Application of ascaroside in anti-aging

Abstract

The invention relates to the technical field of biology, and discloses application of ascaroside in anti-aging, and the ascaroside can be widely applied to the fields of anti-aging foods, nutritional supplements and medicines. The invention reveals that ascaroside asc-C9 can play a cross-species conservative anti-aging effect from insects to mammals to a fermentation system for the first time, and the mechanism of ascaroside asc-C9 comprises promotion of mitochondrial function, enhancement of fat browning, improvement of antioxidant level and maintenance of cell energy steady state; the invention provides an anti-aging active molecule which is natural in source and clear in effect and a fermentation preparation approach thereof.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to the application of ascarid in anti-aging. BACKGROUND

[0002] Ascarid is a small molecule compound, as a pheromone substance, it can induce developmental arrest, increase stress resistance and regulate behavior in various nematodes, these functions are widely concerned; ascarid was first discovered in free-living C. elegans, which induces diapause larvae to form, at this time the larvae do not eat and survive by fat storage; it has been reported that many nematodes can produce ascarid, plant parasitic nematode Bursaphilenchus xylophilus secretes ascarid to promote its vector Monochamus alternutus pupation, and carries and transmits nematodes among pines; previous studies have shown that Asc-C9 can induce diapause of beetles, miR-31-5p regulates the plasticity of the phenotype produced by Asc-C9, improves the synthesis of cryoprotective agents glycol, thereby increasing the supercooling point, and this adaptive strategy of regulating insect growth and development cycle to resist external environmental pressure has potential great application potential in the field of anti-aging, and the application of ascarid in anti-aging has not been reported in the prior art. SUMMARY

[0003] In order to solve the above technical problems, the present application discloses the application of ascarid in anti-aging.

[0004] Preferably, the ascarid includes ascarid asc-C9.

[0005] Preferably, the ascarid is used in anti-aging food, nutritional supplements and medicine.

[0006] Preferably, the ascarid is obtained by purification of pine mushroom fermentation liquor, and the pine mushroom fermentation liquor includes pine mushroom and rhizoma polygonati fermentation liquor and pine mushroom GABA fermentation liquor.

[0007] Preferably, the preparation method of the pine mushroom and rhizoma polygonati fermentation liquor comprises the following steps: The pine mushroom, rhizoma polygonati, lactobacillus acidophilus liquid, carbon source supplement, pure water and L-glutamic acid sodium are weighed according to the proportion; Tricholoma matsutake is crushed to 120 mesh, and Huangjing is cut into 1.5 mm, and then steam sterilized at 110 DEG C for 30 min; the Tricholoma matsutake powder and Huangjing slices are mixed with purified water, 0.4% cellulase is added, and enzymolysis is carried out at 52 DEG C for 1.2 h, then constant temperature extraction is carried out at 90 DEG C for 3 h, after filtration, concentration is carried out to 47 parts; carbon source supplements are added and stirred to dissolve, pH is adjusted to 5.5, sterilization is carried out at 121 DEG C for 20 min, and then cooling is carried out to 34 DEG C; Lactobacillus acidophilus liquid is inoculated, aerobic pre-culture is carried out at 33 DEG C for 5 h, then transfer into anaerobic environment, fermentation is carried out at 35 DEG C for 28 h, stirring is carried out every 8 h for 12 min, L-glutamic acid sodium is added at the 12th hour of fermentation; 4 DEG C cold storage is carried out for 9 h, centrifugation is carried out at 4500 r / min for 18 min, the supernatant is filtered through a 0.22 mu filter membrane, and Tricholoma matsutake Huangjing fermentation liquor is obtained, which is aseptically filled and stored at 4 DEG C.

[0008] Further, the mass ratio of the Tricholoma matsutake, Huangjing, Lactobacillus acidophilus liquid, carbon source supplements, purified water and L-glutamic acid sodium is 8:28:10:6:140:0.2, wherein the carbon source supplements are obtained by mixing glucose and malt dextrin at a mass ratio of 1:2, and the concentration of the Lactobacillus acidophilus liquid is 3*10 9 CFU / mL.

[0009] Preferably, the preparation method of the Tricholoma matsutake GABA fermentation liquor comprises the following steps: Tricholoma matsutake, GABA, Lactobacillus acidophilus liquid, carbon source supplements, purified water and L-glutamic acid sodium are weighed according to the proportion, and the total amount is 140 parts; Tricholoma matsutake is crushed to 120 mesh, and then steam sterilized at 110 DEG C for 30 min; the Tricholoma matsutake powder is mixed with purified water, 0.4% cellulase is added, and enzymolysis is carried out at 52 DEG C for 1.2 h, then constant temperature extraction is carried out at 90 DEG C for 3 h, after filtration, concentration is carried out to 47 parts; carbon source supplements are added and stirred to dissolve, pH is adjusted to 5.5, GABA is added and stirred uniformly, sterilization is carried out at 121 DEG C for 20 min, and then cooling is carried out to 34 DEG C; Lactobacillus acidophilus liquid is inoculated, aerobic pre-culture is carried out at 33 DEG C for 5 h, then transfer into anaerobic environment, fermentation is carried out at 35 DEG C for 28 h, stirring is carried out every 8 h for 12 min, L-glutamic acid sodium is added at the 12th hour of fermentation; 4 DEG C cold storage is carried out for 9 h, centrifugation is carried out at 4500 r / min for 18 min, the supernatant is filtered through a 0.22 mu filter membrane, and Tricholoma matsutake GABA fermentation liquor is obtained, which is aseptically filled and stored at 4 DEG C.

