Application of cinnamyl cinnamate in preparation of anti-aging and life-prolonging product

Abstract

The invention belongs to the technical field of medicines or health foods, and particularly relates to a novel application of cinnamyl cinnamate. The invention provides an application of cinnamyl cinnamate in preparation of a medicine or health food for delaying senescence and prolonging life. The cinnamyl cinnamate can improve senescence-related phenotypes of the nematodes, including prolonging life of the nematodes, improving heat stress resistance and exercise ability of the nematodes, and improving lipofuscin accumulation in vivo. On the molecular level, cinnamyl cinnamate can activate the expression of autophagy related genes on mRNA and protein levels. The invention provides a new way for anti-aging, and has clinical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine or health food, and specifically relates to application of cinnamic acid ester in preparing medicine or health food for delaying aging and prolonging life. Background Art

[0002] Aging is a process in which all living organisms gradually lose their natural balance, characterized by a weakening of the self-renewal and repair capabilities of tissues and organs in the body, and a gradual degenerative change that leads to irreversible death. Functional decline associated with aging may directly cause a variety of chronic diseases, such as cardiovascular disease and neurodegenerative diseases (Alzheimer's disease and Parkinson's syndrome). With the increasing aging of the population and the high incidence of aging-related diseases, the urgent need for "healthy aging" has attracted close attention from the whole society.

[0003] Autophagy is an important regulatory mechanism related to aging that has been recently proposed, and impaired autophagy is one of the important signs of aging. Autophagy refers to the process of encapsulating intracellular substances, including misfolded protein aggregates and damaged organelles (such as mitochondria), by forming autophagosomes with a double-layer membrane structure, and eventually fusing with lysosomes to degrade cell components through lysosomes. Autophagy not only prevents cell damage, but also promotes cell survival in the absence of nutrients and responds to cytotoxic stimuli. With age, the expression of autophagy-related genes is downregulated, leading to autophagy dysfunction, accumulation of protein aggregates and dysfunctional organelles, aggravating inflammation in the body, and accelerating the aging process. Studies have shown that a variety of compounds such as spermidine, rapamycin, and resveratrol can significantly prolong the lifespan of nematodes, fruit flies, mice, and human immune cells by enhancing the body's autophagy. These studies not only suggest the key role of autophagy in the aging process, but more importantly, provide a basis for the development of clinical anti-aging and life-extending drugs and other products.

[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 a molecular formula of C 18 H16O2, its structure is as follows Figure 1 As shown in Figure 1, it is naturally present in balsams such as Styrax balsam and white Peru balsam. Because of its floral and spicy aroma, cinnamate is often used in the cosmetic industry such as spices and flavors. Cinnamate has been approved by the US Food and Drug Administration as a spice for use in food (21CFR 172.515), and is also specified as a permitted edible flavor in my country's GB2760-1996. At present, there is no report on the anti-aging function of cinnamate. Summary of the invention

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

[0006] In certain embodiments, cinnamic acid exerts anti-aging effects by activating the autophagy pathway.

[0007] In certain embodiments, the activation of the autophagy pathway is to increase 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 certain embodiments, the anti-aging is to improve aging-related phenotypes, including extending lifespan, improving exercise capacity and / or stress resistance.

[0009] In certain embodiments, the stress resistance refers to heat stress resistance.

[0010] In certain embodiments, the anti-aging effect is to reduce the accumulation of lipofuscin (ie, age spots) in the intestine.

[0011] In certain embodiments, the product is a food or a medicine; preferably, the food is a health food or a dietary supplement.

[0012] In a second aspect, the present application provides an anti-aging product comprising cinnamic cinnamate.

[0013] In certain embodiments, the product is a food or a medicine; preferably, the food is a health food or a dietary supplement.

[0014] In certain embodiments, the medicament further comprises a pharmaceutically acceptable carrier and / or excipient.

[0015] In certain embodiments, the pharmaceutically acceptable carrier and / or excipient comprises: a filler, a binder, a lubricant, a glidant, a thickener, a flavoring agent, an edible oil, a stabilizer, a suspending agent, a surfactant, or any combination thereof.

[0016] In certain 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), povidone (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, silicon dioxide and its derivatives, hydrolyzates, or any combination thereof.

[0017] In certain embodiments, the dietary supplement or nutraceutical further comprises additional additives.

[0018] In certain embodiments, the additional additives are selected from proteins (eg, enzymes), carbohydrates, fats, vitamins, minerals, dietary fiber, amino acids, or any combination thereof.

