Application of dihydromyricetin in preparation of products for improving physiological status

Abstract

The invention provides an application of dihydromyricetin in preparation of a product for improving physiological status. Improving the physiological status comprises at least one of the following: (1) improving the integrity of ALM neurons; (2) improving the integrity of the PLM neurons; (3) the integrity of GABA neurons is improved; (4) improving touch avoidance response; (5) improving the muscle fiber state; (6) improving the mitochondrial state; and (7) the exercise ability is improved. Namely, dihydromyricetin can improve the specific physiological status, so that the life quality of people is improved, and the health status of people is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of products, and particularly relates to use of dihydromyricetin in preparing products for improving physiological status. Background Art

[0002] Dihydromyricetin (DHM), also known as ampelopsis and ampoules, is a natural flavonoid compound that is mainly found in plants of the genus Ampelopsis in the Vitaceae family, such as Ampelopsis grossedentata, A. cantoniesis, A. chaffanjonii, A. sinica, A. brevipedunculata, and A. sinica var. hancei. The highest content of DHM is in the young stems and leaves of Ampelopsis grossedentata, reaching more than 40%. In addition, it is also found in plants of the Myricaceae, Ericaceae, Garciniaceae, Euphorbiaceae, Oleaceae, Leguminosae, Sapotaceae, and Salicaceae families. The structural formula of DHM is as follows:

[0003]

[0004] Current studies have shown that in addition to the general properties of flavonoids such as liver protection, antioxidant, antibacterial and anti-inflammatory, DHM also has the function of inhibiting the activity of tumors including liver cancer, breast cancer and ovarian cancer, and has an improving effect on hypertension, hyperlipidemia and abnormal blood sugar.

[0005] Although DHM has been proven to have certain improvements or therapeutic effects in many aspects, there are few reports on how it improves some adverse physiological conditions through specific mechanisms or targets. Summary of the Invention

[0006] To address the problems and deficiencies in the prior art, the present invention provides a use of dihydromyricetin in the preparation of a product for improving physiological conditions. Specifically, dihydromyricetin can improve specific physiological conditions, thereby contributing to improving people's quality of life and health.

[0007] According to a first aspect of the present invention, there is provided a use of dihydromyricetin in preparing a product for improving physiological status, wherein the improvement of physiological status includes at least one of the following: (1) improving ALM neuron integrity; (2) improving PLM neuron integrity; (3) improving GABA neuron integrity; (4) improving touch avoidance response; (5) improving muscle fiber status; (6) improving mitochondrial status; and (7) improving exercise capacity.

[0008] The present invention proves through relevant experiments that dihydromyricetin can effectively improve the above-mentioned related physiological states, that is, dihydromyricetin improves various physiological states of the organism by adjusting the homeostatic balance of cells in the organism and optimizing various physiological activities and metabolic conditions in the organism, thereby effectively improving the overall health of the organism and improving the quality of life of the organism.

[0009] It should also be noted that, as previously mentioned, the dihydromyricetin tree originally originated from the genus Ampelopsis in the Vitaceae family. It is safer and healthier for the human body, and even long-term use will not have a significant impact on the human body. Furthermore, the subject of the present invention's experiment is the nematode (Caenorhabditis elegans). From a genetic perspective, some genes in nematodes and humans share similar nucleotide sequences. Although some genes in nematodes and humans may differ in appearance and overall structure, the proteins they encode are functionally similar or even identical. At the same time, nematodes and humans also share some conserved regulatory mechanisms in terms of physiological function. Taking apoptosis (programmed cell death) as an example, this process is precisely regulated by a series of similar genes in both nematodes and humans. Therefore, using nematodes as specific experimental subjects can better simulate relevant physiological processes in the human body, such as the aging process, and has important guiding significance for exploring human physiology and further developing related products such as food, medicine, health products, or cosmetics that improve physiological states (such as the above-mentioned physiological states mentioned in the present invention).

[0010] Preferably, the concentration of dihydromyricetin in the product is 0.01mM-5mM; or, the mass fraction of dihydromyricetin in the product is 20%-85%. At such a low dihydromyricetin concentration, it can effectively improve the physiological conditions mentioned above. That is, at such a low dihydromyricetin concentration, it can effectively improve the integrity of mechanoreceptor neurons, the integrity of GABA neurons, the touch avoidance response, the condition of muscle fibers, the morphology of muscle mitochondria, and motor ability.

[0011] Preferably, the concentration of dihydromyricetin in the product is 1 mM.

[0012] Preferably, the product includes at least one of food, health care products, medicines, and cosmetics.

[0013] Preferably, the food categories include at least one of beverages, candies, and biscuits; the health products and / or pharmaceutical dosage forms include at least one of tablets, ointments, granules, capsules, powders, and oral liquids; and the cosmetic categories include at least one of water, creams, and emulsions.

[0014] Preferably, in the preparation of the dihydromyricetin solution, the solvents used include dimethyl sulfoxide (DMSO) and titrated water (ddH2O).

[0015] Preferably, the subject for improving the above physiological state includes Caenorhabditis elegans. Preferably, the subject for improving the above physiological state includes nematode strain N2.