[0010] Further, the mass ratio of the Tricholoma matsutake, GABA, Lactobacillus acidophilus liquid, carbon source supplements, purified water and L-glutamic acid sodium is 8:1.5:10:6:140:0.2, wherein the carbon source supplements are obtained by mixing glucose and malt dextrin at a mass ratio of 1:2, and the concentration of the Lactobacillus acidophilus liquid is 3*10 9 CFU / mL.

[0011] Compared with the prior art, the present application has the following beneficial effects: The present application discloses a method for preparing a compound of formula (I) and a use of the compound in the preparation of a medicament for treating a disease or disorder associated with the activity of a protein kinase. In the insect system, asc-C9 is generated and accumulated under low temperature conditions, can promote mitochondrial biosynthesis and uncoupling respiration, increase energy metabolism rate and antioxidant defense ability, and exhibit metabolic characteristics similar to mammalian brown fat activation, which suggests that asc-C9 has a "metabolic activation type" anti-aging mechanism. In the mammalian model, through detection of the content of asc-C9 in tissues of mice of different ages, it is found that the endogenous level significantly decreases with age, indicating that asc-C9 is closely related to the aging process of the body; after exogenous injection of asc-C9, the expressions of UCP1 and PGC-1a, the markers of browning of subcutaneous adipose tissue of mice, are significantly up-regulated, and the genes related to fat metabolism, mitochondrial function and antioxidant pathways (such as PPARa, CPT1A, SIRT1, FOXO, SOD, GPX, CAT, etc.) also show an increasing trend, which indicates that asc-C9 can promote fat browning, improve metabolic level and activate multiple anti-aging signaling pathways. In the 15-month-old old mice, the asc-C9 is injected into the abdominal cavity, and the blood indexes such as red blood cell variation coefficient (RDW-CV), neutrophil percentage (NEU), lymphocyte percentage (LYM), mean platelet volume (MPV), platelet distribution width (PDW), red blood cell number (RBC), hemoglobin (HGB) and hematocrit (HCT) are closer to those of 8-10-week-old mice, which indicates that asc-C9 can realize the rejuvenation of the blood system from the multiple dimensions of hematopoietic homeostasis, immune balance and inflammation regulation, has good anti-aging potential, and can be widely applied to the prevention and intervention of aging-related diseases. Further, in the detection of the tricholoma matsutake fermentation broth, it is found that asc-C9 can be effectively produced and accumulated in the tricholoma matsutake fermentation broth (the content of tricholoma matsutake GABA fermentation broth is 2.43 ng / g, and the content of tricholoma matsutake huangjing fermentation broth is 1.03 ng / g), and the tricholoma matsutake fermentation broth shows significant antioxidant activity in DPPH and ABTS free radical scavenging experiments and has a concentration-dependent promoting effect in cell viability detection, which proves that it has overall biological functions of antioxidant and anti-aging; in addition, tricholoma matsutake is rich in polysaccharides and triterpenes, and huangjing contains saponins and polysaccharides, both of which are food and medicine homologous materials and have basic anti-aging effects such as antioxidant and immune regulation; gamma-aminobutyric acid (GABA) can regulate the body's stress response and improve the cell metabolic environment, and cooperates with ascarid to strengthen the anti-aging effect. In summary, the application first discloses that ascaridole asc-C9 can exert a cross-species-conserved anti-aging effect from insects to mammals to fermentation systems, and the mechanism includes promoting mitochondrial function, enhancing fat browning, improving antioxidant levels, and maintaining cellular energy homeostasis; the application provides a natural source, clear anti-aging active molecule and a fermentation preparation method thereof, which can be widely applied in the fields of anti-aging food, nutritional supplements and medicine. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 is a comparison chart of body temperature and survival rate tests of Monochamus alternatus larvae in the control group and the ascaridole asc-C9 treatment group (i.e., the C9 group); Figure 2 is a fat droplet state detection result of Monochamus alternatus larvae in the control group and the ascaridole asc-C9 treatment group; Figure 3 is a comparison chart of triglyceride (TAG) and free fatty acid (FFA) tests of Monochamus alternatus larvae in the control group and the ascaridole asc-C9 treatment group; Figure 4 is an electron microscope graph of fat of Monochamus alternatus larvae in the control group and the ascaridole asc-C9 treatment group; Figure 5 is a comparison chart of mitochondrial quantity tests of fat of Monochamus alternatus larvae in the control group and the ascaridole asc-C9 treatment group; Figure 6 is a comparison chart of carbon dioxide production content tests of Monochamus alternatus larvae in the control group and the ascaridole asc-C9 treatment group; Figure 7 is a comparison chart of adenosine triphosphate (ATP) content tests of fat of Monochamus alternatus larvae in the control group and the ascaridole asc-C9 treatment group; Figure 8 is a comparison chart of relative expression quantity tests of PGC1 and UCP4 of Monochamus alternatus larvae in the control group and the ascaridole asc-C9 treatment group; Figure 9 is a detection result of an energy consumption mechanism of Monochamus alternatus larvae by ascaridole asc-C9; Figure 10 is a test result of an anti-aging mouse model experiment by ascaridole asc-C9; Figure 11 is a detection result of peripheral blood item indicators of old mice by ascaridole asc-C9; Figure 12 is a test result of an anti-aging efficacy experiment of the truffle fermentation liquor prepared in Example 1 and Example 2 of the application. DETAILED DESCRIPTION

[0013] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by a person of ordinary skill in the art without creative effort belong to the scope of the present application.