[0019] In certain 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.

[0020] In certain embodiments, the amino acid is selected from tryptophan, phenylalanine, threonine, leucine, isoleucine, histidine, arginine, or any combination thereof.

[0021] In certain 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.

[0022] In certain embodiments, the mineral is selected from iron, manganese, zinc, copper, selenium, or any combination thereof.

[0023] Advantageous Effects of the Invention

[0024] The present invention provides a new application of the compound cinnamate; studies have shown that cinnamate can significantly improve the physiological function and aging state of nematodes and prolong the lifespan of nematodes, which shows that cinnamate has anti-aging and life-prolonging effects. Further studies have shown that cinnamate can delay aging of nematodes by activating the expression of key autophagy genes bec-1, lgg-1, vps-34, and sqst-1. Since the cinnamate described in the present invention has anti-aging and life-prolonging effects, it has important application value to use it as an active ingredient to prepare products with anti-aging effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the chemical structural formula of cinnamate.

[0026] Figure 2 They are the chemical structural formulas of Nepetoidin B respectively.

[0027] Figure 3 It is the chemical structural formula of 4-hydroxychalcone.

[0028] Figure 4 The figure is a statistical result of the effect of different compounds on the fluorescence intensity of autophagy protein in DA2123 strain of nematodes; wherein, a: P < 0.05 compared with the negative control.

[0029] Figure 5 Western blot experimental results showing that cinnamic acid increases the expression of key autophagy proteins in N2 nematodes.

[0030] Figure 6 This is a statistical chart showing that cinnamic acid increases the expression of key autophagy proteins in N2 nematodes; among them, *P<0.05, **P<0.01, ***P<0.001.

[0031] Figure 7 This is a statistical graph of the qPCR results of the effects of cinnamyl cinnamate on the expression of autophagy and aging-related genes in nematodes.

[0032] Figure 8 This is the experimental result showing that cinnamic acid prolongs the survival time of N2 nematodes under heat stress.

[0033] Fig. 9 This is the experimental result showing that 4-hydroxychalcone can prolong the survival time of N2 nematodes under heat stress.

[0034] Fig.10 This is a graph showing the experimental results of cinnamic acid prolonging the survival time of wild-type N2 nematodes.

[0035] Fig.11This is the experimental result of cinnamic acid increasing the head swing frequency of N2 nematodes; among them, a: P < 0.05 compared with the negative control, b: P < 0.05 compared with the positive control.

[0036] Fig.12 This is a representative experimental result showing that cinnamic acid reduces the fluorescence intensity of lipofuscin in N2 nematodes.

[0037] Fig.13 The figure is a statistical diagram showing that cinnamic acid reduces the fluorescence intensity of lipofuscin in N2 nematodes; wherein, a: P < 0.05 compared with the negative control.

[0038] Fig.14 This is a graph showing the experimental results of the effect of cinnamyl cinnamate on the survival time of bec-1-deficient nematodes. Specific implementation methods

[0040] In order to further explain the technical means and effects adopted by the present invention, the technical solution of the present invention is further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0041] Unless otherwise indicated, the experiments and procedures described in the examples were performed essentially according to conventional methods well known in the art and described in various references.

[0042] In addition, if the specific conditions are not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be obtained commercially. It is known to those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the present invention. All public cases and other references mentioned herein are incorporated herein by reference in their entirety.

[0043] Since the longevity gene was first discovered in Caenorhabditis elegans in 1983, Caenorhabditis elegans (hereinafter referred to as nematodes) has become a mature system for studying aging. Because of its simple individual structure, short lifespan, clear genetic background, and fast reproduction rate, nematodes are often used for laboratory culture and high-throughput screening. In addition, the nematode genome has homology with 60%-80% of human genes, and its signal pathways regulating aging are highly conserved with higher organisms. It is a classic model for anti-aging drug screening and mechanism research. Therefore, the present invention selects nematodes as the research object to explore the effect of cinnamic acid ester on delaying aging and its molecular mechanism, in order to provide a theoretical basis for the use of cinnamic acid ester in the preparation of anti-aging products.

[0044] 1. Instruments, consumables and reagents

[0045] Biological incubator (THERMO FISHER, USA); constant temperature incubator (ICP110, MEMMER, Grammy); stereo microscope (SZX16, OLYMPUS, Japan); clean bench (Esco Airstream, ESCO, Singapore); PCR amplifier (MJReseasch PTC-200, USA); fluorescence quantitative PCR instrument (ROCHE, Switzerland); electrophoresis instrument (BIO-RAD, USA); gel imaging system (ChemiDoc XRS+, MERCK, Grammy).