[0016] Preferably, the aforementioned physiological state is improved by ingesting dihydromyricetin by mixing a dihydromyricetin solution with a biological feed. The specific mixing process is as follows: dihydromyricetin is dissolved in DMSO, diluted to 1 mM with titrated water, and then mixed with the biological feed in equal proportions. Here, "mixed in equal proportions" means that the dihydromyricetin solution and the biological feed are mixed in equal volumes; the biological feed is a solution.

[0017] Preferably, the biological feed comprises an E. coli OP50 solution.

[0018] According to a second aspect of the present invention, there is provided a use of a dihydromyricetin isomer and / or a dihydromyricetin derivative in preparing a product for improving physiological status by increasing vha-3 gene expression to enhance lysosomal acidification, wherein the improved physiological status includes at least one of the following: (1) improving ALM neuron integrity; (2) improving PLM neuron integrity; (3) improving GABA neuron integrity; (4) improving touch avoidance response; (5) improving muscle fiber status; (6) improving mitochondrial status; and (7) improving exercise capacity.

[0019] Similarly, dihydromyricetin isomers and / or dihydromyricetin derivatives are similar to dihydromyricetin and can also effectively improve the above-mentioned related physiological states. By adjusting the homeostatic balance of cells in the organism and optimizing various physiological activities and metabolic conditions in the organism, the various physiological states of the organism are improved, thereby effectively improving the overall health of the organism and improving the quality of life of the organism.

[0020] Preferably, the concentration of dihydromyricetin isomers and / or dihydromyricetin derivatives in the product is 0.01mM-5mM; or, the mass fraction of dihydromyricetin isomers and / or dihydromyricetin derivatives in the product is 20%-85%. At the aforementioned lower concentrations of dihydromyricetin isomers and / or dihydromyricetin derivatives, the aforementioned physiological conditions can be effectively improved. That is, at the aforementioned lower concentrations of dihydromyricetin isomers and / or dihydromyricetin derivatives, the integrity of mechanoreceptor neurons, the integrity of GABA neurons, the touch avoidance response, the condition of muscle fibers, the morphology of muscle mitochondria, and motor ability can be effectively improved.

[0021] Preferably, the concentration of the dihydromyricetin isomers and / or dihydromyricetin derivatives in the product is 1 mM.

[0022] Preferably, the product includes at least one of food, health care products, medicines, and cosmetics.

[0023] Preferably, the food categories include at least one of beverages, candies, and biscuits; the health products and / or pharmaceutical dosage forms include at least one of tablets, ointments, granules, capsules, powders, and oral liquids; and the cosmetic categories include at least one of water, creams, and emulsions.

[0024] Preferably, the solvents used in preparing the dihydromyricetin isomer and / or dihydromyricetin derivative solution include dimethyl sulfoxide (DMSO) and titrated water (ddH2O).

[0025] Preferably, the subject for improving the above physiological state includes Caenorhabditis elegans. Preferably, the subject for improving the above physiological state includes nematode strain N2.

[0026] Preferably, the above-mentioned physiological state is improved by mixing a solution of dihydromyricetin isomers and / or dihydromyricetin derivatives with a biological feed and then ingesting the dihydromyricetin isomers and / or dihydromyricetin derivatives. The specific mixing process is as follows: the dihydromyricetin isomers and / or dihydromyricetin derivatives are dissolved in DMSO, diluted to 1 mM with titrated water, and then mixed with the biological feed in equal proportions. Here, "mixed in equal proportions" means that the dihydromyricetin isomers and / or dihydromyricetin derivatives solution and the biological feed are mixed in equal volumes; the biological feed is a solution.

[0027] Preferably, the biological feed comprises an E. coli OP50 solution.

[0028] According to a third aspect of the present invention, there is provided a use of dihydromyricetin in preparing a product for improving the decline of physiological functions in the elderly by activating atg-7 gene expression to enhance autophagy, and / or by activating vha-3 gene expression to enhance lysosomal acidification.

[0029] The atg-7 gene is a key autophagy-related gene. The encoded atg-7 protein acts as an E1-like ubiquitin-activating enzyme and plays a central regulatory role in autophagosome formation. As an E1-like enzyme, it activates atg-12 and atg-8 (e.g., LC3) to promote the extension and closure of the autophagosome membrane, ensuring the normal progress of the autophagic process. Autophagy is a key mechanism for cells to degrade damaged components and recycle substances, maintaining cellular homeostasis. Furthermore, the atg-7 gene participates in various physiological processes, such as mitophagy, which clears damaged mitochondria (e.g., those affected by oxidative stress or aging); axonal transport, which participates in vesicle transport in neurons to maintain neural signaling; stem cell maintenance, which regulates the self-renewal and differentiation of hematopoietic stem cells; and metabolic regulation, which influences the liver's circadian clock, glucose metabolism, and adipocyte differentiation. atg-7 gene mutations can cause abnormal autophagy function and trigger neuronal damage; atg-7 gene deletion can lead to infertility (such as acrosome developmental abnormalities and premature ovarian failure); atg-7 gene dysfunction may also lead to metabolic disorders such as fatty liver and insulin resistance.

