiting the upregulation of doxorubicin-induced aging markers in mouse liver tissue compared to the model group. These results indicate that isopyram-7-methyl ether has the potential to improve the motor performance of mice and inhibit doxorubicin-induced aging in mice.

[0116] Example 7: Effects of isopyramidal chromogenone-7-methyl ether and its derivatives on T cells (Figure)

[0117] I. Experimental Methods

[0118] Cell surface marker staining:

[0119] 1) Collect and wash PBMC cells:

[0120] PBMCs (human peripheral blood mononuclear cells) were isolated from human peripheral blood samples. The cells were washed twice with pre-cooled PBS. The cells were centrifuged at 500 g at 4°C, and the supernatant was discarded.

[0121] 2) Antibody incubation:

[0122] Resuspend the cell pellet in 100 μL of FACS buffer. Add the CD28-FITC-labeled antibody (eBioscience) at the concentration recommended in the antibody manufacturer's instructions. Gently mix and incubate the cell suspension on ice in the dark for 30 min. Centrifuge at 500 g for 5 min at 4°C. Carefully discard the supernatant, avoiding disturbing the cell pellet.

[0123] 3) Cell washing:

[0124] Cells were washed twice with FACS buffer to remove unbound antibodies.

[0125] 4) Resuspension and detection:

[0126] The washed cells were resuspended in 200 μL of 1× FACS buffer. The cell suspension was then transferred to flow cytometry tubes for flow cytometry analysis.

[0127] T-cell activation:

[0128] This experiment used Dynabeads® Human T-Activator CD3 / CD28 (Thermo) to activate PBMCs.

[0129] 1) Preparation:

[0130] Vigorously shake the glass bottle containing the beans. Transfer the required amount of beans to a 1.5 mL centrifuge tube. Be careful to do this quickly to minimize bean precipitation.

[0131] 2) Clean the beads:

[0132] Add 1 mL of PBS to the centrifuge tube containing the beads. Vigorously vortex for 5 seconds to ensure the beads are fully dispersed in the PBS. Place the centrifuge tube on a magnetic rack and let it stand for 1 minute to allow the beads to aggregate on the tube wall. Discard the supernatant.

[0133] 3) Heavy-suspension beads:

[0134] Add an equal volume of ATCM (appropriate cell culture medium) to a centrifuge tube and resuspend the beads for later use.

[0135] 4) Inoculation with PBMCs:

[0136] Take 10 6 One PBMC was seeded into a 24-well plate. 1 mL of ATCM was added to each well.

[0137] 5) Add beads:

[0138] Based on every 4×10 5 Add 1 μL of beads to each well, and add 25 µL of beads to each well.

[0139] 6) Cell culture:

[0140] Place the 24-well plate in a 37°C cell culture incubator to provide a suitable temperature and environment to promote cell growth and activation. Depending on the experimental purpose and cell type, it may be necessary to keep the plate in the incubator for a certain period of time, such as several hours to several days.

[0141] T cell proliferation analysis

[0142] The CellTrace™ CFSE Cell Proliferation Kit (Invitrogen) staining and tracking method was used in this experiment to analyze the proliferation of suspended cells.

[0143] 1) Prepare a 1mM CFSE stock solution.

[0144] 2) Centrifuge to collect the PBMC cell pellet and resuspend the cells in 1× PBS.

[0145] 3) Add CFSE stock solution to the cell suspension in proportion and mix gently to ensure uniform dye distribution.

[0146] 4) Incubating cells:

[0147] 5) Incubate the cell suspension in a 37°C, light-protected water bath for 20 min.

[0148] 6) Centrifuge at 500 g for 5 min and discard the supernatant. At this point, the cell pellet should be yellow-green, indicating that it has been labeled with CFSE.

[0149] 7) Resuspend the cells in 5 mL of TCM (appropriate cell culture medium).

[0150] 8) Incubate the cell suspension in a 37°C, light-protected water bath for 5 min to remove excess CFSE dye.

[0151] 9) Centrifuge at 500 g for 5 min and discard the supernatant.

[0152] 10) Resuspend the cells using preheated ATCM and perform cell counting to determine the seeding density.

[0153] 11) Seed cells in an appropriate container according to experimental requirements and add the drug to be tested.

[0154] 12) After 5 days of culture, the cells were analyzed using flow cytometry. Cell proliferation was assessed by detecting a decrease in CFSE fluorescence intensity.

[0155] Telomerase activity assay:

[0156] CD28 from PBMC flow cytometry + The telomerase activity of T cells was measured, specifically in the same manner as in Example 1.

[0157] II. Experimental Results

[0158] The effect of isopyramone-7-methyl ether from oxeye fruit on telomerase activity in T cells, as follows: Figure 7 As shown in Figure A, treatment with isopyram-7-methyl ether significantly increased telomerase activity in T cells from two different donors. The effect of isopyram-7-methyl ether on T cell proliferation is as follows: Figure 7 As shown in B, the number of cells with low fluorescence intensity proliferation peaks in the isopyram-7-methyl ether treatment group was significantly higher than that in the control group, indicating that isopyram-7-methyl ether treatment reduced the number of CD28 cells in PBMCs. + The proliferation of T cells was accelerated. These results indicate that isopyramidal chromogenone-7-methyl ether can effectively increase the telomerase activity of T cells and promote T cell proliferation, demonstrating its immunomodulatory capabilities.

[0159] In summary, the isopyramone-7-methyl ether from *Eucalyptus chinensis* and its derivatives of this invention can upregulate the activity of telomerase in human umbilical cord mesenchymal stem cells. This not only reduces the expression levels of cellular senescence markers and delays replicative senescence, but also provides some resistance to drug-induced aging in mice, exhibiting anti-aging or immunomodulatory activity. It improves grip strength in mice, promotes muscle function, and promotes the proliferation of T cells isolated from human peripheral blood mononuclear cells, thus possessing immunomodulatory effects. It can be used alone or in combination as a pharmaceutical or non-pharmaceutical product for delaying aging or immunomodulation, showing promising prospects in both pharmaceutical and dietary supplement applications.

Claims

1. Application of isopyramidal chromogenone-7-methyl ether or its derivatives in the preparation of drugs for delaying aging or regulating immunity; The structural formula of the aforementioned Heteropeucenin 7-methyl ether is shown below: ； The derivative is 8-(dimethylallyl)-7-hydroxy-6-methoxycoumarin, and its structural formula is shown below: 。 2. The application according to claim 1, characterized in that, The drug is used directly or in combination with isopyramidal chromogenone-7-methyl ether or its derivatives, or pharmaceutically acceptable salts thereof, as the active ingredient, with the addition of acceptable excipients or auxiliary ingredients. The derivative is 8-(dimethylallyl)-7-hydroxy-6-methoxycoumarin.

3. The application according to claim 1, characterized in that, The drug may be in the form of oral preparations, injectable preparations, or topical preparations.

4. The application according to claim 1, characterized in that, The drug is in the form of a microcrystalline microneedle formulation.

5. The application according to claim 3, characterized in that, The oral preparations include hard capsules, tablets, oral liquids, granules, soft capsules, and drop pills.

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