[0046] Cinnamyl cinnamate (CAS No.: 122-69-0) is from TargetMol, product No. TN1500, purity 99.65%.

[0047] Rapamycin (R-5000, LC laboratories, USA); floxuridine (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).

[0048] 2. Cultivation of Nematodes

[0049] The three strains of Caenorhabditis elegans used in the present invention are all from the School of Public Health of Tianjin Medical University. The three strains are wild-type N2 nematodes, DA2123 nematodes (genotype adIs2122 [lgg-1p::GFP::lgg-1+rol-6 (su1006)]), and bec-1 deficient nematodes (genotype bec-1 (ok691) IV / nT1 [qls51] (IV; V)). The specific genetic information of the three strains of nematodes can be found on the website https: / / cgc.umn.edu / .

[0050] 2.1 Cultivation and preservation of Escherichia coli OP50

[0051] Caenorhabditis elegans feeds on E.coil OP50, which is a uracil-deficient E.coil. E.coil OP50 is placed in a sterilized fresh LB liquid medium and cultured at 37°C with shaking for 16 hours. Subsequently, the bacterial solution is divided and centrifuged at 4000rpm for 10 minutes, the supernatant is removed and sterile water is added, and it is stored at 4°C for later use.

[0052] 2.2 Preparation of Nematode Growth Medium (NGM)

[0053] Weigh 0.75g of peptone, 0.9g of sodium chloride, and 5.1g of agar into a conical flask, add 291.6mL of deionized water, and sterilize at 121℃ for 30min. After sterilization and cooling, add 0.3mL of 1M calcium chloride solution, 1M magnesium sulfate solution, and 5mg / mL cholesterol anhydrous ethanol solution, respectively, stir well, and then add 7.5mL of pH=6.0 potassium phosphate buffer solution. After thorough mixing, pour an appropriate volume of the above culture medium into the culture dish.

[0054] After the culture medium cools and solidifies, add E.coil OP50 bacterial solution to the surface and evenly spread it. Generally, add 50μL of bacterial solution to a culture dish with a diameter of 30mm. Place it in a 37℃ incubator for 3 hours to form a clear bacterial moss, and place it in a clean bench for ultraviolet sterilization for 20 minutes before use. Before use, check the state of the culture medium in the dish and avoid using culture dishes with mold, too many bubbles, and gaps on the walls.

[0055] 2.3 Subculture of nematodes

[0056] Culture: C. elegans was cultured using a solid culture method. The culture dish with the nematodes on the surface was placed in a constant temperature incubator at 20°C for culture.

[0057] Passaging: Nematodes were passaged using the M9 buffer solution collection and transfer method. The M9 buffer solution avoided the damage of the solvent to the nematodes during the collection process as much as possible. The M9 buffer solution was prepared as follows: 1.5g of potassium dihydrogen phosphate, 2.5g of sodium chloride and 7.56g of disodium hydrogen phosphate dodecahydrate were placed in a conical flask, and then 300mL of deionized water was added and sterilized at high temperature and high pressure for 30min. After the solution cooled, 0.5mL of 1M magnesium sulfate solution was added and mixed, and then placed at room temperature for use.

[0058] 2.4 Synchronization of nematodes

[0059] Synchronization is to obtain the same batch of nematodes with the same life cycle. Nematodes are required to be cultured continuously on the surface of fresh NGM containing food for 2 to 3 generations in a non-starved state. When most nematodes are observed to have dense eggs in their bodies under a stereomicroscope, subsequent synchronization treatment is carried out. The specific steps are as follows: First, collect the nematodes in the egg-laying stage into a 1.5mL centrifuge tube with M9 buffer, remove the supernatant after centrifugation, and then wash with M9 buffer 3 times to remove the culture medium and Escherichia coli on the surface of the worm body. Next, prepare the nematode lysis solution (i.e., a mixture of equal volumes of sodium hypochlorite solution and 5M sodium hydroxide solution), and pay attention to preparing it before use. Take 1mL of the above lysis solution and act on the nematodes in the egg-laying stage for 6.5min. During this period, gently pour it repeatedly, then immediately centrifuge it quickly, and wash it with M9 buffer 3 times to remove the lysis solution. Finally, add less than 100μL of the residual solution to an appropriate amount of M9 buffer and incubate it at 20℃ overnight. The next day, the above solution was collected in a 1.5 mL centrifuge tube, and the supernatant was removed after rapid centrifugation. The L1 stage nematode pellet was added to fresh NGM containing food and incubated at 20°C for 36 hours to obtain L4 stage nematodes. FUdR was added to the NGM medium to block nematode reproduction and maintain a synchronized nematode population.

