Use of salabioside in the preparation of products for prolonging life

Abstract

This invention discloses the application of glycyrrhizin in the preparation of products that extend lifespan. In a specific embodiment of this invention, *C. elegans*, a model organism for lifespan and aging, was used as the research subject. It was found that glycyrrhizin can extend the lifespan of *C. elegans*, demonstrating that glycyrrhizin has good anti-aging effects and can be applied to the preparation of products that delay aging or prevent or treat age-related diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of glycyrrhizin in the preparation of products that extend lifespan. Background Technology

[0002] Aging is a complex, progressive, and irreversible process occurring at the molecular, cellular, and overall organismal levels. It is the comprehensive result of the continuous interaction and mutual influence between genetic factors and environmental factors, ultimately leading to a corresponding decline in individual physiological functions, adaptability, and resistance. my country has approximately 280 million people aged 65 and above, making them a high-risk group for cardiovascular diseases, tumors, Alzheimer's disease, cataracts, macular degeneration, and other aging-related conditions. Therefore, developing new drugs to delay aging and elucidating their mechanisms of action provides new evidence and directions for delaying aging and treating age-related diseases.

[0003] Over the past nearly 40 years, research on the mechanisms of aging has made tremendous progress. In particular, research findings in model organisms have greatly broadened our understanding of the molecular mechanisms regulating aging and longevity. After years of meticulous research, modern science believes that the aging process, like other biological processes, is regulated by classical signaling pathways and transcription factors. These signaling pathways regulate the aging process through environmental or physiological signals, such as the insulin / insulin-like growth factor 1 (IGF-1) signaling pathway, dietary restriction signaling pathway, reactive oxygen species (ROS) signaling, reproductive system signaling pathways, epigenetic pathways, and environmental factor-related signaling pathways.

[0004] Heat shock factor 1 (HSF1) and its downstream heat shock protein family protect cells from stress-induced apoptosis and senescence by regulating the orderly folding, transport, and degradation of proteins; this protective stress response is called the heat shock response. In aged tissues and senescent cells, the capacity for the heat shock response is reduced. HSF1 and its downstream heat shock proteins have been used as anti-aging targets in research on the prevention and treatment of Alzheimer's disease and cardiovascular disease. These heat shock proteins can maintain protein homeostasis by remodeling damaged proteins accumulated during aging, thus delaying animal aging. Overexpression of the central regulatory protein HSF-1 in nematodes effectively enhances their resistance to thermal stress, slows senescence, and prolongs their lifespan; overexpression of mitochondrial Hsp22 in Drosophila promotes longevity; mutant mice with a common partner (CHIP) exhibit a faster aging process, while certain HSP genes are upregulated in long-lived mutant mice.

[0005] In summary, HSF1 can serve as a target for anti-aging drugs. While drugs targeting the HSF1 signaling pathway have been reported, they are still in the preclinical research stage. Therefore, developing suitable drugs that regulate the HSF1-mediated heat shock response pathway and elucidating their mechanisms of action is of paramount importance in the prevention and treatment of age-related diseases.

[0006] Caenorhabditis elegans (C. elegans) Caenorhabditis.elegans Nematodes are a classic model organism for studying lifespan and aging, possessing the following advantages: 1) Adults are approximately 1 mm long and fed with E. coli, making them easy to culture in the laboratory; 2) They have a short life cycle, with a reproductive cycle of about 3 days, and a total lifespan of only a few weeks, significantly shortening research time; 3) Nematodes are completely transparent, allowing for accurate imaging and easy observation and detection; 4) They have a clear genetic background and were the first organisms to have their entire genome sequence obtained, sharing 40% homology with mammals, which is beneficial for reverse genetics research; 5) They have a small cell count, exhibiting clear cell differentiation and functionally specific tissue classification, enabling them to perform a variety of biological behaviors; 6) They exhibit stability between lineages, and synchronization treatment of nematodes allows for strict control of experimental conditions, improving comparability. Furthermore, nematodes can recover from starvation in less than a year; they can be permanently preserved at -80 degrees Celsius in liquid nitrogen; new strains can be obtained through genetic modification in about a week; any gene can be knocked out to obtain the desired new gene in about two months; and a large number of non-lethal mutants can be obtained through mutagenesis. Based on the above advantages, this invention selects Caenorhabditis elegans as a model to study drugs that can affect animal lifespan. Summary of the Invention

[0007] The purpose of this invention is to provide a product that can delay aging or prevent or treat aging-related diseases.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In one aspect, the present invention provides a composition for delaying aging or preventing or treating aging-related diseases, the composition comprising glycyrrhizin.