[0030] The present invention has verified through relevant experiments that dihydromyricetin can increase the expression of the atg-7 gene to enhance autophagy and improve the decline of physiological functions in the elderly, thereby helping to improve the homeostasis of cells in the organism, reduce stress response and reduce disease susceptibility, improve the quality of life of the organism, and prolong the life of the organism.

[0031] The vha-3 gene (Vacuolar H+-ATPase subunit 3) encodes a subunit of the vacuolar H+-ATPase (V-ATPase), a member of a highly conserved family of multisubunit proton pumps. This gene participates in a variety of physiological and pathological processes by regulating the acidification of intracellular organelles. This acidification primarily involves pumping protons (H+) from the cytoplasm into organelles such as lysosomes, endosomes, and vacuoles, maintaining an acidic environment. This acidic environment is crucial for the activity of various hydrolases within lysosomes and for the fusion of autophagosomes with lysosomes. (After fusion, Vha-3-driven acidification is a key step in activating lysosomal enzymes and degrading autophagic substrates, such as damaged organelles and misfolded proteins.) Mutations in the vha-3 gene impair V-ATPase function, thereby impairing lysosomal acidification.

[0032] The present invention has verified through relevant experiments that dihydromyricetin can increase the expression of vha-3 gene to enhance lysosomal acidification and improve the decline of physiological functions in the elderly, that is, by activating the vacuolar ATPase vha-3, starting lysosomal acidification to improve the decline of physiological functions in the elderly. Specifically, dihydromyricetin activates the vacuolar ATPase vha-3 and starts lysosomal acidification, which can ensure that the pH value in the lysosome is within a lower range, increase the activity of lysosomal hydrolases, optimize lysosomal acidification, and then optimize the normal progress of the autophagy process, improve the smoothness of the autophagy flux, and enable harmful substances in the cell to be cleared in time, reducing the obstruction and damage of these harmful substances to cell functions, thereby further improving the homeostasis of cells in the organism, reducing stress response and reducing disease susceptibility, improving the quality of life of the organism, and prolonging the life of the organism.

[0033] Therefore, under the action of the above two specific pathways, dihydromyricetin can effectively improve the decline of physiological functions of the organism due to aging, thereby improving the various physiological states of the organism, improving the quality of life of the organism, and prolonging the life of the organism.

[0034] Preferably, the concentration of dihydromyricetin in the product is 0.01 mM-5 mM; or, the mass fraction of dihydromyricetin in the product is 20%-85%.

[0035] Preferably, the concentration of dihydromyricetin in the product is 1 mM.

[0036] Preferably, the product includes at least one of food, health care products, medicines, and cosmetics.

[0037] Preferably, the food categories include at least one of beverages, candies, and biscuits; the health products and / or pharmaceutical dosage forms include at least one of tablets, ointments, granules, capsules, powders, and oral liquids; and the cosmetic categories include at least one of water, creams, and emulsions.

[0038] Preferably, in the preparation of the dihydromyricetin solution, the solvents used include dimethyl sulfoxide (DMSO) and titrated water (ddH2O).

[0039] Preferably, the subject for improving the above physiological state includes Caenorhabditis elegans. Preferably, the subject for improving the above physiological state includes nematode strain N2.

[0040] Preferably, the aforementioned physiological state is improved by ingesting dihydromyricetin by mixing a dihydromyricetin solution with a biological feed. The specific mixing process is as follows: dihydromyricetin is dissolved in DMSO, diluted to 1 mM with titrated water, and then mixed with the biological feed in equal proportions. Here, "mixed in equal proportions" means that the dihydromyricetin solution and the biological feed are mixed in equal volumes; the biological feed is a solution.

[0041] Preferably, the biological feed comprises an E. coli OP50 solution.

[0042] According to a fourth aspect of the present invention, there is provided a use of a dihydromyricetin isomer and / or a dihydromyricetin derivative in the preparation of a product for improving the decline of physiological functions in aging by activating atg-7 gene expression to enhance autophagy, and / or by activating vha-3 gene expression to enhance autophagy.

[0043] Preferably, the concentration of dihydromyricetin isomers and / or dihydromyricetin derivatives in the product is 0.01 mM-5 mM; or, the mass fraction of dihydromyricetin isomers and / or dihydromyricetin derivatives in the product is 20%-85%.

[0044] Preferably, the concentration of the dihydromyricetin isomers and / or dihydromyricetin derivatives in the product is 1 mM.

[0045] Preferably, the product includes at least one of food, health care products, medicines, and cosmetics.

[0046] Preferably, the food categories include at least one of beverages, candies, and biscuits; the health products and / or pharmaceutical dosage forms include at least one of tablets, ointments, granules, capsules, powders, and oral liquids; and the cosmetic categories include at least one of water, creams, and emulsions.

[0047] Preferably, the solvents used in preparing the dihydromyricetin isomer and / or dihydromyricetin derivative solution include dimethyl sulfoxide (DMSO) and titrated water (ddH2O).

[0048] Preferably, the subject for improving the above physiological state includes Caenorhabditis elegans. Preferably, the subject for improving the above physiological state includes nematode strain N2.