[0060] 3. Experimental groups and intervention methods

[0061] Grouping: According to different treated compounds, the cells were divided into negative control group, 50 μM cinnamate treatment group, nepetoidin B treatment group and 4-hydroxychalcone treatment group, and positive control group was 100 μM rapamycin treatment group.

[0062] Preparation of E. coli OP50 bacterial solution in each intervention group: In order to prevent E. coli OP50 from metabolically changing the biochemical properties of cinnamic acid ester, all experiments in the present invention use high-temperature inactivated E. coli OP50 bacterial solution as food for nematodes. The OP50 bacterial solution was placed in a 65°C constant temperature box for 30 minutes to achieve the inactivation effect. The mother solutions of cinnamic acid ester, Nepetoidin B and 4-hydroxychalcone were added to the E. coli OP50 bacterial solution as needed to ensure that the final concentration of AST after constant volume was 50 μM. The E. coli bacterial solution in the negative control group contained an equal volume of DMSO as that in the intervention group, and the final concentration of DMSO in all E. coli OP50 bacterial solutions was less than 0.2% (v / v).

[0063] The preparation of cinnamate mother solution (20mM) is as follows: weigh 105.7mg of cinnamate and dissolve it in 20mL of DMSO; the preparation of nepetoidin B mother solution (20mM) is as follows: weigh 125.7mg of nepetoidin B and dissolve it in 20mL of DMSO; the preparation of 4-hydroxychalcone mother solution (20mM) is as follows: weigh 89.7mg of 4-hydroxychalcone and dissolve it in 20mL of DMSO; the preparation of rapamycin mother solution (10mM) is as follows: dissolve 182.8mg of rapamycin in 10mL of DMSO; the above mother solutions are filtered through a 0.22μm filter and then packaged and stored at -20℃ for short term. Dilute to the required working concentration before use.

[0064] 4. Data Processing and Analysis

[0065] All lifespan experiments were repeated 2 or more times, and at least 60 nematodes were counted in each experiment. Survival analysis was performed using the Kaplan Meier method, and the significance was calculated using the log-rank test. The remaining experiments were repeated 3 times independently, and the data are expressed as mean ± standard deviation (Mean ± SD). Unless otherwise specified, each treatment group included at least 20 nematodes. The differences between the two groups were evaluated using an unpaired t-test. One-way or two-way ANOVA with Bonferroni's multiple comparison test was used to analyze the differences between groups of more than two groups, with a significance level of α = 0.05. GraphPad Prism 8.0 software, Excel 2013 software, and Image J software were used to analyze and plot the experimental data.

[0066] Example 1 Detection of the Effect of Cinnamyl Cinnamate Structural Analogs on the Fluorescence Intensity of Autophagy Protein in DA2123 Strain Nematodes

[0067] As a conservative cell cycle mechanism, autophagy eliminates unnecessary macromolecules or damaged organelles through lysosomes and recycles decomposition products to maintain cell homeostasis and renewal. Dysfunction of autophagy can induce ubiquitination inhibition, increased ROS levels, mitochondrial dysfunction, and genetic material changes, destroying the body's homeostasis and causing a variety of diseases. Increasing evidence shows that autophagy regulates the aging process. As the organism ages, the production of autophagosomes decreases, and organelles and metabolic substances damaged by ROS accumulate, thereby accelerating death. The activation of autophagy is essential for the extension of lifespan in a variety of model organisms. Therefore, increasing the level of autophagy is an important way to delay aging.

[0068] The present invention selects the following two compounds with similar chemical structures to cinnamate to compare the effects of compounds with similar structures on the activation of autophagy in nematodes, including Nepetoidin B and 4-hydroxychalcone. Nepetoidin B is a natural product isolated from many herbs, with a chemical structure such as Figure 2 4-Hydroxychalcone (4-hydroxyacetophenone, referred to as 4-HC in the present invention) is a chalcone isolated from the root of licorice, and its chemical structure is as follows: Figure 3 As shown, it can play multiple roles such as antioxidant, anti-inflammatory and antibacterial.