[0010] In the specification and claims of this invention, the words "comprising," "including," and "containing" mean "including but not limited to" and are not intended to exclude other parts, additives, components, or steps.

[0011] As described herein, the compositions provided herein for delaying aging, or for preventing or treating age-related diseases, refer to any anti-aging treatment. Anti-aging treatments include (but are not limited to) treatments that result in the prevention, improvement, or mitigation of the effects of aging; reduction or delay of the increase in biological age; treatment, prevention, improvement, or mitigation of the effects of frailty or age-related diseases and conditions or decline; slowing the progression of such decline, conditions, or diseases; extension of healthy periods or lifespan; restoration of vitality; increased stress or resilience; improved recovery rates or other enhancements after surgery, radiation therapy, illness, and / or any other stress; prevention and / or treatment of menopausal syndrome; restoration of reproductive function; elimination or reduction of the spread of senescent cells; reduction of all or multiple risks of death or all or multiple causes of death associated with at least one or more age-related diseases or conditions, or delaying such risk increases; and reduction of the risk of disease. Treatments that modulate at least one biomarker of aging to a younger state or slow its transition to an "older" state are also considered anti-aging treatments, including but not limited to biomarkers of aging that show visible signs of aging, such as wrinkles, gray hair, etc. In some implementation schemes, age-related diseases or disorders are selected from: atherosclerosis, cardiovascular disease, arthritis, cataracts, osteoporosis, type 2 diabetes, hypertension, neurodegenerative diseases (including but not limited to Alzheimer's disease, Huntington's disease and other age-related dementias; Parkinson's disease; and amyotrophic lateral sclerosis [ALS]), stroke, atrophic gastritis, osteoarthritis, NASH, trunk prodrome, chronic obstructive pulmonary disease, coronary artery disease, dopamine dysregulation syndrome, metabolic syndrome, exertional urinary incontinence, Hashimoto's thyroiditis, heart failure, geriatric depression, immunosenescence (including but not limited to age-related decline in immune response to vaccines and age-related decline in response to immunotherapy), myocardial infarction, acute coronary syndrome, sarcopenia, sarcopenic obesity, geriatric osteoporosis, urinary incontinence, etc. Cancer survivors, patients undergoing chemotherapy and radiation therapy, and other comparable stressors, as well as HIV patients, may experience accelerated aging. Treatments targeting this accelerated aging or its consequences are also considered anti-aging therapies and preventative measures.

[0012] In some embodiments, the composition may also include a pharmaceutically acceptable buffer, excipient, or carrier.

[0013] The term “pharmaceutically acceptable” is defined herein as those compounds, materials, compositions and / or dosage forms that are suitable, to the extent of reasonable medical judgment, for contact with the tissues of a subject without excessive toxicity, irritation, allergic reactions and other problematic complications, and that are commensurate with a reasonable benefit / risk ratio.

[0014] The term "subject" refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, dogs, cats, rodents, etc. Furthermore, a subject is a human subject. The terms "subject," "individual," and "patient" are used interchangeably herein. Therefore, the terms "subject," "individual," and "patient" encompass individuals suffering from age-related diseases. "Subject" also includes the model organism for lifespan and aging—*Caenorhabditis elegans*.

[0015] In some embodiments, the buffer includes Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazole lactate, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO, and TES.

[0016] In some embodiments, the carrier includes any and all solvents, diluents or other liquid media, dispersants or suspending agents, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc., suitable for preparing the desired specific dosage form. Some examples of materials that can be used as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth gum; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol and phosphate buffers; and, at the discretion of the formulation personnel, other non-toxic and compatible lubricants such as sodium dodecyl sulfate and magnesium stearate, as well as colorants, release agents, coating agents, sweeteners, flavorings and aromas, preservatives, and antioxidants.

[0017] In some embodiments, the excipients include carbohydrates, polymers, lipids, or minerals.