[0014] Embodiment 1

[0015] The present embodiment discloses a preparation method of Tricholoma matsutake and Rhizoma Polygonati fermenting liquid, which is specifically as follows: Tricholoma matsutake 18 parts, Rhizoma Polygonati 28 parts, Lactobacillus acidophilus liquid (3×10 9 CFU / mL) 10 parts, carbon source supplement (glucose: malt dextrin = 1:2) 6 parts, pure water 140 parts, and L-glutamic acid sodium 0.2 part are weighed by weight fraction. Tricholoma matsutake is crushed to 120 mesh, and Rhizoma Polygonati is sliced to 1.5 mm, and then steamed sterilization is performed at 110℃ for 30 min; Tricholoma matsutake powder, Rhizoma Polygonati slices and pure water are mixed, 0.4% cellulase is added, and enzymolysis is performed at 52℃ for 1.2 h, and then constant temperature extraction is performed at 90℃ for 3 h, and after filtration, concentration is performed to 47 parts; carbon source supplement is added and stirred to dissolve, pH is adjusted to 5.5, 121℃ sterilization is performed for 20 min, and then cooling is performed to 34℃; Lactobacillus acidophilus liquid is inoculated, aerobic pre-culture is performed at 33℃ for 5 h, and then transfer is performed to anaerobic environment, fermentation is performed at 35℃ for 28 h, stirring is performed every 8 h for 12 min, L-glutamic acid sodium is added at the 12th hour of fermentation; 4℃ cold storage is performed for 9 h, 4500 r / min centrifugation is performed for 18 min, the supernatant is filtered through a 0.22 μm filter membrane, and Tricholoma matsutake and Rhizoma Polygonati fermenting liquid containing ascaroside is obtained, which is aseptically filled and stored at 4℃.

[0016] Embodiment 2

[0017] The present embodiment discloses a preparation method of Tricholoma matsutake GABA fermenting liquid, which is specifically as follows: Tricholoma matsutake 18 parts, GABA (gamma-aminobutyric acid) 1.5 parts, Lactobacillus acidophilus liquid (3×10 9 CFU / mL) 10 parts, carbon source supplement (glucose: malt dextrin = 1:2) 6 parts, pure water 140 parts, and L-glutamic acid sodium 0.2 part are weighed by weight fraction. Tricholoma matsutake was pulverized to 120 mesh, and sterilized at 110°C for 30 min. The Tricholoma matsutake powder was mixed with purified water, 0.4% cellulase was added, and enzymolysis was performed at 52°C for 1.2 h, followed by constant temperature extraction at 90°C for 3 h. After filtration, the solution was concentrated to 47 parts. A carbon source supplement was added, stirred and dissolved, the pH was adjusted to 5.5, GABA was added and stirred uniformly, sterilized at 121°C for 20 min, and cooled to 34°C. Lactobacillus acidophilus was inoculated, pre-cultured aerobically at 33°C for 5 h, transferred to an anaerobic environment, and fermented at 35°C for 28 h. Stirring was performed every 8 h for 12 min, and L-glutamic acid sodium was added after 12 h of fermentation. The solution was stored at 4°C for 9 h, centrifuged at 4500 r / min for 18 min, the supernatant was filtered through a 0.22 μm filter membrane, and Tricholoma matsutake GABA fermentation liquor containing ascaroside was obtained. The fermentation liquor was aseptically filled and stored at 4°C.

[0018] Experimental Example Experimental Example 1: Effect of ascaroside asc-C9 on body temperature and survival rate of Monochamus alternatus larvae (1) Effect of asc-C9 on body temperature Monochamus alternatus larvae that had just completed diapause after overwintering were collected from the wild, injected with 900 nM asc-C9, and then placed in a -10°C constant temperature incubator for 10 min to adapt, and then the body temperature was measured. Subsequently, the larvae were transferred to 10°C, 15°C, 20°C and 25°C constant temperature incubators, respectively, and the body temperature was measured after 10 min of adaptation at each gradient. The results are shown in Figure 1 a to Figure 1 b, wherein, Figure 1 a is a temperature difference diagram of the control group and the C9 group (i.e., ascaroside asc-C9 treatment, the same below) at -10°C, Figure 1 b is a temperature difference diagram of the control group and the C9 group at different environmental temperatures; From Figure 1 a and Figure 1 b, it can be seen that the body temperature of the larvae after injection of asc-C9 was significantly higher than that of the control group in each experimental temperature zone.

[0019] (2) Effect of asc-C9 on survival rate In the survival rate experiment, the larvae were first placed in a -10°C constant temperature incubator for 17 h 30 min, and then placed in a 25°C constant temperature incubator (L:D=12:12 h) for 1 d. The larvae were stimulated with a needle, and if there was no obvious writhing or curling of the mandible or body, they were evaluated as dead. The results are shown in Figure 1 c; From Figure 1 c, it can be seen that the survival rate of the control group larvae was significantly lower than that of the asc-C9 treatment group (i.e., the C9 group).

