Use of lauric acid lauryl ester in the preparation of anti-aging, life-extending products

Abstract

The application belongs to the technical field of medicine or health food, and particularly relates to a new use of cinnamic acid guaiacyl ester. The application provides application of cinnamic acid guaiacyl ester in preparation of a medicine or health food for delaying aging and prolonging life. The cinnamic acid guaiacyl ester can improve aging-related phenotypes of nematodes, including prolonging the life of nematodes, improving the heat stress resistance and movement ability of nematodes, and improving the accumulation of lipofuscin in the body. On a molecular level, the cinnamic acid guaiacyl ester can activate the expression of autophagy-related genes at mRNA and protein levels. The application provides a new way for anti-aging and has clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical or health food technology, specifically relating to the application of cinnamic acid ester in the preparation of drugs or health foods that delay aging and prolong life. Background Technology

[0002] Aging is a process in which all living organisms gradually lose their natural balance. It is characterized by a weakening of the body's tissues and organs' ability to self-renew and repair themselves, leading to irreversible degenerative changes and eventual death. Age-related functional decline can directly cause various chronic diseases, such as cardiovascular diseases and neurodegenerative diseases (Alzheimer's disease and Parkinson's syndrome). With the increasing aging population and the high incidence of age-related diseases, the urgent need for "healthy aging" has attracted close attention from the entire society.

[0003] Autophagy is a recently proposed important regulatory mechanism related to aging, and impaired autophagy is one of the key markers of aging. Autophagy refers to the process by which autophagosomes, with their double-membrane structure, encapsulate intracellular materials, including misfolded protein aggregates and damaged organelles (such as mitochondria), ultimately fusing with lysosomes and degrading cellular components. Autophagy not only prevents cell damage but also promotes cellular survival under nutrient-deficient conditions and enables responses to cytotoxic stimuli. With increasing age, the expression of autophagy-related genes is downregulated, leading to autophagy dysfunction, the accumulation of protein aggregates and dysfunctional organelles, exacerbating inflammation and accelerating the aging process. Studies have shown that various compounds, such as spermidine, rapamycin, and resveratrol, can significantly extend the lifespan of immune cells in nematodes, fruit flies, mice, and humans by enhancing autophagy. These studies not only highlight the crucial role of autophagy in the aging process but, more importantly, provide a basis for the development of drugs and products for clinical anti-aging and life-extending purposes.

[0004] In recent years, the search for new anti-aging substances from natural plants or traditional Chinese medicine has become a hot topic and breakthrough in aging research. Cinnamyl cinnamate, also known as cinnamic acid cinnamate, has the molecular formula C64-C ... 18 H16O2 has the following structure: Figure 1 As shown, cinnamyl cinnamate is naturally found in balsams such as styrax and white balsam. Due to its floral and spicy aroma, cinnamyl cinnamate is commonly used in the fragrance and cosmetics industry. Cinnamyl cinnamate has been approved by the U.S. Food and Drug Administration for use as a flavoring agent in food (21 CFR 172.515) and is also permitted for use as an edible flavoring agent in my country's GB2760-1996 standard. Currently, there are no reports on the anti-aging functions of cinnamyl cinnamate. Summary of the Invention

[0005] In the first aspect, this application provides a new use of cinnamyl cinnamate, specifically relating to its application in the preparation of anti-aging products.

[0006] In some implementations, cinnamyl cinnamate exerts its anti-aging effect by activating the autophagy pathway.

[0007] In some embodiments, the activation of the autophagy pathway involves increasing the expression of autophagy-related genes and proteins; the autophagy-related genes are selected from bec-1, lgg-1, vps-34, sqst-1, or any combination thereof.

[0008] In some implementations, the anti-aging effect is to improve age-related phenotypes, including extending lifespan, improving physical fitness, and / or stress resistance.

[0009] In some implementations, the stress resistance refers to the resistance to heat stress.

[0010] In some implementations, the anti-aging effect refers to reducing the accumulation of lipofuscin (i.e., age spots) in the gut.

[0011] In some implementations, the product is a drug.

[0012] In some embodiments, the drug further comprises a pharmaceutically acceptable carrier and / or excipient.

[0013] In some embodiments, the pharmaceutically acceptable carrier and / or excipient includes: fillers, binders, lubricants, thickeners, flavoring agents, edible oils, stabilizers, suspending agents, surfactants, or any combination thereof.