[0018] In some implementations, the age-related diseases include atherosclerosis, cardiovascular disease, cachexia, arthritis, cataracts, osteoporosis, type 2 diabetes, hypertension, neurodegeneration, stroke, atrophic gastritis, osteoarthritis, NASH, trunk prodrome, chronic obstructive pulmonary disease, coronary artery disease, dopamine dysregulation syndrome, metabolic syndrome, exertional urinary incontinence, Hashimoto's thyroiditis, heart failure, geriatric depression, immunosenescence, myocardial infarction, acute coronary syndrome, sarcopenia, sarcopenic obesity, geriatric osteoporosis, and urinary incontinence.

[0019] In another aspect, the present invention provides applications as described in any of the following:

[0020] (1) The use of the above-described composition in the preparation of products for delaying aging or preventing or treating aging-related diseases;

[0021] (2) The application of the aforementioned composition in the preparation of HSF1 phosphorylation reagent;

[0022] (3) The use of the above-described composition in the preparation of reagents that enhance the expression levels of HSP70 and HSP27;

[0023] (4) Application of the above-described composition in the preparation of reagents that enhance the expression level of oxidative stress-related genes.

[0024] The terms "level of expression" or "expression level" are generally used interchangeably and generally refer to the amount of polynucleotides, mRNA, or amino acid products or proteins in a biological sample. "Expression" generally refers to the process by which the information encoded by a gene is converted into structures present and functioning in the cell. Therefore, according to this invention, gene "expression" can refer to transcription into polynucleotides, translation into proteins, or even post-translational modifications of proteins. Fragments of transcribed polynucleotides, translated proteins, or post-translational modified proteins should also be considered expressed, whether they originate from transcripts generated by alternative splicing or degraded transcripts, or from post-translational processing of proteins (e.g., through proteolysis). "Expressed genes" include genes transcribed into polynucleotides (such as mRNA) and then translated into proteins, as well as genes transcribed into RNA but not translated into proteins (e.g., miRNA).

[0025] In some embodiments, the HSF1 phosphorylation is the phosphorylation of HSF1 at site S326;

[0026] In some embodiments, the oxidative stress-related genes are oxidative stress-related genes of nematodes, preferably including hsp12.6, hsp16.2, hsp17, hsp25, and hsp70.

[0027] In some implementations, the product is a drug.

[0028] In some embodiments, the dosage form of the drug includes solutions, suspensions, emulsions, extracts, elixirs, powders, granules, tablets, and capsules.

[0029] The route of administration of the drug described in this invention is not limited, as long as it can achieve the desired therapeutic or preventive effect, including (but not limited to): intravenous, intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, intravesical, intramuscular, intratracheal, subcutaneous, through skin, through pleura, local, inhalation, through mucous membranes, skin, gastrointestinal tract, intra-articular, intraventricular, rectal, vaginal, intraskull, intraurethral, ​​intrahepatic, intratumoral. In some cases, it can be administered systemically, and in some cases, it can be administered locally.

[0030] Furthermore, the dosage of the drug described in this invention is not limited, as long as the desired effect of delaying aging or preventing or treating aging-related diseases is achieved. It can be appropriately determined based on symptoms, gender, age, etc. The dosage of the therapeutic or preventive drug of this invention can be determined using, for example, the therapeutic or preventive effect on the disease as an indicator.

[0031] In some implementations, the age-related diseases include atherosclerosis, cardiovascular disease, cachexia, arthritis, cataracts, osteoporosis, type 2 diabetes, hypertension, neurodegeneration, stroke, atrophic gastritis, osteoarthritis, NASH, trunk prodrome, chronic obstructive pulmonary disease, coronary artery disease, dopamine dysregulation syndrome, metabolic syndrome, exertional urinary incontinence, Hashimoto's thyroiditis, heart failure, geriatric depression, immunosenescence, myocardial infarction, acute coronary syndrome, sarcopenia, sarcopenic obesity, geriatric osteoporosis, and urinary incontinence.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] This invention is the first to discover the effect of glycyrrhizin in willow leaf water on lifespan, and it can be used to prepare products that delay aging or prevent or treat aging-related diseases, and has good practical application value. Attached Figure Description