[0049] Preferably, the above-mentioned physiological state is improved by mixing a solution of dihydromyricetin isomers and / or dihydromyricetin derivatives with a biological feed and then ingesting the dihydromyricetin isomers and / or dihydromyricetin derivatives. The specific mixing process is as follows: the dihydromyricetin isomers and / or dihydromyricetin derivatives are dissolved in DMSO, diluted to 1 mM with titrated water, and then mixed with the biological feed in equal proportions. Here, "mixed in equal proportions" means that the dihydromyricetin solution and the biological feed are mixed in equal volumes; the biological feed is a solution.

[0050] Preferably, the biological feed comprises an E. coli OP50 solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1The effect of dihydromyricetin on the lifespan of the common C. elegans strain N2 in Example 1. A is the survival curve, and B is the average lifespan. Vehicle represents the solvent control group (i.e., administered only with the solvent in the dihydromyricetin solution), and DHM represents the dihydromyricetin-administered group.

[0052] Figure 2 This is the survival curve of the experiment on the effect of dihydromyricetin on the lifespan of Caenorhabditis elegans strains daf-16 (mu86), eat-2 (sd1116), aak-2 (gt33), vha-3 (ok1501), glp-1 (e2144), rsks-1 (ok1255), clk-1 (qm30), and atg-7 (bp422) in Example 2, as well as the lifespan effect curve of feeding with heat-killed Escherichia coli HK E.coli. Among them, A is the survival curve of daf-16 (mu86) nematode strain and N2 common nematode strain, B is the survival curve of eat-2 (sd1116) nematode strain and N2 common nematode strain, C is the survival curve of aak-2 (gt33) nematode strain and N2 common nematode strain, D is the survival curve of vha-3 (ok1501) nematode strain and N2 common nematode strain, E is the survival curve of glp-1 (e2144) nematode strain and N2 common nematode strain, F is the survival curve of rsks-1 (ok1255) nematode strain and N2 common nematode strain, G is the survival curve of clk-1 (qm30) nematode strain and N2 common nematode strain, H is the survival curve of atg-7 (bp422) nematode strain and N2 common nematode strain, and I is the survival curve of nematodes fed with heat-killed Escherichia coli HK Survival curves of E. coli and the N2 common nematode strain. Vehicle represents the solvent control group (i.e., administered only with the solvent in the dihydromyricetin solution), and DHM represents the dihydromyricetin-administered group.

[0053] Figure 3 This is the observation of the morphological effects of dihydromyricetin on ALM and PLM neurons of the nematode strain zdIs5[mec-4p::GFP+lin-15(+)] in Example 3. Mock represents the blank group, and DHM represents the dihydromyricetin-treated group.

[0054] Figure 4 This is the observation of the effect of dihydromyricetin on the GABA neuron morphology of the nematode strain oxls12 (unc-47::GFP) in Example 4. Mock represents the blank group, and DHM represents the dihydromyricetin-treated group.

[0055] Figure 5 The score of dihydromyricetin on the touch avoidance reaction of nematodes in Example 5. Mock represents the blank group, and DHM represents the dihydromyricetin-administered group.

[0056] Figure 6 This is the observation of the effect of dihydromyricetin on the muscle morphology of the nematode strain raIs5 [myo-3p::GFP::myo-3+rol-6(su1006)] in Example 6. Mock represents the blank group, and DHM represents the dihydromyricetin-treated group.

[0057] Figure 7 This is the observation of the muscle mitochondrial morphology of the nematode strain foxSi16 [myo-3p::tomm-20::mKate2::HA::tbb-2 3'UTR] treated with dihydromyricetin in Example 7. Mock represents the blank group, and DHM represents the dihydromyricetin-treated group.

[0058] Figure 8 The effect of dihydromyricetin on the motility of nematodes in Example 8. Mock represents the blank group, and DHM represents the dihydromyricetin-administered group.

[0059] Figure 9 Schematic diagram of ALM and PLM neurons in Example 3. DETAILED DESCRIPTION

[0060] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0061] Example 1: Effect of dihydromyricetin on the lifespan of nematodes

[0062] Follow the steps below to explore how dihydromyricetin improves nematode lifespan:

[0063] Step 1: prepare nematode growth medium (NGM) plates and nematode egg-laying restriction medium (FUdR) plates coated with a mixture of dihydromyricetin solution and OP50 E. coli and OP50 E. coli alone, as well as a common nematode strain N2; in the mixture of dihydromyricetin solution and OP50 E. coli, the dihydromyricetin solution and OP50 E. coli are mixed in equal proportions, where equal proportion mixing means that the dihydromyricetin solution and the OP50 E. coli solution are mixed in equal volumes, and the OD value of the OP50 E. coli solution is 1;

[0064] The dihydromyricetin solution was dissolved in DMSO and diluted to 1 mM with titrated water;

[0065] NGM plates were prepared by weighing 3.5 g NaCl, 2.0 g peptone, and 25 g agar powder, adding 800 mL deionized water, fully dissolving, and then sterilizing by autoclaving (121° C., 20 min); cooling to 55° C., adding 1 mL 1 mol / L CaCl 2 , 1 mL 1 mol / L MgSO 4 , and 1 mL 5 mg / L cholesterol in a clean bench, and then adding 25 mL 1 mol / L potassium phosphate solution (pH = 6.0), and adjusting the volume to 1 L with ddH 2 O; mixing well, and then quickly dispensing into culture dishes to prepare the plate;