[0014] In some embodiments, the pharmaceutically acceptable carrier and / or excipient includes: sugars (such as xylose, sucrose, fructose, lactose, trehalose), sugar alcohols (such as glycerol, xylitol, sorbitol, mannitol, erythritol), polysaccharides (such as cellulose and its derivatives, starch and its derivatives, chitosan, gum, maltodextrin), polyethers (such as polypropylene glycol, polyethylene glycol, polybutylene glycol), povidones (such as povidone K30, K60, K90), oils (such as rapeseed oil, sunflower oil, soybean oil, sesame oil, olive oil), surfactants (such as Tween20, Tween40, Tween60, Tween80, fatty acids, polyoxyethylene fatty alcohol ethers), inorganic salts (such as phosphates, carbonates, citrates, chlorides, sulfates, borates, citrates), talc, silica and its derivatives, hydrolysis products, or any combination thereof.

[0015] In some implementations, the dietary supplement or health food also includes additional additives.

[0016] In some embodiments, the additional additive is selected from proteins (e.g., enzymes), carbohydrates, fats, vitamins, minerals, dietary fiber, amino acids, or any combination thereof.

[0017] In some embodiments, the carbohydrate is selected from monosaccharides (glucose, fructose, xylose), disaccharides (maltose, lactose, sucrose, trehalose), sugar alcohols (such as glycerol, xylitol, sorbitol, mannitol, erythritol), polysaccharides (such as cellulose and its derivatives, starch and its derivatives, chitosan, gum, maltodextrin), or any combination thereof.

[0018] In some embodiments, the amino acid is selected from tryptophan, phenylalanine, threonine, leucine, isoleucine, histidine, arginine, or any combination thereof.

[0019] In some embodiments, the vitamin is selected from vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B9, vitamin B12, vitamin C, vitamin D, vitamin E, vitamin K, or any combination thereof.

[0020] In some embodiments, the mineral is selected from iron, manganese, zinc, copper, selenium, or any combination thereof.

[0021] Beneficial effects of the invention

[0022] This invention provides a novel application of the compound cinnamyl cinnamate. Studies have shown that cinnamyl cinnamate can significantly improve the physiological function and aging state of nematodes, and prolong their lifespan, thus demonstrating its anti-aging and lifespan-extending effects. Further research indicates that cinnamyl cinnamate can delay nematode aging by activating the expression of key autophagy genes bec-1, lgg-1, vps-34, and sqst-1. Because the cinnamyl cinnamate described in this invention has anti-aging and lifespan-extending effects, its use as an active ingredient in the preparation of products with anti-aging properties has significant application value. Attached Figure Description

[0023] Figure 1 The chemical structural formula of cinnamyl cinnamate is given.

[0024] Figure 2 These are the chemical structural formulas of Nepetoidin B.

[0025] Figure 3 The chemical structural formula of 4-hydroxychalcone is given.

[0026] Figure 4The figure shows the statistical results of the effects of different compounds on the fluorescence intensity of autophagy proteins in the DA2123 strain of nematodes; where a: P < 0.05 compared with the negative control.

[0027] Figure 5 Figure 1. Western blot results showing how cinnamyl cinnamate enhances the expression of key autophagy proteins in N2 nematodes.

[0028] Figure 6 Statistical graph showing the effect of cinnamyl cinnamate on the expression of key autophagy proteins in N2 nematodes; where *P<0.05, **P<0.01, ***P<0.001.

[0029] Figure 7 This is a statistical graph showing the effects of cinnamyl cinnamate on the expression of autophagy and aging-related genes in nematodes using qPCR.

[0030] Figure 8 Figure showing the experimental results of cinnamyl cinnamate prolonging the survival time of N2 nematodes under heat stress.

[0031] Figure 9 Figure showing the experimental results of 4-hydroxychalcone prolonging the survival time of N2 nematodes under heat stress.

[0032] Figure 10 Figure showing the experimental results of using cinnamyl cinnamate to prolong the survival time of wild-type N2 nematodes.

[0033] Figure 11 Figure 1 shows the experimental results of cinnamyl cinnamate increasing the head twitching frequency of N2 nematodes; where a: P < 0.05 compared with the negative control, b: P < 0.05 compared with the positive control.

[0034] Figure 12 A representative experimental result showing how cinnamyl cinnamate reduces the fluorescence intensity of lipofuscin in N2 nematodes.

[0035] Figure 13 Statistical graph showing the reduction of lipofuscin fluorescence intensity in N2 nematodes by cinnamyl cinnamate; where a: P < 0.05 compared with the negative control.

[0036] Figure 14 The figure shows the experimental results of the effect of cinnamyl cinnamate on the survival time of the BEC-1 defective nematode strain. Specific implementation methods

[0037] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0038] Unless otherwise specified, the experiments and methods described in the embodiments are performed in accordance with conventional methods well known in the art and described in various references.

[0039] Furthermore, unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Those skilled in the art will understand that the examples are described by way of illustration and are not intended to limit the scope of protection claimed by the invention. All disclosures and other references mentioned herein are incorporated herein by reference in their entirety.