[0034] Figure 1The results of the screening of HSF1 activators are shown in Figure A, which is a schematic diagram of plasmid construction of Hela / HSPA1A- / - / GFP monoclonal cell line; Figure B is a flow cytometry result of Hela / HSPA1A- / - / GFP monoclonal cell line sorting; Figure C is a Western blot result of GFP protein level detection in monoclonal cell lines after heat shock, 17AAG or MG132 treatment; Figure D is a qRT-PCR result of GFP mRNA level detection in monoclonal cell lines after heat shock, 17AAG or MG132 treatment; Figure E is a flow cytometry result of GFP fluorescence intensity detection; Figure F is a flow cytometry result of GFP fluorescence intensity statistics; and Figure G is a Western blot result of HSP70 expression level detection in Hela cells after treatment with glycyrrhizin.

[0035] Figure 2 Figure A shows the experimental results demonstrating that glycyrrhizin activates HSF1 and thus upregulates the expression of HSP27 and HSP70. Figure B shows the results of a nucleocytoplasmic separation experiment demonstrating that glycyrrhizin can promote HSF1 nucleation. Figure B shows the results of Western blotting analysis of the phosphorylation level of HSF1 at S326 and the expression levels of HSP7 and HSP27 proteins in cells treated with glycyrrhizin.

[0036] Figure 3 It is the structural formula of glycyrrhizin;

[0037] Figure 4 Figure 1 shows the experimental results of detecting the effect of low concentrations of glycyrrhizin on the lifespan of nematodes. Figure 2 shows the results of continuous stimulation of nematodes with 0.1 μM / L glycyrrhizin starting from stage L4; Figure 3 shows the results of continuous stimulation of nematodes with 1 μM / L glycyrrhizin starting from stage L4; Figure 4 shows the results of continuous stimulation of nematodes with 0.1 μM / L glycyrrhizin starting from stage 1; Figure 5 shows the results of continuous stimulation of nematodes with 1 μM / L glycyrrhizin starting from stage 1; Figure 6 shows the results of continuous stimulation of nematodes with 0.1 μM / L glycyrrhizin starting from stage 7; Figure 7 shows the results of continuous stimulation of nematodes with 1 μM / L glycyrrhizin starting from stage 7.

[0038] Figure 5 Figure 1 shows the experimental results of detecting the effect of glycyrrhizin on the lifespan of HSF-1 mutant nematode hsf-1(sy441) using HSF-1 as a model. Figure 2 shows the results of treating HSF-1 mutant nematode with 0.1 μM / L glycyrrhizin and Figure 3 shows the results of treating HSF-1 mutant nematode with 1 μM / L glycyrrhizin.

[0039] Figure 6 This is a graph showing the results of real-time quantitative PCR detection of the expression levels of oxidative stress-related genes in wild-type nematodes after treatment with glycyrrhizin in willow leaf water;

[0040] Figure 7 This is a schematic diagram illustrating the mechanism by which glycyrrhizin in willow leaf water prolongs life. Detailed Implementation

[0041] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the embodiments are generally performed under conventional conditions or as recommended by the manufacturer.

[0042] Experimental materials

[0043] Reagents and materials: Sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, calcium chloride, cholesterol, agar powder, tryptone, yeast extract, peptone, carbenicillin, 5-fluorodeoxyuridine (Floxuridine, FUDR) (Sigma), glycyrrhizin (MCE); cDNA reverse transcription kit (Thermo Scientific), qRT-PCR kit (Vazyme), 6cm culture dishes; tert-butyl hydroperoxide (t-BHP, Sigma); DMEM-F12 medium, hydrogen peroxide, β-galactosidase staining kit, β-action, HSF1, P-HSF1 (S326), HSP70, HSP27 antibody (CST);

[0044] Experimental strains and insect strains: uracil-deficient Escherichia coli (E. coli) OP50; wild-type N2 strain; HSF-1 mutant strain hsf-1 (sy441);

[0045] Mammalian cell lines: HeLa cells, ARPE-19 retinal pigment epithelial cells;

[0046] Experimental instruments: dissecting microscope (Jiangnan NSZ608T), biochemical incubator (Wuhan Ruihua), TI-Sβ inverted fluorescence microscope (Nikon, Japan), Q3 quantitative PCR instrument (ABI), flow cytometer, shaker, clean bench, glass plates and electrophoresis tank for SDS-PAGE electrophoresis, etc.