[0069] The present invention applies the above three compounds with similar chemical structures to cinnamic acid laurate, and compares the effects of various compounds on the fluorescence intensity of autophagy proteins in DA2123 strain nematodes at the same concentration. LGG-1 is an important protein for autophagy regulation in nematodes, similar to LC3 in mammals, and is a key molecule for the formation of autophagic vesicles and autophagic lysosomes. The expression of fluorescent marker protein in LGG-1::GFP is often used as a marker for autophagosomes to characterize the level of autophagy. Since the LGG-1 reporter gene (LGG-1:GFP) marked with green fluorescent protein (GFP) is stably expressed in the DA2123 strain nematodes, it is often used to visually detect the activation of autophagy in nematodes. If autophagy is induced and the number of autophagosomes increases, the number of fluorescent dot structures of LGG-1::GFP nematodes will increase significantly.

[0070] Studies have reported that rapamycin can activate autophagy in nematodes and significantly prolong the life span of nematodes. Therefore, in the present invention, the rapamycin (100 μM) treatment group is set as the positive control group. Specifically, the nematodes are grouped in the following manner: the negative control group only adds an appropriate amount of DMSO; the positive control group contains rapamycin at a concentration of 100 μM; the experimental group contains three compounds at a concentration of 50 μM (the OP50 bacterial solution contains different compounds). In the present invention, the DA2123 strain nematodes synchronized to the L4 stage are transferred to the corresponding NGM culture dish (adding FUdR) containing or not containing the compound, and the fluorescence intensity of the nematode autophagy protein after treatment with different concentrations is detected. The nematodes in different treatment groups are cultured at 20 ° C. After 5 days, the nematodes in different treatment groups are picked onto the agarose pad and anesthetized with levamisole hydrochloride solution. The fluorescence expression of nematodes in different treatment groups is observed under a laser confocal microscope, and the fluorescence intensity is analyzed by ImageJ software. The DA2123 strain of nematodes treated with only solvent DMSO was used as a negative control and subjected to the above-mentioned operation.

[0071] The average fluorescence intensity refers to the quantitative statistics of the fluorescence expression intensity of the autophagy marker LGG-1::GFP in the nematode body. The experimental results are as follows Figure 4As shown. Figure 4 It can be seen that compared with the negative control group, the fluorescence intensity of LGG-1::GFP in the nematodes treated with cinnamyl cinnamate at a concentration of 50 μM 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 the nematodes treated with Nepetoidin B and 4-hydroxychalcone, which have similar structures to cinnamyl cinnamate, did not show an enhancement. The above experimental results show that cinnamyl cinnamate can activate the occurrence of autophagy in nematodes.

[0072] Example 2 Detection of the Effect of Cinnamyl Cinnamate on the Expression of Key Autophagy Proteins in DA2123 Strain Nematodes

[0073] The present invention adds synchronized L4 stage DA2123 strain nematodes to NGM culture medium containing different compounds, and collects each group of nematodes after culturing at 20°C for 5 days. After repeated washing with M9 solution, the protein in the nematodes is extracted by ultrasonic lysis, and the protein concentration of different treatment groups is detected by BCA kit. The expression of GFP protein in the nematodes in the negative control group, the rapamycin treatment positive group and the cinnamic acid ester treatment experimental group is detected by protein immunoblotting experiment (Western blot). GFP protein is used to indicate the expression of LGG-1 protein in the nematodes. Subsequently, Image J is used to quantify and count the results of three independent repeated experiments.

[0074] In this embodiment, the negative control group is a group treated with only the solvent DMSO, the positive control group is a group treated with rapamycin at a concentration of 100 μM; and the experimental group is a group treated with cinnamyl laurate (50 μM).

[0075] like Figure 5 As shown, taking the expression level of β-actin (i.e., the depth of the band color) as the standard, the expression level of GFP protein was significantly increased in the cinnamic acid 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 cinnamic acid ester treatment group was significantly higher than that in the negative control group and the positive control group, both of which were statistically significant (P<0.05). Figure 5 and Figure 6 The results collectively indicate that cinnamic acid ester intervention can effectively activate the occurrence of autophagy in nematodes.

[0076] Example 3 Detection of the Effect of Cinnamyl Cinnamate on the Expression of Autophagy-Related Genes in Wild-Type N2 Nematodes

[0077] The nematode genes bec-1, lgg-1 and sqst-1 are homologs of the mammalian autophagy-related genes BECN1, MAP1LC3 and P62, respectively. A large number of studies have confirmed that bec-1 is a key protein that induces the initiation of autophagy, lgg-1 is involved in regulating the formation of autophagosomes, 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 vacuoles and plays an important role in the degradation and clearance of mitochondria during the autophagy process. Therefore, the present invention uses bec-1, lgg-1, sqst-1 and vps-34 genes as autophagy markers to indicate the autophagy situation in nematodes.