[0066] The FUdR plate is prepared as follows: 3.5 g NaCl, 2.0 g peptone, and 25 g agar powder are weighed, added to 800 mL deionized water, fully dissolved, and then sterilized by autoclaving (121° C., 20 min); after cooling to 55° C., 1 mL 1 mol / L CaCl2, 1 mL 1 mol / L MgSO4, 1 mL 5 mg / L cholesterol, and 5 mL 10 mg / mL 5-fluoro-2'-deoxyguanosine (FUdR) are added in a clean bench to a final concentration of 50 μg / mL of FUdR; 25 mL 1 mol / L potassium phosphate solution (pH = 6.0) is then added, and the volume is adjusted to 1 L with ddH2O; after mixing, the plate is quickly dispensed into a culture dish to prepare the plate;

[0067] NGM plates and FUdR plates were wrapped with sealing film and stored at 4°C;

[0068] Step 2: Synchronize nematodes. Culture the nematodes on NGM plates for 2-3 days. Obtain a plate rich in eggs without exhausting OP50. Use a pipette to draw 1-2 mL of M9 buffer onto the NGM plate and rinse repeatedly to ensure that the eggs do not remain on the plate. Transfer the liquid containing the eggs to a 1.5 mL centrifuge tube. Centrifuge at 4,000 rcf for 1 minute and discard the supernatant. Add 500 μL of bleach buffer to the remaining 100 μL of liquid, fasten the tube cap, and shake for 3-5 minutes. The time control should ensure that the whole nematode cannot be seen under the microscope, and at the same time, the eggs are not soaked for too long and inactivated. Centrifuge at 4,000 rcf for 0.5 minutes and discard the supernatant. Add 1 mL of Resuspend the pellet in M9 buffer, centrifuge at 4,000 rcf for 1 min, discard the supernatant, and repeat this step once more. Pipette the remaining 100 μL of liquid several times to evenly distribute the pellet, then transfer it to a new NGM plate near the bacterial lawn. After drying, invert the plate and incubate it in a 20°C incubator.

[0069] M9 buffer was prepared as follows: 0.25 g MgSO4·7H2O, 3.0 g KH2PO4, 6.0 g Na2HPO4, and 5.0 g NaCl were weighed and fixed to 1 L with ddH2O. The solution was filtered through a 0.45 μm filter in a laminar flow hood, aliquoted into sterile containers, and stored at room temperature until use.

[0070] Bleach buff was prepared as follows: 10 mL of NaClO (4%), 10 mL of NaOH (1 M), and 20 mL of ddH2O were mixed evenly, transferred to a brown centrifuge tube, and stored at room temperature in the dark;

[0071] Step 3: After the eggs were cultured to the L4 stage, 50 nematodes were picked and placed on the corresponding fresh FUdR plates, with 3 replicates for each feeding group; there were two different feeding groups, one for the nematode growth medium (FUdR) plates coated with a mixture of dihydromyricetin solution and OP50 E. coli in step 1 (NGM medium was used for synchronization of the L4 stage), and the other for the nematode growth medium (FUdR) plates with only OP50 E. coli (NGM medium was used for synchronization of the L4 stage);

[0072] Step 4: Count the number of dead nematodes every day. Those that do not react after being touched are dead; continue counting until all the nematodes in the plate are dead.

[0073] like Figure 1 As shown, it can be seen that dihydromyricetin has the effect of significantly extending the lifespan of Caenorhabditis elegans.

[0074] Example 2: Mechanism of Dihydromyricetin in Prolonging the Lifespan of Nematodes

[0075] The mechanism of action of dihydromyricetin was determined by analyzing the survival curves of nematode strains with knockdown genes in different pathways after feeding them with dihydromyricetin. The mechanism of action of dihydromyricetin was also determined by mixing dihydromyricetin with HK E. coli (to confirm whether the anti-aging effect was caused by low food intake and caloric restriction) and then feeding it to the normal nematode N2.

[0076] The CRISPR / Cas9 technology was used to edit the genome of Caenorhabditis elegans and knock down the genes daf-16, eat-2, aak-2, vha-3, glp-1, rsks-1, clk-1, and atg-7. The corresponding gene knockdown strains daf-16 (mu86), eat-2 (sd1116), aak-2 (gt33), vha-3 (ok1501), glp-1 (e2144), rsks-1 (ok1255), clk-1 (qm30), and atg-7 (bp422) were obtained.

[0077] Among them, the daf-16 (mu86) strain represents the insulin / IGF-1 signaling pathway. The eat-2 (sd1116) strain represents the caloric restriction pathway. The aak-2 (gt33) strain represents the AMPK signaling pathway. The vha-3 (ok1501) strain represents the lysosomal acidification and autophagy pathway. The glp-1 (e2144) strain represents the germline stem cell Notch signaling pathway. The rsks-1 (ok1255) strain represents the TOR signaling pathway. The clk-1 (qm30) strain represents the mitochondrial function pathway. The atg-7 (bp422) strain represents the autophagy pathway. The heat-killed HK E. coli feeding represents the microbial host interaction pathway.