[0047] Example 1: Glycyrrhizin in willow leaf extract is a novel HSF1 activator

[0048] 1) Construction of HeLa / HSPA1A- / - / GFP recombinant monoclonal cell line:

[0049] This invention first utilizes CRISPR / Cas9 gene editing technology to construct the PX333-HSPA1A-gRNA expression plasmid and the HSPA1A- / - / GFP recombinant fragment plasmid; then, positive cells are enriched through transfection, pressurization, and enrichment; finally, single cells expressing GFP fluorescence are sorted using a high-speed flow cytometry cell sorting system; and two positive cell clones with successful insertion of the GFP gene before the HSPA1A promoter in the genome are verified by genome sequencing and qRT-PCR, named cells #3 and #4, respectively. Figure 1 A, B).

[0050] Western blot and qRT-PCR techniques were used to demonstrate that after heat shock treatment (43℃, 1 h), treatment with the Hsp90 specific inhibitor 17AAG (0.2 µM / L), or treatment with the proteasome inhibitor MG132 (5 µM / L), the expression level of GFP protein and the mRNA level of this cell line were significantly increased compared with the control group. Figure 1 (C, D). These results demonstrate that the present invention successfully obtained the monoclonal cell line Hela / HSPA1A- / - / GFP, and the fluorescence intensity of GFP in this cell line can characterize the activity of HSF1 and the expression level of Hsp70.

[0051] 2) A new HSF1 activator, glycyrrhizin, was identified:

[0052] Using the Hela / HSPA1A- / - / GFP cell line as a screening model, and stimulating the cells with approximately 400 available Chinese herbal medicine monomers, the fluorescence intensity of GFP was detected by flow cytometry. It was found that glycyrrhizin from willow leaves significantly enhanced the fluorescence intensity of GFP. Figure 1 E and F (tabs in the figure represent glycyrrhizin), and Western blotting experiments have demonstrated that glycyrrhizin can indeed upregulate the expression of HSP70 in HeLa cells. Figure 1 G).

[0053] Example 2: Glycyrrhizin in willow leaves activates HSF1, thereby upregulating the expression of HSP27 and HSP70.

[0054] 1) Nucleocytoplasmic separation experiment: using HeLa and HeLa hsf-1 _ / _ Using cells as a model, cells were treated with 15 μM glycyrrhizin aqueous solution for 6 h, and then nuclear-cytoplasmic separation experiments were performed to demonstrate that glycyrrhizin aqueous solution could promote HSF1 nucleation. Figure 2 A).

[0055] 2) Using HeLa cells as a model, cells were treated with DMSO and 10 μM glycyrrhizin for 24 h. The results showed that glycyrrhizin activated HSF1 by phosphorylating the S326 site, thereby upregulating the expression of HSP70 and HSP27 to delay aging. Figure 2 B).

[0056] Example 3: Effects of low concentrations of glycyrrhizin in willow leaf water on the lifespan of nematodes

[0057] 1) Preparation of NGM (Nematode Growth Media, NGM) culture medium:

[0058] In the laboratory, nematodes are cultured on non-glucan-medium (NGM) agar, which is aseptically poured into petri dishes. The preparation method is as follows: 1) Add 3g NaCl, 17g agar, and 2.5g peptone to a 2L Erlenmeyer flask, then add 975mL of water, seal with aluminum foil, and autoclave for 30 minutes; 2) After the autoclave pressure gauge drops to zero, remove the flask and cool it in a 55°C water bath for 15 minutes to lower the temperature to approximately 55°C; 3) Add 1mL of 1M MgSO4, 1mL of 1M CaCl2, 1mL of 5mg / mL cholesterol solution, and 25mL of 1M K2PO4 buffer, and shake well; 4) Using an aseptic procedure, dispense the NGM culture medium into petri dishes using a peristaltic pump, filling the dishes to 2 / 3 full; 5) Before use, place the plates at room temperature for 2-3 days to allow for contamination detection and evaporation of excess moisture. Plates stored in a sealed container at room temperature can be used for several weeks. Excess boards can be stored at 4 degrees Celsius.