[0020] Experimental Example 2: Effect of asc-C9 on fat metabolism of Monochamus alternatus larvae The fat droplet state, triglyceride (TAG) and free fatty acid (FFA) of the dissected fat of the larvae in Experimental Example 1 (the larvae were dissected, and the head, epidermis, intestine, digestive tract, etc. were removed, and the fat was collected for determination) were determined; the specific determination method is as follows: Hematoxylin and eosin staining: the fat of the larvae was fixed with 4% paraformaldehyde at room temperature overnight, washed with 75% ethanol for multiple times, dehydrated with 80%, 85%, 90%, 95% and 100% ethanol, and the tissue fat was sequentially placed in 50% / 50% ethanol / dimethylbenzene mixture, dimethylbenzene, dimethylbenzene I (another new dimethylbenzene solution), dimethylbenzene II (another new dimethylbenzene solution) for 15 min each time, until the tissue was transparent and translucent amber, and then sequentially immersed in 50% / 50% dimethylbenzene / paraffin, paraffin (I), paraffin (II), and then the tissue was cut into 5m thin sections after being fixed with paraffin, stained with hematoxylin and eosin (H&E), and then sealed with neutral resin, and observed and imaged analyzed using the CaseViewer system (version 2.3), and the results are shown in Figure 2 ; TAG determination: 100 μL PBST buffer was added to the fat body of the longicorn beetle larvae, and then homogenized, incubated in a 70°C water bath for 5 min, 20 μL of the sample containing precipitates after heat treatment was taken, 20 μL Triglyceride Reagent was added and mixed well, and then incubated in a 37°C constant temperature incubator for 30 min, centrifuged at 13000g for 3 min, 30 μL supernatant was taken to a UV transparent 96-well plate, 100 μL Free Glycerol Reagent was added and mixed well, and then incubated in a 37°C constant temperature incubator for 5 min, and finally the absorbance value at 540 nm wavelength was detected by an enzyme marker, and the results are shown in Figure 3 b; FFA determination: 5 μL of methyl tridecanoate was added to the larval fat body as an internal control, followed by 1 mL of a 2% H₂SO₄ / 98% Methanol mixture. The mixture was thoroughly homogenized and incubated in an 80℃ metal bath for 1 h. After cooling to room temperature, 1.5 mL of double-distilled water and 0.3 mL of n-hexane were added. The fatty acid methyl ester was extracted to the n-hexane layer by shaking. The sample was centrifuged at 5000 g for 10 min, and the upper organic phase was collected. GC-MS analysis was performed using an Agilent Technologies 6890N GC-5973N mass spectrometer with an HP-5MS capillary column (60 m × 0.25 mm × 0.25 μm, J&W Scientific, Folsom, CA). The GC-MS transfer line temperature was 280℃, the ion source temperature was 230℃, the quadrupole temperature was 50℃, the component analysis electron energy was 70 eV, the scan range was 35–400 amu, the solvent delay was 3 min, and the column temperature program was 50℃. The temperature was increased to 200℃ at a rate of 5℃ / min for 0.5 min, then to 240℃ at a rate of 2℃ / min, then to 250℃ at a rate of 5℃ / min, held for 10 min, and finally increased to 280℃ at a rate of 3℃ / min, held for 3 min. The fatty acid compounds were identified by comparing their retention times with those of the internal reference compounds and by alignment with the NIST02 mass spectrometry library (Rev. D.04.00; Agilent Technologies). The results are as follows: Figure 3 As shown in c; Measurements revealed that after injection of asc-C9, lipid droplets in the fat of longhorn beetle larvae became smaller, and the fat became fragmented. Figure 2 Furthermore, after injection of asc-C9, the TAG and FFA contents of larvae decreased significantly, and lipid metabolism accelerated. Figure 3 (b and 3c) The above results indicate that asc-C9 promotes lipid metabolism and decomposition in the larvae of the pine sawyer beetle.

[0021] Experiment Example 3: Effect of asc-C9 on uncoupling respiration in larvae of the pine sawyer beetle The mitochondrial inner membrane transport protein-uncoupling protein (UCP1) can mediate mitochondrial uncoupling thermogenesis, converting the hydrogen potential energy accumulated in the electron transport chain into internal energy, and finally releasing the energy as heat. During thermogenesis of fat, energy consumption increases. Therefore, this invention explores the effect of asc-C9 on uncoupling respiration by observing fat electron microscopy and detecting mitochondrial number, larval respiratory metabolism, and larval ATP changes. Specifically, this is achieved through the following method: Fat electron microscope: fresh fat body (cut open the larvae, remove the head, epidermis, intestine, digestive tract, etc., collect fat for determination) was soaked in 2.5% glutaraldehyde solution at 4℃ for 2 days, then washed with PBS solution for 15 min, repeated 3 times, then fixed with 1% osmium acid, washed after 2h, dehydrated with 30%, 50% ethanol for 15 min, then transferred to saturated uranyl acetate solution prepared with 70% ethanol, soaked at 4℃ in the dark for 12h, then dehydrated with 80%, 90%, 100% ethanol solution for 15 min, then transferred to 100% acetone for 20 min, 50% acetone / 50% paraffin I embedding agent (Kanglang Biology, KL130888-50) for 1h, paraffin II (new paraffin) 33% / acetone 67% embedding agent for 3h, paraffin III (new paraffin) 100% embedding agent for 5h, then heated at 65℃ for polymerization, and then placed for 1-2d, then sliced with an ultramicrotome, and then placed at 65℃ for 1-2d (baking slices, in order to make the tissue fully adhere to the glass slide), then the ultrathin sections were dyed with lead citrate hematoxylin and eosin, and then observed under an electron microscope, and the results are shown in Figure 4 ; Mitochondrial number: the mitochondrial DNA copy number was evaluated by qPCR using primers for COXII and DNA of standardized RpL32 as a template, and the results are shown in Figure 5 ; Determination of carbon dioxide production: the cerambycid larvae were placed in an insect respiratory metabolism measurement system (Beijing Yiketaisheng Ecological Technology Co., Ltd.) for oxygen consumption determination, and the results are shown in Figure 6 ; Determination of ATP: the larval fat was ground in a glass tube with mitochondrial isolation buffer (210mM d-mannitol, 70mM sucrose, 5mM HEPES, 1mM EGTA and 0.5% (w / v) fatty acid-free BSA, pH 7.2), then centrifuged at 4℃, 800g for 10 min, then the supernatant was taken and centrifuged at 4℃, 8000g for 10 min, and then the relative content of ATP was determined by Bradford method, and the results are shown in Figure 7 ; Meanwhile, from the results of the research on the regulation of fat metabolism that have been reported, the corresponding heat production functional genes in the fat metabolism related pathway were screened, the sequences of close relatives were downloaded from NCBI, and PGC1 and UCP4 were found in M. alternatus by Blast comparison, and the relative expression was detected at the gene level by RT-qPCR technology, and the results are shown in Figure 8 ; As can be seen from Figure 4 , compared with the control group, the asc-C9 treatment group (i.e. C9 group) larvae fat contains more mitochondria; as can be seen from Figure 5It can be seen that the relative number of mitochondria in the fat of the Monochamus alternatus larvae after asc-C9 treatment is significantly increased; from Figure 6 to Figure 7 It can be seen that the respiration rate of the Monochamus alternatus larvae after injection of asc-C9 is significantly accelerated, but it is found through ATP determination that the relative content of ATP in the asc-C9 treatment group is significantly less; from Figure 8 It can be seen that the relative expression of PGC1 and UCP4 of the larvae is significantly up-regulated after injection of asc-C9; The above results all show that asc-C9 can promote the uncoupling of mitochondria for heat production in the fat utilization of Monochamus alternatus larvae.