[0040] Since the discovery of longevity genes in *Caenorhabditis elegans* in 1983, *Caenorhabditis elegans* has become a mature system for studying aging. Due to its simple structure, short lifespan, clear genetic background, and rapid reproduction rate, nematodes are commonly used for laboratory culture and high-throughput screening. Furthermore, the nematode genome shares 60%-80% homology with human genes, and its aging-regulating signaling pathways are highly conserved in higher organisms, making it a classic model for screening anti-aging drugs and studying their mechanisms of action. Therefore, this invention selects nematodes as the research subject to explore the anti-aging effects and molecular mechanisms of cinnamyl cinnamate, aiming to provide a theoretical basis for the use of cinnamyl cinnamate in the preparation of anti-aging products.

[0041] 1. Instruments, consumables and reagents

[0042] Biological incubator (THERMO FISHER, USA); Incubator (ICP110, MEMMER, Grammy); Stereo microscope (SZX16, OLYMPUS, Japan); Clean bench (Esco Airstream, ESCO, Singapore); PCR amplification instrument (MJ Reseasch PTC-200, USA); Real-time PCR instrument (ROCHE, Switzerland); Electrophoresis apparatus (BIO-RAD, USA); Gel imaging system (ChemiDoc XRS+, MERCK, Grammy).

[0043] Cinnamyl cinnamate (CAS No.: 122-69-0) is derived from TargetMol, product number TN1500, with a purity of 99.65%.

[0044] Rapamycin (R-5000, LC laboratories, USA); Fluorouracil (FUdR, HY-B0097, MedChemExpress, USA); Levamisole hydrochloride (196142, Sigma-Aldrich, USA); Dimethyl sulfoxide (DMSO) (07-4875, Sigma-Aldrich, USA); GFP antibody (HY-P80141, MedChemExpress, USA).

[0045] 2. Nematode culture

[0046] The three strains of *Caenorhabditis elegans* used in this invention were all obtained from the School of Public Health, Tianjin Medical University. The three strains are wild-type N2 nematode, DA2123 nematode (genotype adIs2122 [lgg-1p::GFP::lgg-1 +rol-6(su1006)]), and bec-1 defective nematode (genotype bec-1(ok691) IV / nT1[qls51](IV;V)). Specific genetic information for all three strains can be found at https: / / cgc.umn.edu / .

[0047] 1.1 Culture and preservation of Escherichia coli OP50

[0048] *Caenorhabditis elegans* feeds on *Escherichia coli* E. coil OP50, a uracil-deficient *E. coli*. *E. coil OP50* was placed in sterile, fresh LB broth and cultured with shaking at 37°C for 16 h. Subsequently, the bacterial culture was aliquoted, centrifuged at 4000 rpm for 10 min, the supernatant was removed, sterile water was added, and the culture was stored at 4°C for later use.

[0049] 1.2 Preparation of Nematode Growth Medium (NGM)

[0050] Weigh 0.75 g peptone, 0.9 g sodium chloride, and 5.1 g agar into an Erlenmeyer flask, add 291.6 mL of deionized water, and sterilize at 121°C for 30 min. After sterilization and cooling, add 0.3 mL each of 1 M calcium chloride solution, 1 M magnesium sulfate solution, and 5 mg / mL cholesterol anhydrous ethanol solution, and stir thoroughly. Then add 7.5 mL of pH 6.0 potassium phosphate buffer solution. After thorough mixing, pour an appropriate volume of the above culture medium into a petri dish.

[0051] After the culture medium has cooled and solidified, apply E. coil OP50 bacterial suspension evenly to the surface. Generally, add 50 μL of bacterial suspension to a 30 mm diameter petri dish. Incubate at 37°C for 3 hours to allow a distinct bacterial growth to form. Then, sterilize under UV light in a clean bench for 20 minutes before use. Before use, check the condition of the culture medium in the dish and avoid using dishes with mold, excessive air bubbles, or gaps in the walls.

[0052] 1.3 Subculture of Nematodes

[0053] Culture: *C. elegans* was cultured using a solid-state culture method. Culture dishes containing nematodes were placed in a constant-temperature incubator at 20°C for cultivation.

[0054] Subculturing: Nematodes were subcultured using the M9 buffer solution collection and transfer method. The M9 buffer solution minimizes solvent damage to the nematodes during collection. The M9 buffer solution was prepared as follows: 1.5 g potassium dihydrogen phosphate, 2.5 g sodium chloride, and 7.56 g disodium hydrogen phosphate dodecahydrate were placed in an Erlenmeyer flask, followed by 300 mL of deionized water. The flask was then autoclaved for 30 min. After cooling, 0.5 mL of 1 M magnesium sulfate solution was added, mixed thoroughly, and stored at room temperature until ready for use.