[0078] The present invention utilizes ultrasound to lyse the nematodes of different treatment groups, and then uses Trizol to extract RNA from each group of nematodes, and reversely transcribes RNA into cDNA using a PrimeScript RT kit. Next, a real-time fluorescence quantitative PCR (qPCR) kit is used to detect the expression level of autophagy-related genes on a CFX96TM system. The group setting of this embodiment is the same as that of Example 2.

[0079] 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 the nematode treated with cinnamyl cinnamate was significantly upregulated. In addition, the expression of bec-1 and lgg-1 genes was significantly higher than that in the positive control group. The above results confirmed that cinnamyl cinnamate can upregulate the expression of autophagy-related genes in nematodes and activate autophagy.

[0080] As a self-protection mechanism of cells, autophagy can degrade damaged intracellular proteins and organelles and other structures, and participate in aging and various pathological processes related to aging. Existing literature studies have reported that autophagy plays an important role in different longevity models. It is important that the knockout of autophagy-related genes in nematodes will lead to a significant shortening of the life span of nematodes, indicating that the activation of autophagy function in vivo is indispensable for extending the life span of nematodes. The results of the above examples 1-3 suggest that cinnamic acid may prolong the life span of nematodes by activating nematode autophagy. Therefore, subsequent experiments further studied the effect of cinnamic acid acid intervention of 50 μM on the life span of nematodes.

[0081] Example 4 Detection of the effect of cinnamate intervention on the life span of wild-type N2 nematodes under heat stress

[0082] The extension of the lifespan of Caenorhabditis elegans is often accompanied by the improvement of multiple aging-related phenotypes, including the nematode's ability to resist stress, lifespan, motility, and the accumulation of lipofuscin in the body.

[0083] 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 body to a certain extent. Therefore, the present invention uses heat stress experiments to explore the effect of cinnamic acid on the health of nematodes during aging. OP50 bacteria containing different compounds are added to NGM culture dishes (with FUdR added), and 10 wild-type N2 nematodes synchronized to the L4 stage (i.e., the first day of adulthood) are placed on each culture dish, and the culture dish is placed in a 35°C incubator for heat activation. The survival rate of the nematodes is observed and recorded every 2 hours until all the nematodes in the culture dish die.

[0084] In this example, a positive control group treated with rapamycin (100 μM), an experimental group treated with cinnamate (50 μM), an experimental group treated with 4-hydroxychalcone (50 μM), and a negative control group treated with only the solvent DMSO were set up. Figure 8 , Fig. 9 As shown in Table 1 and Table 2.

[0085] Figure 8 The results showed that under 35°C (ie, heat stress), the life curve of N2 nematodes in the cinnamate-treated group shifted significantly to the right compared with the negative control group, and the degree was significantly higher than that in the positive control group (P=0.028). Fig. 9 The results showed that under heat stress conditions, the survival time of the 4-hydroxychalcone-treated group was significantly longer than that of the negative control group, but the extent was similar to that of the positive control (P=0.755).

[0086] Table 1 and Table 2 are Figure 8 and Fig. 9 Kaplan-Meier analysis was performed, and the statistical P value was calculated using the log-rank test. The data in Tables 1 and 2 respectively show that cinnamic acid ester and 4-hydroxychalcone intervention can significantly increase the lifespan of nematodes under heat stress, but only cinnamic acid ester has a significantly better effect on improving 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 cinnamate can significantly improve the ability of nematodes to resist heat stress. Combined with the result of cinnamate activating autophagy in nematodes in Example 1, the above results collectively suggest that cinnamate can significantly improve the ability of nematodes to resist heat stress and anti-aging, making the nematodes healthy.