[0078] Follow the steps below to explore how dihydromyricetin improves the lifespan of different types of nematodes:

[0079] Step 1: Prepare nematode strains daf-16 (mu86), eat-2 (sd1116), aak-2 (gt33), vha-3 (ok1501), glp-1 (e2144), rsks-1 (ok1255), clk-1 (qm30), atg-7 (bp422), and heat-killed Escherichia coli (HK E. coli) (fed with normal nematode N2); prepare NGM and FUdR plates;

[0080] Step 2: As in Example 1, a large number of synchronized nematode eggs were prepared and cultured on NGM plates to the L4 stage, and then 50 were picked and placed on corresponding FUdR plates and cultured in an incubator; three replicates were performed for each of the different feeding groups of nematodes; here, the different feeding groups corresponded to the different types of nematode strains in step 1 and the common nematodes N2 fed with heat-killed Escherichia coli (HK E.coli); and each different type of nematode strain and the common nematode N2 fed with heat-killed Escherichia coli (HK E.coli) also included a sample group and a solvent control group, the sample group was a dihydromyricetin solution group, and the solvent control group was only administered with the solvent in the dihydromyricetin solution, and the dihydromyricetin solution group and the solvent control group were generally added with an equal volume of mixed OP50 Escherichia coli solution, that is, the volume ratio of the dihydromyricetin solution and the solvent alone to the OP50 Escherichia coli solution was 1:1, and the OD value of the OP50 Escherichia coli solution was 1;

[0081] Step 3: Count the number of dead nematodes every day. Those that do not react after being touched are dead; continue until all the nematodes in the plate are dead.

[0082] like Figure 2As shown, dihydromyricetin extends the lifespan of C. elegans mainly through the regulation of vha-3 and atg-7 genes, that is, dihydromyricetin extends the lifespan of C. elegans by relying on two pathways: lysosomal acidification and autophagy. Specifically, we can see that only in the nematode strains vha-3 and atg-7, the survival curves of nematodes treated with dihydromyricetin and those not treated with dihydromyricetin are similar, indicating that dihydromyricetin does not improve the lifespan or other physiological states of nematodes by affecting other genes, but rather by affecting the regulation of vha-3 and atg-7 genes.

[0083] Example 3: Dihydromyricetin improves mechanoreceptor neuron integrity

[0084] The zdIs5[mec-4p::GFP+lin-15(+)] nematode strain was used to observe changes in mechanoreceptor neurons in the nematode C. elegans. The mec-4 gene promoter drives the expression of green fluorescent protein (GFP), resulting in mec-4::GFP expression in touch-sensitive neurons. Mec-4p::GFP labeling allows for clear visualization of the morphology and distribution of mechanoreceptor neurons. For example, neurons such as the anterior lateral microtubule (ALM) and posterior lateral microtubule (PLM) were successfully labeled in this strain. These neurons are responsible for sensing mechanical stimuli. ALM and PLM neurons undergo significant morphological changes during aging, affecting not only their structure (such as increased branch curling, looping, and outgrowth processes), but also impaired sensory function (such as slowed touch response), decreased mobility (such as slow crawling and difficulty turning), and diseases (such as neurodegeneration and protein homeostasis imbalance).

[0085] The following steps were used to investigate the effect of dihydromyricetin on improving the integrity of mechanoreceptor neurons:

[0086] Step 1: Prepare the nematode strain zdIs5[mec-4p::GFP+lin-15(+)]; prepare NGM and FUdR plates;

[0087] Step 2: Prepare a large number of synchronized nematode eggs as in Example 1 and culture them on NGM plates until the L4 stage. Then transfer them to FUdR plates for culture and start drug administration (1mM dihydromyricetin solution and OP50 Escherichia coli solution are mixed in equal volumes, and the OD value of the OP50 Escherichia coli solution is 1). Use a stereo fluorescence microscope to take fluorescent photos on day 3, day 5, and day 10 to observe the integrity of neurons; the neurons here include ALM and PLM neurons. ALM neurons and PLM neurons are anterior lateral microtubule cells and posterior lateral microtubule cells, respectively. Figure 9 shown.

[0088] Complete mechanosensory neurons are characterized by being long, smooth, and without breakpoints. The aging of mechanosensory neurons will affect the nematode's response to touch avoidance and external stimuli.

[0089] Depend on Figure 3 As shown, it can be seen that dihydromyricetin has the effect of significantly improving the integrity of the mechanoreceptor neurons of the nematode on the 5th day, that is, on the 5th day, the mechanoreceptor neurons of the nematode can still remain intact, and the breakpoints and losses are significantly less than those of the nematodes not administered with dihydromyricetin.

[0090] Example 4: Dihydromyricetin improves GABA neuron integrity

[0091] GABA neuron degeneration leads to abnormal changes such as increased branching, curling, and looping, which also affect the nematode's motor coordination, foraging, and olfactory perception. The oxls12(unc-47::GFP) nematode strain was used to observe GABA neuron aging. The unc-47 gene (encoding the GABA transporter) promoter drives the expression of green fluorescent protein (GFP), making all GABA neurons clearly visible under a fluorescence microscope.