[0055] 1.4 Synchronization treatment of nematodes

[0056] Synchronization is used to obtain nematodes of the same life cycle from the same batch. The nematodes are first cultured continuously for 2-3 generations on fresh NGM containing food, in a non-starved state. When most nematodes show densely packed eggs under a stereomicroscope, subsequent synchronization treatment is performed. The specific steps are as follows: First, collect oviposition-stage nematodes into 1.5 mL centrifuge tubes using M9 buffer. After centrifugation, remove the supernatant and wash three times with M9 buffer to remove culture medium and E. coli from the nematode surface. Next, prepare nematode lysis buffer (i.e., equal volumes of sodium hypochlorite solution and 5 M sodium hydroxide solution), preparing it fresh each time. Take 1 mL of the above lysis buffer and incubate it on oviposition-stage nematodes for 6.5 min. Gently pour the buffer repeatedly during incubation, then immediately centrifuge rapidly and wash three times with M9 buffer to remove the lysis buffer. Finally, add less than 100 μL of the residual solution to an appropriate amount of M9 buffer and incubate overnight at 20°C. The next day, the above solution was collected in a 1.5 mL centrifuge tube, centrifuged rapidly, and the supernatant was removed. The L1 stage nematode pellet was added to fresh NGM containing food and incubated at 20°C for 36 h to obtain L4 stage nematodes. Fluorouracil (FUdR) was added to the NGM medium to inhibit nematode reproduction and maintain the synchronized nematode population.

[0057] 3. Experimental grouping and intervention methods

[0058] Grouping: Based on the different treatments, compounds were divided into a negative control group, a cinnamyl cinnamate treatment group, a Nepetoidin B treatment group, and a 4-hydroxychalcone treatment group, all with a concentration of 50 μM. The positive control group was a rapamycin treatment group with a concentration of 100 μM.

[0059] Preparation of E. coli OP50 bacterial suspensions for each intervention group: To prevent E. coli OP50 metabolism from altering the biochemical properties of cinnamyl cinnamate, all experiments in this invention used E. coli OP50 bacterial suspensions inactivated at high temperature as food for the nematodes. The OP50 bacterial suspensions were placed in a 65 °C incubator for 30 min to achieve inactivation. Stock solutions of cinnamyl cinnamate, Nepetoidin B, and 4-hydroxychalcone were added to the E. coli OP50 bacterial suspensions as needed to ensure a final AST concentration of 50 μM after volume adjustment. The negative control group's E. coli bacterial suspension contained the same volume of DMSO as the intervention group, and the final DMSO concentration in all E. coli OP50 bacterial suspensions was less than 0.2% (v / v).

[0060] The cinnamyl cinnamate stock solution (20 mM) was prepared as follows: 105.7 mg of cinnamyl cinnamate was dissolved in 20 mL of DMSO; the Nepetoidin B stock solution (20 mM) was prepared as follows: 125.7 mg of Nepetoidin B was dissolved in 20 mL of DMSO; the 4-hydroxychalcone stock solution (20 mM) was prepared as follows: 89.7 mg of 4-hydroxychalcone was dissolved in 20 mL of DMSO; the rapamycin stock solution (10 mM) was prepared as follows: 182.8 mg of rapamycin was dissolved in 10 mL of DMSO. All stock solutions were filtered through a 0.22 μm filter and then dispensed, and stored at -20 °C for short-term storage. Dilute to the required working concentration before use.

[0061] 4. Data Processing and Analysis

[0062] All lifespan experiments were repeated at least twice, with at least 60 nematodes included in each experiment. Survival analyses were performed using the Kaplan-Meier method, and significance was calculated using the log-rank test. All other experiments were independently repeated three times, and data are expressed as mean ± standard deviation (Mean ± SD). Unless otherwise specified, each treatment group included at least 20 nematodes. Unpaired t-tests were used to assess differences between groups. One-way or two-way ANOVA with Bonferroni multiple comparisons was used to analyze differences between more than two groups at a significance level of α = 0.05. GraphPadPrism 8.0, Excel 2013, and ImageJ software were used for data analysis and plotting.

[0063] Example 1: Detection of the effect of cinnamyl cinnamate structural analogs on the fluorescence intensity of autophagy proteins in the DA2123 strain of nematodes.