[0087] Table 1 Effects of cinnamate on the life span of wild-type N2 nematodes under heat stress

[0088]

[0089] Table 2 Effects of 4-hydroxychalcone on the life span of wild-type N2 nematodes under heat stress

[0090]

[0091] Example 5 Detection of the effect of cinnamate on the life span of wild-type N2 nematodes

[0092] Aging is time-dependent, and lifespan is one of the most intuitive and meaningful biological indicators for measuring the aging process. The present invention detects the effect of cinnamic acid on the lifespan of wild-type nematodes. A certain number of L4 wild-type N2 nematodes were transferred to the surface of NGM (added with FUdR) containing different compounds using a nematode pick. Counting and transfer were performed under a stereomicroscope every day. Transfers were performed every other day after the egg-laying period. The number of surviving and dead nematodes was counted until all individuals died. Nematodes that failed to respond to slight touch were considered dead. Among them, nematodes produced by embryos hatched from adult hermaphrodites and nematodes that escaped from NGM (such as climbing the dish wall and drilling into the culture medium, etc.) were not included in the statistics.

[0093] In this example, the rapamycin (100 μM) treatment group was set as the positive control group. In addition, the experimental group was the cinnamate (50 μM) treatment group, and the negative control group was the solvent DMSO treatment group. Fig.10 And as shown in Table 3.

[0094] Fig.10 The results showed that compared with the negative control group, the life curves of nematodes in the cinnamate group and the positive control group were significantly shifted to the right. Figure 5 The results were analyzed by Kaplan-Meier, and the statistical P value was calculated by log-rank test. The number of nematodes presented in the table is the number of nematodes finally counted in the experiment. As can be seen from Table 3, the intervention of cinnamic acid ester extended the average lifespan of wild-type N2 nematodes by 38.951%, and significantly exceeded the positive control. The above results show that cinnamic acid ester can extend the lifespan of Caenorhabditis elegans.

[0095] Table 3 Effects of cinnamate on the life span of wild-type N2 nematodes

[0096]

[0097] Example 6 Effect of cinnamate on head swing frequency of wild-type N2 nematodes

[0098] In adult Caenorhabditis elegans, the characteristics of aging can be reflected by changes such as changes in behavioral patterns and decline in neural function. Similar to the decline in motor ability and nervous system function shown in humans during aging, as nematodes age, their nervous system function and motor ability also decrease significantly, specifically manifested as a decrease in the frequency of head swings. This change provides an important model reference for studying the degeneration of neural and motor behaviors associated with aging. When the head of the nematode swings from one side to the other and back again, it is counted as a head swing. The effect of cinnamon ester intervention on the nervous system activity and motor behavior control of N2 nematodes was observed by counting the number of head swings per minute.

[0099] In the present invention, 10 wild-type N2 nematodes were randomly selected from each group at the 2nd, 4th, 6th, 8th, 10th, and 12th day after the intervention, and placed in 50 μL of M9 buffer. After the nematodes were adapted to M9 for 1 minute, the number of head swings of the nematodes within 1 minute was recorded. The experiment needed to be repeated 3 times.

[0100] As shown in Example 4, this example set up a positive control group treated with rapamycin (100 μM), an experimental group treated with cinnamate (50 μM), and a negative control group treated with only the solvent DMSO. Fig.11 shown.

[0101] Fig.11 The results showed that starting from the eighth day after intervention, the head swing frequency of nematodes in the cinnamic acid ester treatment group was significantly increased compared with the negative control group (P < 0.05). And starting from the tenth day, the head swing frequency of N2 nematodes treated with cinnamic acid ester was significantly higher than that of the positive control group (P < 0.05). The above experimental results show that the intervention of cinnamic acid ester can improve the nervous system function and motor ability of nematodes.

[0102] Example 7 Detection of the Effect of Cinnamyl Cinnamate on Lipofuscin Content in Wild-Type N2 Nematodes

[0103] 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 tissues. Due to its highly polymerized and cross-linked chemical properties, lipofuscin is difficult to degrade or clear through cellular metabolic pathways and continues to accumulate with age. Therefore, the content of lipofuscin is often used as a reliable indicator to assess the degree of aging of organisms (including nematodes).

[0104] The present invention transfers L4 wild-type N2 nematodes to NGM medium containing rapamycin or cinnamic acid ester (with FUdR added). After 10 days, the nematodes are picked onto an agarose pad, and an appropriate amount of levamisole hydrochloride is added to anesthetize the nematodes. Then, the autofluorescence of the nematodes is observed and recorded on a laser confocal microscope. At least 20 nematodes are measured in each group. The results are shown in FIG. Fig.12 and Fig.13 shown.

[0105] from Fig.12 It can be seen that the fluorescence intensity of lipofuscin in the intestine of nematodes in the cinnamate treatment group and the positive control group was significantly weakened compared with the negative control group. Fig.13 This is a statistical analysis of the fluorescence intensity of lipofuscin in the intestine of nematodes in different treatment groups. Fig.13 The results further confirmed that cinnamic acid can significantly reduce the accumulation of lipofuscin in nematodes.