[0022] Experimental Example 4: Mechanism of asc-C9 for increasing energy consumption of Monochamus alternatus larvae In order to explore the mechanism of asc-C9 for increasing energy consumption of Monochamus alternatus larvae, the functional gene UCP4 is further verified by the present application, and immunofluorescence and immunohistochemistry are realized by the following method: Immunofluorescence: the fat of the larvae is embedded with paraffin, and after the section is developed at 37 DEG C, it is fixed with acetone at 4 DEG C for 10 min, then washed with PBS, repeated three times, then blocked with 0.1% Triton X-100 and 5% BSA mixed solution for 1 h, incubated with UCP4 containing custom rabbit source antibody (Jin Si Rui, polypeptide antibody) at 4 DEG C overnight, alpha-tubulin (1:5000, Cell Signalling Technology, USA) as an internal reference gene, washed with PBS three times, incubated with secondary antibody (Alexa Fluor 594 goat anti-rabbit IgG, Thermo Fisher, A-11012) for 30 min, 1 ug / mL DAPI staining for 10 min, using anti-fading adhesive medium (invitrogen) for adhesion, finally using confocal microscope (Zeiss LSM710) for analysis; Immunohistochemistry: the fat section of the larvae is blocked with 2.5% goat serum-PBST mixed solution at room temperature for 1 h, incubated with UCP4 containing custom rabbit source antibody at 4 DEG C overnight, washed with PBST three times, incubated with rabbit source secondary antibody (Signal Stain Boost IHC, Cell Signaling, 8114) for 1 h, washed with DAB Kit (Vector Laboratories), stained with hematoxylin-eosin, and photographed the stained slide using EVOS FL color imaging system, and the slice is quantified for lipid area and immunofluorescence brightness analysis using Image J2; From the experimental results, it can be seen that the expression of UCP4 is increased after injection of asc-C9 Figure 9a); When the expression of UCP4 or PGC1 in larvae was interfered with (dsGFP was the control group, and dsUCP4 and dsPGC1 were the post-interference treatment groups), the body temperature and survival rate at -10℃ were significantly reduced. Figure 9 b), and the resilience (head oscillation frequency within 1 minute) of larvae in the treatment group with interfered expression after being transferred from -10℃ to 25℃ for 1 day was also significantly lower than that in the control group. Figure 9 d); the respiratory rate and mitochondrial number in the interfered treatment group were significantly inhibited ( Figure 9 b and 9c), but the corresponding lipid metabolism slowed down, and the contents of TAG and ATP both increased significantly ( Figure 9 c), which is consistent with the trend after interfering with the upstream activator PGC1; however, after interfering and injecting asc-C9 (dsUCP4+C9, dsPGC1+C9 are the treatment groups after interfering with C9), the previous changes were not restored. Figure 9 c), which indicates that UCP4 is a key gene in asc-C9-mediated uncoupling thermogenesis; In summary, the generation and accumulation of ascaroside ASC-C9 under low-temperature conditions can promote mitochondrial generation and uncoupling respiration in the pine sawyer beetle, improve metabolic levels and antioxidant capacity, thereby enhancing the individual's low-temperature survival rate. This mechanism is consistent with the energy metabolism regulation and antioxidant and anti-aging effects observed during the activation of brown adipose tissue in mammals, suggesting that ascaroside ASC-C9 may have potential biological functions in delaying aging, maintaining cellular energy homeostasis, and antioxidant balance. It could serve as a candidate substance for anti-aging-related active molecules. This adaptive response centered on mitochondrial function activation is highly consistent with the mechanism of maintaining energy metabolism and redox balance during anti-aging, suggesting that ascaroside ASC-C9 may be a natural "anti-aging metabolic factor" in insects. This mechanism provides new molecular evidence for the intrinsic link between low-temperature adaptation and delaying physiological decline.