[0064] As a conserved cellular recycling mechanism, autophagy eliminates unwanted macromolecules or damaged organelles via lysosomes and recycles degradation products to maintain cellular homeostasis and renewal. Dysfunction of autophagy induces ubiquitination inhibition, increased ROS levels, mitochondrial dysfunction, and alterations in genetic material, disrupting homeostasis and triggering various diseases. Increasing evidence suggests that autophagy regulates the aging process. With increasing age, the production of autophagosomes decreases, leading to the accumulation of organelles and ROS-damaged metabolites, thus accelerating death. Conversely, activation of autophagy is essential for lifespan extension in various model organisms. Therefore, increasing autophagy levels is a crucial pathway to delaying aging.

[0065] In this invention, two compounds with chemical structures similar to cinnamyl cinnamate were selected to compare the effects of structurally similar compounds on the autophagy activation effect in nematodes, including Nepetoidin B and 4-hydroxychalcone. Nepetoidin B is a natural product isolated from many herbs, with the chemical structure shown below. Figure 2 As shown, it possesses anti-inflammatory and antibacterial activities. 4-hydroxyacetophenone (hereinafter referred to as 4-HC) is a chalcone isolated from licorice root, with the chemical structural formula shown below. Figure 3 As shown, it can play a variety of roles such as anti-oxidation, anti-inflammation, and antibacterial.

[0066] This invention utilizes three compounds with chemical structures similar to cinnamyl cinnamate to compare the effects of various compounds at the same concentration on the fluorescence intensity of autophagy proteins in the DA2123 strain of nematodes. LGG-1 is an important protein regulating autophagy in nematodes, similar to LC3 in mammals, and is a key molecule in the formation of autophagic vesicles and autolysosomes. The expression of fluorescently labeled proteins in LGG-1::GFP is often used as a marker for autophagosomes to characterize the level of autophagy. Because the DA2123 strain of nematodes stably expresses the LGG-1 reporter gene (LGG-1:GFP) labeled with green fluorescent protein (GFP), it is often used to visually detect the activation of autophagy in nematodes. If autophagy is induced, the number of autophagosomes increases, and the number of fluorescent dot structures in LGG-1::GFP nematodes increases significantly.

[0067] Previous studies have reported that rapamycin can activate autophagy in nematodes and significantly prolong their lifespan. Therefore, in this invention, the rapamycin (100 μM) treatment group was set as the positive control group. Specifically, nematodes were grouped as follows: the negative control group was treated with only an appropriate amount of DMSO; the positive control group was treated with rapamycin at a concentration of 100 μM; and the experimental groups were treated with three compounds at a concentration of 50 μM (OP50 bacterial culture contained different compounds). In this invention, DA2123 strain nematodes synchronized to the L4 stage were transferred to NGM culture dishes (with added FUdR) containing or without the corresponding compounds, and the fluorescence intensity of autophagy proteins in nematodes after different concentration treatments was detected. The nematodes in different treatment groups were cultured at 20 °C. After 5 days, the nematodes in different treatment groups were picked onto agarose pads and anesthetized with levamisole hydrochloride solution. The fluorescence expression of the nematodes in different treatment groups was observed under a laser confocal microscope, photographed and recorded, and the fluorescence intensity was analyzed using ImageJ software. The DA2123 strain of nematodes treated with only DMSO solvent was used as a negative control, and the above procedures were performed.

[0068] The average fluorescence intensity refers to the quantitative statistical analysis of the fluorescence expression intensity of the autophagy marker LGG-1::GFP in nematodes. Experimental results are as follows: Figure 4 As shown. From Figure 4 As can be seen, compared with the negative control group, the fluorescence intensity of LGG-1::GFP in nematodes treated with 50 μM cinnamyl cinnamate was significantly enhanced, and the average value was higher than that in the positive control group. In particular, the fluorescence intensity of LGG-1::GFP in nematodes treated with Nepetoidin B and 4-hydroxychalcone, which have a similar structure to cinnamyl cinnamate, did not show any enhancement. These experimental results indicate that cinnamyl cinnamate can activate autophagy in nematodes.

[0069] Example 2: Detection of the effect of cinnamyl cinnamate on the expression of key autophagy proteins in the DA2123 strain of nematodes.

[0070] This invention involves adding synchronized L4-stage DA2123 strain nematodes to NGM medium containing different compounds. After culturing at 20 °C for 5 days, nematodes from each group are collected. After repeated washing with M9 solution, proteins are extracted from the nematodes using ultrasonic lysis, and the protein concentrations in different treatment groups are detected using a BCA kit. Western blot analysis is used to detect the expression of GFP protein in nematodes in the negative control group, the rapamycin-treated positive group, and the cinnamyl cinnamate-treated experimental group. GFP protein is used to indicate the expression of LGG-1 protein in nematodes. Subsequently, the results of three independent replicate experiments are quantified and statistically analyzed using ImageJ.