[0106] Example 8: Detection of the effect of cinnamyl cinnamate on the lifespan of bec-1-deficient nematodes

[0107] In order to further determine the important regulatory role of autophagy in the extension of nematode lifespan by cinnamic acid, the present invention adds synchronized L4 bec-1 defective nematodes to NGM (FUdR added) culture dishes containing different compounds, and counts the number of nematodes that survive / die in different treatment groups (as shown in Example 2), and detects the effect of cinnamic acid on the lifespan of bec-1 defective nematodes. The results are shown in Figure 2. Fig.14 And as shown in Table 4.

[0108] Table 4 Effect of cinnamate intervention on the lifespan of bec-1-deficient strains

[0109] Group Number of nematodes Average life span (days) P-value Negative control group 68 15.853±0.364 Cinnamyl cinnamate treatment group 65 16.954±0.288 0.062

[0110] Fig.14 The survival curves of cinnamate showed that cinnamate could not further extend the lifespan of the mutant, indicating that cinnamate delayed nematode aging by acting on bec-1. Fig.14 The quantitative statistics (as shown in Table 1 in Example 2) indicate that the life-extending effect of cinnamic acid ester depends on the activation of autophagy.

[0111] The results of the above examples show that cinnamic acid can improve aging-related phenotypes of nematodes, including extending the lifespan of nematodes, improving the nematodes' resistance to heat stress, nervous system function and motor ability, and improving the accumulation of lipofuscin in the body. At the molecular level, cinnamic acid can activate the expression of autophagy-related genes at the mRNA and protein levels; individual survival experiments further show that cinnamic acid mainly extends the lifespan of nematodes by activating the autophagy pathway.

[0112] The above results further support that cinnamic acid has a good anti-aging effect, and cinnamic acid can be used to prepare products for delaying or resisting aging.

[0113] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. Application of cinnamate in the preparation of anti-aging products.

2. Use of cinnamate in the preparation of products for prolonging life span, improving nervous system function, increasing motor ability, and / or anti-stress ability.

3. Application of cinnamate in the preparation of products for reducing lipofuscin (age spots) in the intestines of the body.

4. The use according to any one of claims 1 to 3, characterized in that: The product is a food or a medicine; preferably, the food is a health food or a dietary supplement.

5. An anti-aging product, characterized in that: Includes cinnamate.

6. The anti-aging product according to claim 5, characterized in that: The product is food or medicine; preferably, the food is health food or dietary supplement.

7. The anti-aging product according to claim 6, characterized in that: The medicament further comprises a pharmaceutically acceptable carrier and / or excipient.

8. The product according to claim 7, characterized in that The pharmaceutically acceptable carrier and / or excipient includes: a filler, a binder, a lubricant, a glidant, a thickener, a flavoring agent, an edible oil, a stabilizer, a suspending agent, a surfactant, or any combination thereof; Preferably, in certain embodiments, the pharmaceutically acceptable carrier and / or excipient includes: sugar (such as xylose, sucrose, fructose, lactose, trehalose), sugar alcohol (such as glycerol, xylitol, sorbitol, mannitol, erythritol), polysaccharides (such as cellulose and its derivatives, starch and its derivatives, chitosan, gum, maltodextrin), polyether (such as polypropylene glycol, polyethylene glycol, polybutylene glycol), povidone (such as povidone K30, K60, K90), oil (such as rapeseed oil, sunflower oil, soybean oil, sesame oil, olive oil), surfactant (such as Tween20, Tween40, Tween60, Tween80, fatty acid, polyoxyethylene fatty alcohol ether), inorganic salt (such as phosphate, carbonate, citrate, chloride, sulfate, borate, citrate), talc, silicon dioxide and its derivatives, hydrolyzate, or any combination thereof.

9. The product according to claim 6, characterized in that The dietary supplement or health food may also include additional additives.

10. The product according to claim 9, characterized in that The additional additives are selected from proteins (e.g., enzymes), carbohydrates, fats, vitamins, minerals, dietary fibers, amino acids, or any combination thereof; Preferably, 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. Preferably, the amino acid is selected from tryptophan, phenylalanine, threonine, leucine, isoleucine, histidine, arginine, or any combination thereof. Preferably, 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. Preferably, the mineral is selected from iron, manganese, zinc, copper, selenium, or any combination thereof.

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