[0023] Experimental Example: Ascaroside ASC-C9 Anti-aging Mouse Model Experiment Based on the results obtained in the insect system, it was found that ascaridin asc-C9 can achieve "anti-aging metabolic activation" by promoting mitochondrial function and enhancing antioxidant defense, exhibiting physiological characteristics similar to the activation of brown adipose tissue in mammals. It is speculated that the metabolic regulation mechanism of asc-C9 may be conserved among different species. To further verify its anti-aging mechanism in mammals, this invention conducted in vivo verification experiments in mouse models. (1) Correlation analysis between asc-C9 content and aging Specifically, the asc-C9 content in mice of different ages was determined. Three male SPF-grade C57BL / 6J mice aged 6, 12, 18, and 24 months (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were purchased. The mice were allowed free access to food and water. The room temperature was 22±2℃, the relative humidity was 45%-65%, and the day / night light / dark cycle was 12h / 12h. The mice were housed in cages of 3-4. The asc-C9 content was determined by dissecting and combining the tissues. Experimental results showed that the asc-C9 content in 24-month-old aged mice was significantly lower than that in 6-, 12-, and 18-month-old mice. Figure 10 a) indicates that the endogenous level of asc-C9 is closely related to the aging process.

[0024] (2) Verification of the anti-aging effect of ASC-C9 administration A 100 mg / kg saline solution of ascaroside ASC-C9 was prepared, with saline as the control group. Ten male C57 / B mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) aged 6-8 weeks were intraperitoneally injected with the solution. The weight of each group was measured. The injection dose was 10 μL / g, administered once every two days for two months. Subcutaneous adipose tissue was collected for transcriptome analysis. Western blot experiments were performed on subcutaneous adipose tissue to verify the protein expression levels of browning marker proteins UCP4 and PGC1α. Furthermore, after injection of ASC-C9 into mice, the transcription and protein expression levels of browning-related genes, as well as the transcription levels of antioxidant and anti-aging marker genes, were detected to verify its physiological effects in promoting browning and anti-aging. The results showed that after asc-C9 treatment, the browning marker genes UCP1 (uncoupling protein 1) and PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1α) in mouse subcutaneous adipose tissue were significantly upregulated at both the transcriptional and protein levels. Figure 10 b) The above results indicate that asc-C9 can promote browning of adipose tissue and enhance mitochondrial activity and energy metabolism. The UCP1 is an uncoupling protein located on the mitochondria, which can eliminate the transmembrane proton concentration difference on both sides of the inner membrane of the mitochondria, slow down the oxidative phosphorylation process driven by the proton concentration difference, hinder the normal production of adenosine triphosphate (ATP), decouple ATP synthesis and oxidative phosphorylation, and release energy in the form of heat; the PGC1a is a peroxisome proliferator-activated receptor gamma coactivator-1a, which is a main medium for coordinating 15 mitochondrial biogenesis, cell respiration and energy metabolism, is a coactivator of the PPARs (PPAR-alpha, PPAR-delta and PPAR-gamma) transcription factor family and the retinoid X receptor, and can promote mitochondrial biosynthesis and promote UCP1 expression in cooperation with the transcription factor; Transcriptome analysis further reveals that a plurality of lipid metabolism and anti-aging pathway genes in adipose tissue are significantly up-regulated after asc-C9 treatment Figure 10 c), including: lipid metabolism and energy regulation pathway genes: PPARa, CPT1A (carnitine palmitoyltransferase 1A), ACADM (medium-chain acyl-coenzyme A dehydrogenase), FABP4 (fatty acid binding protein 4), ATGL (adipose triacylglycerol lipase), HSL (hormone-sensitive lipase), etc., which indicates that asc-C9 can enhance fatty acid oxidation and energy metabolism; Anti-aging and antioxidant-related genes: deacetylase Sirtuins (SIRT1-7), transcription factor FOXO (Forkhead Box O), Telomerase telomerase (TERT), nuclear lamina protein Lamin A / C, autophagy-related gene ATG gene, and antioxidant-related genes: superoxide dismutase SOD, glutathione peroxidase GPX, peroxidase Prdx, catalase Cat are up-regulated after administration of ascotin, which indicates that ascotin has the effects of anti-aging and antioxidant; In summary, the application first discloses the anti-aging mechanism of ascotin asc-C9 in mammals: asc-C9 activates mitochondrial biosynthesis and lipid metabolism pathways, enhances antioxidant defense and maintains cell energy homeostasis, thereby realizing the anti-aging metabolic effect similar to the activation of mammalian brown adipose tissue, which provides a new theoretical basis and application prospect for the development of anti-aging active substances and metabolic regulators based on natural products.

[0025] Experimental Example Six In the application, the peripheral blood indicators of 15-month-old old mice after intraperitoneal injection of asc-C9 or normal saline for one month are compared with the baseline levels of 8-10-week-old normal mice, and the results show that Figure 11), asc-C9 treatment significantly reversed multiple hematological parameters deteriorated with age, restoring them to the normal range of young mice; Specifically, red blood cell count (RBC), hemoglobin (HGB) and hematocrit (HCT) were significantly increased, suggesting that the impaired hematopoietic function of old mice was improved; At the same time, the red blood cell distribution width coefficient of variation (RDW-CV) decreased significantly, indicating that the heterogeneity of red blood cell volume decreased, and the hematopoietic homeostasis tended to be younger; In terms of platelet indicators, mean platelet volume (MPV) and platelet distribution width (PDW) decreased to young levels, suggesting that platelet activation and volume heterogeneity associated with aging were corrected; In terms of immune cell composition, the proportion of neutrophils, which was characteristically increased in old mice, was significantly reduced, while the proportion of lymphocytes and absolute number increased, indicating that the systemic inflammatory state (inflammaging) was reduced and adaptive immune function was restored. In summary, asc-C9 can improve red blood cell hematopoiesis, platelet homeostasis and immune cell composition with age in multiple dimensions, and overall shows a significant blood system rejuvenation effect, supporting its potential anti-aging effect.