[0071] In this embodiment, the negative control group was treated with DMSO solvent only, the positive control group was treated with rapamycin at a concentration of 100 μM, and the experimental group was treated with cinnamyl cinnamate (50 μM).

[0072] like Figure 5 As shown, using the expression level of β-actin (i.e., the intensity of the band color) as the standard, the expression level of GFP protein was significantly higher in the cinnamyl ester treatment group compared with the negative control group. Figure 6 The quantitative statistical graph of GFP protein expression using Image J confirmed that the expression of GFP protein in the cinnamyl ester treatment group was significantly higher than that in the negative control group and the positive control group, and the differences were statistically significant (P < 0.05). Figure 5 and Figure 6 The results collectively indicate that intervention with cinnamyl cinnamate can effectively activate autophagy in nematodes.

[0073] Example 3: Detection of the effect of cinnamyl cinnamate on the expression of autophagy-related genes in wild-type N2 nematodes

[0074] The nematode genes bec-1, lgg-1, and sqst-1 are homologs of mammalian autophagy-related genes BECN1, MAP1LC3, and P62, respectively. Numerous studies have confirmed that bec-1 is a key protein in inducing autophagy initiation, lgg-1 is involved in regulating autophagosome formation, and sqst-1, as a selective autophagy adaptor protein, plays an important regulatory role in clearing proteins to be degraded and maintaining intracellular protein homeostasis. In nematodes, vps-34 can induce the formation of primary autophagic vesicles and plays a crucial role in the degradation and clearance of mitochondria during autophagy. Therefore, this invention uses the bec-1, lgg-1, sqst-1, and vps-34 genes as autophagy markers to indicate autophagy status in nematodes.

[0075] In this invention, nematodes in different treatment groups were lysed using ultrasound, and RNA was extracted from each group using the Trizol method. The RNA was then reverse transcribed into cDNA using a PrimeScript RT kit. Subsequently, the expression levels of autophagy-related genes were detected using a real-time quantitative PCR (qPCR) kit on a CFX96TM system. The group setup in this embodiment is the same as in Example 2.

[0076] Figure 7 The results showed that, compared with the negative control group, the expression of autophagy-related genes bec-1, lgg-1, vps-1, and sqst-1 in nematodes treated with cinnamyl cinnamate was significantly upregulated. Furthermore, the expression of bec-1 and lgg-1 genes was significantly higher than that in the positive control group. These results confirm that cinnamyl cinnamate can upregulate the expression of autophagy-related genes in nematodes and activate autophagy.

[0077] As a cellular self-protection mechanism, autophagy degrades damaged intracellular proteins and organelles, participating in aging and various age-related pathological processes. Existing literature reports that autophagy plays a crucial role in different longevity models. Importantly, knockout of autophagy-related genes in nematodes leads to a significant shortening of nematode lifespan, indicating that activation of autophagy is indispensable for extending nematode lifespan. The results of Examples 1-3 above suggest that cinnamyl cinnamate may extend nematode lifespan by activating autophagy. Therefore, subsequent experiments further investigated the effect of 50 μM cinnamyl cinnamate intervention on nematode lifespan.

[0078] Example 4: Detection of the effect of cinnamyl cinnamate intervention on the lifespan of wild-type N2 nematodes under heat stress.

[0079] The extended lifespan of *C. elegans* is often accompanied by improvements in various age-related phenotypes, including stress resistance, lifespan, motility, and the accumulation of lipofuscin in the nematode.

[0080] Heat stress experiments can reflect the environmental adaptability and stress resistance of nematodes under high-temperature stimulation conditions, and can reflect the physiological state of the organism to a certain extent. Therefore, this invention uses heat stress experiments to investigate the effects of cinnamyl cinnamate on the health of nematodes during aging. OP50 bacteria containing different compounds were added to NGM culture dishes (with added FUdR). Ten wild-type N2 nematodes synchronized to the L4 stage (i.e., the first day of adulthood) were placed on each culture dish. The culture dishes were placed in a 35°C incubator for heat activation treatment. The survival rate of the nematodes was observed and recorded every 2 hours until all the nematodes in the culture dishes died.

[0081] This example included a positive control group treated with rapamycin (100 μM), an experimental group treated with cinnamyl cinnamate (50 μM), an experimental group treated with 4-hydroxychalcone (50 μM), and a negative control group treated with DMSO only. The experimental results are as follows: Figure 8 , Figure 9 As shown in Table 1 and Table 2.

[0082] Figure 8 The results showed that, under 35℃ (i.e. heat stress) conditions, the life curve of N2 nematodes in the cinnamyl cinnamate treatment group shifted significantly to the right compared with the negative control group, and the degree of shift was significantly greater than that in the positive control group (P=0.028). Figure 9 The results showed that under heat stress, the survival time of the 4-hydroxychalcone treatment group was significantly prolonged compared with the negative control group, but the extent was similar to that of the positive control (P=0.755).