[0092] The following steps were used to investigate the effect of dihydromyricetin on improving the integrity of GABA neurons:

[0093] Step 1: Prepare the nematode strain oxls12 (unc-47::GFP) and prepare NGM and FUdR plates;

[0094] Step 2: As in Example 1, a large number of synchronized nematode eggs were prepared and cultured on NGM plates until the L4 stage. The eggs were then transferred to FUdR plates and drug administration was initiated (1 mM dihydromyricetin solution and OP50 E. coli solution were mixed in equal volumes, and the OD value of the OP50 E. coli solution was 1). Fluorescence images were taken using a stereo fluorescence microscope on day 3, day 5, and day 10 to observe the integrity of the neurons; the neurons here are GABA neurons.

[0095] The intact GABA neurons have many short stripes and are smooth and flat. Figure 4 As shown, it can be seen that dihydromyricetin has the effect of significantly improving the integrity of GABA neurons, that is, on the 3rd, 5th and 10th days, the GABA neurons of nematodes can still remain intact, and the number of broken and spotted neurons is significantly less than that of nematodes not administered with dihydromyricetin.

[0096] Example 5: Dihydromyricetin improves nematode touch avoidance response

[0097] Follow the steps below to investigate how dihydromyricetin improves the touch avoidance response in nematodes:

[0098] Step 1: Prepare nematode strain N2 and prepare NGM and FUdR plates;

[0099] Step 2: As in Example 1, a large number of synchronized nematode eggs were prepared and cultured on NGM plates until the L4 stage. They were then transferred to FUdR plates and drug administration was initiated (a mixture of equal volumes of 1 mM dihydromyricetin solution and OP50 E. coli culture, with an OD value of 1 for the OP50 E. coli culture). Touch response experiments were conducted on days 3, 5, and 10. Nematodes naturally tend to avoid harm and avoid advantages. When the head and tail of a nematode are touched with an eyebrow, the nematode will flee away from the touched area. This characteristic was used here to study the speed and loss of touch response. For the touch response sample, n = 15, each of which was touched three times on the head and tail to observe whether there was a response, with a response counted as 1 and no response as 0.

[0100] Depend on Figure 5 It can be seen that dihydromyricetin can significantly improve the touch avoidance response of nematodes on the 5th day, which is reflected in the delayed aging of mechanoreceptor neurons.

[0101] Example 6: Dihydromyricetin improves the muscle fiber state of nematodes

[0102] The nematode strain raIs5 [myo-3p::GFP::myo-3+rol-6(su1006)] can be used to study muscle morphology and function in C. elegans. The myo-3 gene promoter drives the expression of GFP-tagged MYO-3 protein, enabling real-time observation of changes in muscle morphology in living C. elegans and understanding degenerative changes in muscle during aging. For example, muscle fiber thinning and breakage, and myofilament disorganization, can be observed.

[0103] Follow the steps below to investigate how dihydromyricetin improves the muscle fiber condition in nematodes:

[0104] Step 1: Prepare the nematode strain raIs5 [myo-3p::GFP::myo-3+rol-6(su1006)] and prepare NGM and FUdR plates;

[0105] Step 2: As in Example 1, a large number of synchronized nematode eggs were prepared and cultured on NGM plates until the L4 stage. The eggs were then transferred to FudR plates and drug administration was initiated (equal volumes of 1 mM dihydromyricetin solution and OP50 E. coli solution were mixed; the OD value of the OP50 E. coli solution was 1). Fluorescence images were taken using a stereo fluorescence microscope on day 3, day 5, and day 10 to observe the condition of the muscle fibers.

[0106] Muscle fibers with normal morphology and function are arranged regularly, and the shape of myofilaments is obvious. Figure 6It can be seen that the muscle fibers in the nematode body wall change from regular arrangement to disordered arrangement and knotting with age, and the intervention of dihydromyricetin can significantly improve the reduction and knotting of muscle fibers in nematodes (fluorescence aggregation) on the 10th day.

[0107] Example 7: Dihydromyricetin improves mitochondrial morphology in nematode muscle

[0108] The nematode strain foxSi16 [myo-3p::tomm-20::mKate2::HA::tbb-2 3'UTR] was used to study the morphology and distribution of muscle mitochondria. The myo-3 promoter drove the expression of TOMM-20 (mitochondrial outer membrane translocase complex subunit 20) fused with mKate2 (a red fluorescent protein), an HA tag, and the tbb-2 3'UTR. The morphology and distribution of muscle mitochondria were observed under a fluorescence microscope. With aging, adverse environmental influences, or changes in physical condition, muscle mitochondrial morphology becomes irregular, their number decreases, and their function gradually declines. These changes are closely related to the weakening of muscle strength and decreased exercise capacity.

[0109] Follow the steps below to investigate how dihydromyricetin improves mitochondrial morphology in C. elegans muscle:

[0110] Step 1: Prepare the nematode strain foxSi16[myo-3p::tomm-20::mKate2::HA::tbb-2 3'UTR]I and prepare NGM and FUdR plates;

[0111] Step 2: As in Example 1, a large number of synchronized nematode eggs were prepared and cultured on NGM plates until the L4 stage. Then, the plates were transferred to FudR plates and drug administration was initiated (equal volumes of 1 mM dihydromyricetin solution and OP50 E. coli solution were mixed, and the OD value of the OP50 E. coli solution was 1). Fluorescence images were taken using a stereo fluorescence microscope on day 3, day 5, and day 10 to observe the muscle mitochondrial morphology.