[0026] Example Seven Anti-aging efficacy experiment of Tricholoma matsutake fermentation liquor In experimental examples 1-5, the biological effects of ascarid asc-C9 in promoting mitochondrial function, enhancing antioxidant defense and delaying aging have been verified in insect systems and mouse models. In order to further confirm whether the key active ingredient ascarid asc-C9 can be generated in the Tricholoma matsutake fermentation liquor of the present application, and to verify the anti-aging efficacy of the fermentation product as a whole, the Tricholoma matsutake fermentation liquor of the present application was subjected to component detection and function evaluation: First, the content of asc-C9 in the Tricholoma matsutake fermentation liquor of the present application was detected by high performance liquid chromatography or mass spectrometry to determine whether it was produced and accumulated during the fermentation process. Subsequently, in order to evaluate the antioxidant and cell protection activity of the Tricholoma matsutake fermentation liquor, a cell model in vitro was used for function verification; Secondly, the free radical scavenging ability of the Tricholoma matsutake fermentation liquor was evaluated by conventional DPPH free radical scavenging assay and ABTS free radical cation scavenging assay; and the effect of the Tricholoma matsutake fermentation liquor on cell viability and anti-aging function was evaluated by cell viability assay based on cell metabolic activity; The above experiments all set blank control and known antioxidant / active positive control, and were repeated multiple times to ensure the repeatability and statistical reliability of the results; DPPH radical scavenging activity assay: The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical system was used to evaluate the antioxidant capacity of the Tricholoma matsutake fermentation broth samples of Example 2; the method principle is: the Tricholoma matsutake fermentation broth sample to be tested is contacted with the DPPH radical system, so that the antioxidant active ingredients in the Tricholoma matsutake fermentation broth sample react with the DPPH radical, causing the characteristic absorbance of the DPPH solution to decrease as the free radicals are scavenged; the system without sample is used as a blank control, and a known antioxidant is used as a positive control; the DPPH radical scavenging capacity of the sample is evaluated by measuring the change in absorbance before and after the reaction and calculating the inhibition rate or clearance rate compared with the control, and the results are expressed as mean ± standard deviation, and the clearance rate or equivalent antioxidant activity index of the sample in the system can be reported (for example, the relative potency compared with the positive control); ABTS radical cation scavenging assay: The ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diamine) radical cation (ABTS•⁺) system was used to evaluate the antioxidant activity of the Tricholoma matsutake fermentation broth samples of Example 2; the method principle is: the Tricholoma matsutake fermentation broth sample to be tested is contacted with the pre-prepared ABTS•⁺ system, and the antioxidant components in the Tricholoma matsutake fermentation broth sample reduce ABTS•⁺, thereby causing the characteristic absorbance of the system to decrease; the system without sample is used as a blank control, and a known antioxidant is used as a positive control; the ABTS•⁺ clearance rate or equivalent antioxidant activity of the sample is calculated by comparing the absorbance change before and after treatment of the Tricholoma matsutake fermentation broth sample or with the control, and the data is reported as the average of repeated measurements and the corresponding statistical indicators, and can be presented in a standardized manner as needed (for example, the relative activity based on the positive control); Cell viability / cell survival rate evaluation: To evaluate the effect of Tricholoma matsutake fermentation broth on cell physiological activity, a cell viability assay based on cell metabolic activity or staining indicators (such as commonly used color reactions or fluorescence methods to detect cell metabolic enzyme activity or membrane integrity to reflect survival / viability levels) was used; the method principle is: cells are cultured under suitable conditions and treated with the Tricholoma matsutake fermentation broth sample of Example 2, and the control group includes the untreated (or carrier-treated) group and the positive / negative control group; after the treatment is completed, the cell viability or survival rate is reflected by measuring the metabolic markers (such as the conversion / staining intensity of the indicator substrate or the fluorescence signal) of the cells, and is normalized to the untreated control, and is reported as the relative cell viability (percentage) or statistical representation of the absolute absorbance / fluorescence value; all results should be based on biological repeats and appropriate statistical analysis to evaluate significance; The experimental results show that: LC / MS detection of ascaridol content in Tricholoma matsutake fermentation broth shows that the ascaridol content in Tricholoma matsutake GABA fermentation broth is 2.43 ng / g, and the ascaridol content in Tricholoma matsutake Huangjing fermentation broth is 1.03 ng / g Figure 12 a), indicating that the Tricholoma matsutake fermentation broth can effectively produce ascaridol; The cell experiment results show that, compared with the blank control, the Tricholoma matsutake fermentation liquor has significant free radical scavenging activity in DPPH and ABTS systems Figure 12 b-c), which indicates that the Tricholoma matsutake fermentation liquor has antioxidant effect, and the antioxidant effect increases with the increase of the concentration; In the cell viability detection, the treatment group shows the cell viability increase relative to the blank / vector control, the Tricholoma matsutake fermentation liquor has concentration-dependent effect on the cell viability increase, and the cell viability increase trend is more obvious with the increase of the Tricholoma matsutake fermentation liquor concentration Figure 12 d), which proves that the Tricholoma matsutake fermentation liquor has the effect of antioxidant and cell viability promotion; In summary of the above results, the Tricholoma matsutake fermentation liquor can produce the ascarid asc-C9 with physiological activity in the fermentation process, and realize the anti-aging effect by increasing the antioxidant level and cell metabolic activity, and the Tricholoma matsutake fermentation liquor has significant advantages in maintaining cell function, delaying tissue degradation and improving oxidative stress of the body, and provides a new technical approach and theoretical basis for developing natural anti-aging active substances, health foods or pharmaceutical preparations.