[0083] Tables 1 and 2 respectively show the... Figure 8 and Figure 9 Kaplan-Meier analysis was performed, and statistical p-values ​​were calculated using the log-rank test. The data in Tables 1 and 2 show that intervention with cinnamyl cinnamate and 4-hydroxychalcone significantly increased the lifespan of nematodes under heat stress, but only cinnamyl cinnamate showed a significantly better effect on increasing the lifespan of nematodes under heat stress than the positive control group (P=0.028). Figure 8 Both Table 1 and Table 2 show that cinnamyl cinnamate can significantly improve the heat stress resistance of nematodes. Combined with the results of cinnamyl cinnamate activating autophagy in nematodes in Example 1, the above results suggest that cinnamyl cinnamate can significantly enhance the heat stress resistance and anti-aging ability of nematodes, enabling them to maintain a healthy state.

[0084] Table 1. Effects of cinnamyl cinnamate on the lifespan of wild-type N2 nematodes under heat stress.

[0085] Table 2. Effects of 4-hydroxychalcone on lifespan of wild-type N2 nematodes under heat stress.

[0086]

[0087] Example 5: Detection of the effect of cinnamyl cinnamate on the lifespan of wild-type N2 nematodes

[0088] Aging is time-dependent, and lifespan is one of the most direct and meaningful biological indicators for measuring the aging process. This invention investigated the effect of cinnamyl cinnamate on the lifespan of wild-type nematodes. A certain number of L4-stage wild-type N2 nematodes were transferred to a surface of NGM (with added FUdR) containing different compounds using a nematode picker. Counting and transfer were performed daily under a stereomicroscope. After the oviposition period, transfers were performed every other day. The number of surviving and dead nematodes was counted until all individuals had died. Nematodes that did not respond to slight touch were considered dead. Nematodes produced from hermaphroditic embryos and nematodes escaping from the NGM (such as those climbing the dish walls or burrowing into the culture medium) were not included in the count.

[0089] In this embodiment, the rapamycin (100 μM) treatment group was used as the positive control group. Additionally, the experimental group was the cinnamyl cinnamate (50 μM) treatment group, and the negative control group was the group treated with DMSO only. The experimental results are as follows: Figure 10 As shown in Table 3.

[0090] Figure 10 The results showed that, compared with the negative control group, the life curves of nematodes in both the cinnamyl cinnamate group and the positive control group shifted significantly to the right. Table 3 shows the results. Figure 5 The results were analyzed using Kaplan-Meier analysis, and the statistical p-value was calculated using the log-rank test. The number of nematodes presented in the table represents the final number included in the statistical analysis. Table 3 shows that the intervention with cinnamyl cinnamate extended the average lifespan of wild-type N2 nematodes by 38.951%, significantly exceeding that of the positive control. These results indicate that cinnamyl cinnamate can prolong the lifespan of *C. elegans*.

[0091] Table 3. Effects of cinnamyl cinnamate on the lifespan of wild-type N2 nematodes.

[0092]

[0093] Example 6: Effect of cinnamyl cinnamate on head oscillation frequency of wild-type N2 nematodes

[0094] In adult *C. elegans*, aging characteristics are manifested through changes in behavioral patterns and decline in neural function. Similar to the decreased motor ability and nervous system function observed in human aging, nematodes exhibit a significant reduction in nervous system function and motor ability with increasing age, specifically a decrease in head-shaking frequency. This change provides an important model for studying age-related neural and motor behavioral degeneration. A head-shaking motion is defined as the nematode's head swinging from one side to the other and back. The effects of cinnamyl cinnamate intervention on the nervous system activity and motor behavior control of *N. elegans* were observed by calculating the number of head-shaking motions per minute.

[0095] In this invention, at the start of intervention on days 2, 4, 6, 8, 10, and 12, 10 wild-type N2 nematodes were randomly selected from each group and placed in 50 μL of M9 buffer. After the nematodes had adapted to the M9 buffer for 1 minute, the number of times their heads twitched within that 1 minute was recorded. This experiment needs to be repeated 3 times.

[0096] Similar to Example 4, this example included a positive control group treated with rapamycin (100 μM), an experimental group treated with cinnamyl cinnamate (50 μM), and a negative control group treated only with DMSO. The results are as follows: Figure 11 As shown.

[0097] Figure 11 The results showed that, starting from the eighth day after intervention, the head-shaking frequency of nematodes in the cinnamic acid ester treatment group was significantly increased compared with the negative control group (P < 0.05). Furthermore, starting from the tenth day, the head-shaking frequency of N2 nematodes treated with cinnamic acid ester was significantly higher than that in the positive control group (P < 0.05). These experimental results indicate that cinnamic acid ester intervention can improve the nervous system function and motor ability of nematodes.