[0112] Normal mitochondria are microporous tubular structures with remarkable continuity. Mitochondria that have aged or been damaged have fragmented structures and are obviously broken. Figure 7 It can be seen that dihydromyricetin can significantly improve the mitochondrial state of nematodes, especially on the 5th day.

[0113] Example 8: Dihydromyricetin improves nematode motility

[0114] Follow the steps below to investigate how dihydromyricetin improves mitochondrial morphology in C. elegans muscle:

[0115] Step 1: Prepare common nematodes N2, prepare NGM and FUdR plates;

[0116] Step 2: As in Example 1, a large number of synchronized nematode eggs were prepared and cultured on NGM plates until the L4 stage. The eggs were then transferred to FudR plates and drug administration was initiated (1 mM dihydromyricetin solution and OP50 E. coli culture were mixed in equal volumes, with the OD value of the OP50 E. coli culture being 1). Locomotor activity was measured on days 3, 5, and 10. Specifically, the nematodes were transferred to a fresh NGM plate on a bacterial lawn. Timing began when the nematodes contacted the lawn and began to move. Each head swing exceeding 90° during crawling was counted as one swing, and the number of head swings within 30 seconds was recorded. The sample size, n, was ≥ 15.

[0117] Depend on Figure 8 It can be seen that after the intervention of dihydromyricetin, the nematodes can still maintain a good movement state on the 5th and 10th days.

[0118] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents, but these modifications or replacements are all within the scope of protection of the present invention.

Claims

1. Use of dihydromyricetin in preparing a product for improving physiological status, characterized in that: Improving physiological status includes at least one of the following: (1) Improve ALM neuronal integrity; (2) improve PLM neuronal integrity; (3) Improve GABA neuron integrity; (4) Improve touch avoidance response; (5) Improve muscle fiber condition; (6) Improve mitochondrial status; (7) Improve athletic ability.

2. The use of dihydromyricetin as claimed in claim 1 in preparing a product for improving physiological status, characterized in that: The concentration of dihydromyricetin in the product is 0.01mM-5mM; Alternatively, the mass fraction of the dihydromyricetin in the product is 20%-85%.

3. The use of dihydromyricetin in preparing a product for improving physiological status according to claim 2, characterized in that: The concentration of the dihydromyricetin in the product is 1 mM.

4. The use of dihydromyricetin as claimed in claim 1 in preparing a product for improving physiological status, characterized in that: The product includes at least one of food, health care products, medicine, and cosmetics; The food categories include at least one of beverages, candies, and biscuits; the health products and / or drug dosage forms include at least one of tablets, ointments, granules, capsules, powders, and oral liquids; the cosmetic categories include at least one of water, creams, and lotions.

5. Use of dihydromyricetin isomers and / or dihydromyricetin derivatives in the preparation of products for improving physiological status, characterized in that: Improving physiological status includes at least one of the following: (1) Improve ALM neuronal integrity; (2) improve PLM neuronal integrity; (3) Improve GABA neuron integrity; (4) Improve touch avoidance response; (5) Improve muscle fiber condition; (6) Improve mitochondrial status; (7) Improve athletic ability.

6. Use of dihydromyricetin in preparing a product for improving the decline of physiological functions in the elderly by activating atg-7 gene expression to enhance autophagy, and / or activating vha-3 gene expression to enhance lysosomal acidification.

7. Use of dihydromyricetin as claimed in claim 6 in preparing a product for improving aging physiological function decline by activating atg-7 gene expression to enhance autophagy, and / or activating vha-3 gene expression to enhance lysosomal acidification, characterized in that: The concentration of dihydromyricetin in the product is 0.01mM-5mM; Alternatively, the mass fraction of the dihydromyricetin in the product is 20%-85%.

8. Use of dihydromyricetin as claimed in claim 7 in preparing a product for improving aging physiological function decline by activating atg-7 gene expression to enhance autophagy, and / or activating vha-3 gene expression to enhance lysosomal acidification, characterized in that: The concentration of the dihydromyricetin in the product is 1 mM.

9. Use of dihydromyricetin as claimed in claim 6 in preparing a product for improving aging physiological function decline by activating atg-7 gene expression to enhance autophagy, and / or activating vha-3 gene expression to enhance lysosomal acidification, characterized in that: The product includes at least one of food, health care products, medicine, and cosmetics; The food categories include at least one of beverages, candies, and biscuits; the health products and / or drug dosage forms include at least one of tablets, ointments, granules, capsules, powders, and oral liquids; the cosmetic categories include at least one of water, creams, and lotions.

10. Use of dihydromyricetin isomers and / or dihydromyricetin derivatives in the preparation of products for improving the decline of physiological functions in the elderly by activating atg-7 gene expression to enhance autophagy, and / or activating vha-3 gene expression to enhance lysosomal acidification.

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