[0027] In summary, the application discloses an anti-aging active substance based on ascarid asc-C9 and a fermentation product thereof, research finds that asc-C9 can be generated and accumulated under low temperature conditions in an insect system, and by promoting mitochondrial biosynthesis and uncoupling respiration, the energy metabolism and antioxidant level are improved, and the "metabolic activation type" anti-aging effect is shown; in a mammalian model, the asc-C9 content significantly decreases with the increase of age, and exogenous supplementation can up-regulate the expressions of brown-related genes UCP1, PGC-1a and antioxidant and anti-aging pathway genes (SIRT1, FOXO, SOD, GPX, CAT, etc.), and enhance the mitochondrial function and fat browning; further detection finds that asc-C9 can be effectively generated in the Tricholoma matsutake fermentation liquor, and significant antioxidant and activity promotion effects are shown in DPPH and ABTS free radical scavenging experiments and cell viability detection; the results show that asc-C9 and the fermentation product thereof have the cross-species conserved anti-aging effect, and provide a new technical approach for the development of natural anti-aging active substances and functional foods.

[0028] Although the embodiments of the application have been shown and described, it is to be understood that for the purpose of the present application, the embodiments can be substantially changed without departing from the spirit and the scope of the present application, the scope of the present application being defined by the appended claims and their equivalents.

Claims

1. Application of ascaroside in anti-aging.

2. The application according to claim 1, characterized in that, The ascaroside includes ascaroside asc-C9.

3. The application according to claim 1, characterized in that, The ascaroside is used in anti-aging foods, nutritional supplements, and medicines.

4. The application according to any one of claims 1-3, characterized in that, The ascaroside is obtained by purifying matsutake fermentation liquid, which includes matsutake polygonatum fermentation liquid and matsutake GABA fermentation liquid.

5. The application according to claim 4, characterized in that, The preparation method of the matsutake and Solomon's seal fermentation liquid: Weigh out the following ingredients in the specified proportions: matsutake mushroom, polygonatum, Lactobacillus acidophilus bacterial solution, carbon source supplement, purified water, and monosodium glutamate (MSG). Matsutake mushrooms were pulverized to 120 mesh, and Polygonatum sibiricum slices were sliced ​​to 1.5 mm. The mixtures were then steam-sterilized at 110℃ for 30 min. The matsutake powder, Polygonatum sibiricum slices, and purified water were mixed, and 0.4% cellulase was added. The mixture was enzymatically hydrolyzed at 52℃ for 1.2 h, followed by extraction at 90℃ for 3 h. After filtration, the mixture was concentrated to 47 portions. A carbon source supplement was added and stirred to dissolve the mixture. The pH was adjusted to 5.5, and the mixture was sterilized at 121℃ for 20 min, then cooled to 34℃. Lactobacillus acidophilus culture was inoculated and pre-cultured aerobically at 33℃ for 5 h. The mixture was then transferred to an anaerobic environment and fermented at 35℃ for 28 h, with stirring for 12 min every 8 h. L-Glutamate sodium was added after 12 h of fermentation. The mixture was then refrigerated at 4℃ for 9 h, centrifuged at 4500 r / min for 18 min, and the supernatant was filtered through a 0.22 μm filter membrane to obtain the matsutake and Polygonatum sibiricum fermentation broth. This broth was aseptically packaged and stored at 4℃.

6. The application according to claim 5, characterized in that, The mass ratio of matsutake mushroom, polygonatum, Lactobacillus acidophilus bacterial solution, carbon source supplement, purified water, and L-glutamate is 8:28:10:6:140:0.

2. The carbon source supplement is obtained by mixing glucose and maltodextrin at a mass ratio of 1:2, and the concentration of the Lactobacillus acidophilus bacterial solution is 3 × 10⁻⁶. 9 CFU / mL.

7. The application according to claim 4, characterized in that, The method for preparing the matsutake mushroom GABA fermentation broth: Weigh out the following ingredients in the specified proportions: matsutake mushroom, GABA, Lactobacillus acidophilus bacterial solution, carbon source supplement, purified water (140 parts), and monosodium glutamate (0.2 parts). Matsutake mushrooms were pulverized to 120 mesh and steam-sterilized at 110℃ for 30 min. The matsutake powder was mixed with purified water, 0.4% cellulase was added, and the mixture was enzymatically hydrolyzed at 52℃ for 1.2 h, followed by extraction at 90℃ for 3 h. After filtration, the mixture was concentrated to 47 parts. A carbon source supplement was added and stirred to dissolve the mixture. The pH was adjusted to 5.5, and GABA was added and stirred evenly. The mixture was sterilized at 121℃ for 20 min and cooled to 34℃. Lactobacillus acidophilus culture was inoculated and pre-cultured aerobically at 33℃ for 5 h. The mixture was then transferred to an anaerobic environment and fermented at 35℃ for 28 h, with stirring for 12 min every 8 h. L-Glutamate sodium was added after 12 h of fermentation. The mixture was refrigerated at 4℃ for 9 h, centrifuged at 4500 r / min for 18 min, and the supernatant was filtered through a 0.22 μm filter membrane to obtain matsutake GABA fermentation broth. The broth was aseptically packaged and stored at 4℃.

8. The application according to claim 7, characterized in that, The mass ratio of matsutake mushroom, GABA, Lactobacillus acidophilus bacterial solution, carbon source supplement, purified water, and L-glutamate is 8:1.5:10:6:140:0.

2. The carbon source supplement is obtained by mixing glucose and maltodextrin at a mass ratio of 1:2, and the concentration of the Lactobacillus acidophilus bacterial solution is 3 × 10⁻⁶. 9 CFU / mL.

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