[0098] Example 7: Detection of the effect of cinnamyl cinnamate on the lipofuscin content in wild-type N2 nematodes

[0099] During human aging, the accumulation of lipofuscin in neurons is one of the most significant morphological changes. This autofluorescent substance is a complex formed by cross-linking reactions of oxidized lipids, proteins, a small amount of carbohydrates, and metals, and is widely present in nerve cells, cardiomyocytes, and skin tissue. Due to its highly polymerized and cross-linked chemical properties, lipofuscin is difficult to degrade or eliminate through cellular metabolic pathways and continues to accumulate with age. Therefore, the content of lipofuscin is often used as a reliable indicator for assessing the degree of aging in organisms (including nematodes).

[0100] In this invention, L4 stage wild-type N2 nematodes were transferred to NGM medium containing rapamycin or cinnamyl cinnamate (with added FUdR). After 10 days, nematodes were picked onto agarose pads, and anesthetized with an appropriate amount of levamisole hydrochloride. The autofluorescence of the nematodes was then observed and recorded using a laser confocal microscope. At least 20 nematodes were measured in each group. The results are as follows: Figure 12 and Figure 13 As shown.

[0101] from Figure 12 It can be seen that the fluorescence intensity of lipofuscin in the intestines of nematodes in the cinnamic acid ester treatment group and the positive control group was significantly reduced compared with that in the negative control group. Figure 13 This is a statistical analysis of the fluorescence intensity of lipofuscin in the intestines of nematodes in different treatment groups. Figure 13The results further confirmed that cinnamyl cinnamate can significantly reduce the accumulation of lipofuscin in nematodes.

[0102] Example 8: Detection of the effect of cinnamyl cinnamate on the lifespan of BEC-1 defective nematodes

[0103] To further determine the important regulatory role of autophagy in the lifespan extension of nematodes by cinnamyl cinnamate, this invention added synchronized L4-stage bec-1 defective nematodes to NGM (with added FUdR) culture dishes containing different compounds, and counted the number of surviving / dead nematodes in different treatment groups (as shown in Example 2), thus detecting the effect of cinnamyl cinnamate on the lifespan of bec-1 defective nematodes. The results are as follows: Figure 14 As shown in Table 4.

[0104] Table 4. Effects of cinnamyl cinnamate intervention on the lifespan of BEC-1 defective strains.

[0105]

[0106] Figure 14 The survival curves showed that cinnamyl cinnamate could not further prolong the lifespan of this mutant strain, indicating that cinnamyl cinnamate delays nematode senescence by acting on bec-1. Table 4 shows the results. Figure 14 The quantitative statistics (as shown in Table 1 of Example 2) indicate that the life-extending effect of cinnamate depends on the activation of autophagy.

[0107] The results of the above examples indicate that cinnamyl cinnamate can improve aging-related phenotypes in nematodes, including prolonging lifespan, enhancing heat stress resistance, nervous system function, and motor ability, as well as improving lipofuscin accumulation. At the molecular level, cinnamyl cinnamate can activate the expression of autophagy-related genes at both the mRNA and protein levels; individual survival experiments further demonstrate that cinnamyl cinnamate primarily prolongs nematode lifespan by activating the autophagy pathway.

[0108] The above results further support the claim that cinnamyl cinnamate has a good anti-aging effect and can be used in the preparation of products that delay or resist aging.

[0109] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. Application of cinnamyl cinnamate as the sole active ingredient in the preparation of anti-aging products.

2. The application as described in claim 1, characterized in that, Cinnamyl cinnamate is used as the sole active ingredient in the preparation of products that prolong lifespan, improve nervous system function, enhance motor ability, and / or stress resistance.

3. The application as described in claim 1, characterized in that, The application of cinnamyl cinnamate as the sole active ingredient in the preparation of products that reduce lipofuscin in the human gut.

4. The application as described in any one of claims 1-3, characterized in that, The product in question is a medicine.

5. The application as described in claim 4, characterized in that, The drug also includes a pharmaceutically acceptable carrier and / or excipient.

6. The application as described in claim 5, characterized in that, The pharmaceutically acceptable carriers and / or excipients include: fillers, binders, lubricants, thickeners, flavoring agents, edible oils, stabilizers, suspending agents, surfactants, or any combination thereof.

7. The application as described in claim 5, characterized in that, The pharmaceutically acceptable carriers and / or excipients include: disaccharides, sugar alcohols, polysaccharides, polyethers, povidones, oils, talc, silica and their derivatives, or any combination thereof.

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