[0059] 2) Preparation of food for nematodes:

[0060] Under laboratory conditions, nematodes feed on *E. coli* OP50. Using a fermentation agent, single colonies were isolated onto striped plates of a richer medium (such as LB agar) (LB agar: 10 g bacterial tryptone, 5 g bacterial yeast, 5 g NaCl, 15 g agar, diluted to 1 liter with water, pH 7.5). Single colonies were then picked and aseptically inoculated into LB liquid medium (LB liquid medium: 10 g bacterial tryptone, 5 g bacterial yeast, 5 g NaCl, diluted to 1 liter with water, pH adjusted to 7.0 with 1 M NaOH). The medium was incubated overnight at 37°C in a shaker. Fresh OP50 was concentrated three times, inactivated by adding carbenicillin, and then incubated at 4°C for one day before use.

[0061] 3) Synchronization of nematodes:

[0062] Synchronization of nematodes is essential for experimental accuracy. There is a simple method for synchronizing a small number of nematodes: select 20-25 day 1 nematodes and place them on an NGM plate containing bacteria, allow them to lay eggs for about 2 hours, then remove and kill the adult nematodes. This will yield about 200 eggs, which will then develop into synchronized nematodes.

[0063] 4) Preparation of plate preparations containing glycyrrhizin:

[0064] Spread 250 μL of glycyrrhizin aqueous solution (final concentrations of 0.1 μM and 1 μM, respectively) evenly in the center of a plate containing OP50, gently shake to ensure even distribution, and let stand overnight for use the next day.

[0065] 5) Life test experiment:

[0066] Once the nematodes reached the late L4 stage and day 1, they were divided into seven prepared plates containing DMSO (control) and glycyrrhizin, with 16 nematodes per plate. For the first seven days, the nematodes were transferred daily to new plates containing fresh food and medication. Then, on days 10, 13, and 15, they were transferred to new plates containing fresh food and medication. Simultaneously, the survival status of the nematodes was monitored, and experimental data were recorded. Nematodes that did not move or contract when gently touched by a platinum wire were considered dead. Therefore, in this experiment, the late L4 stage nematodes were designated as the beginning of the adult stage, recorded as day 0, and the first day of adult nematodes was recorded as day 1 of the experiment. Nematodes that crawled out of the petri dish, burst, or had larvae hatching inside were considered to have died abnormally. After collecting the lifespan observation data, this invention used GraphPad Prism7 (GraphPad Software, Inc) and IBM SPSS Statistics 22 (IBM, Inc) to perform statistical analysis on the obtained lifespan observation data. The p-value is calculated using Log-rank (Kaplan-Meier).

[0067] The results showed that continuous stimulation with low concentrations of glycyrrhizin from willow leaf water at different stages of nematode development could prolong the lifespan of the nematodes. Figure 4AB: Continuous stimulation of nematodes with glycyrrhizin from stage L4 resulted in a 10.1% and 10.3% increase in nematode lifespan for 0.1 μM / L (A) and 1 μM / L (B), respectively. CD: Continuous stimulation of nematodes with glycyrrhizin from stage 1 resulted in a 16.4% and 16.3% increase in nematode lifespan for 0.1 μM / L (C) and 1 μM / L (D), respectively. EF: Continuous stimulation of nematodes with glycyrrhizin from stage 7 resulted in a 12.5% ​​and 7.9% increase in nematode lifespan for 0.1 μM / L (E) and 1 μM / L (F), respectively. (A) and (B), (C) and (D), and (E) and (F) shared the same WT;DMSO and were performed at the same time, with each experiment repeated twice.

[0068] Example 4: The role of HSF1 factor in the life-extending effect of glycyrrhizin in willow leaf extract.

[0069] When organisms are exposed to protein-inhibiting stressors such as heat, the heat shock response (HSR) is involved in managing protein damage and restoring protein homeostasis. The HSR is controlled by a series of heat shock factors (HSFs), among which HSF1 is crucial for the regulation of protein toxic stress and heat shock proteins (HSPs). The heat shock protein (HSP) family primarily acts as molecular chaperones and is associated with lifespan and aging in many species. These heat shock proteins can maintain protein homeostasis and delay animal aging by remodeling damaged proteins accumulated during aging. Overexpression of the central regulatory protein HSF-1 of the heat shock response in nematodes effectively enhances their resistance to thermal stress, slows aging, and prolongs their lifespan; overexpression of mitochondrial Hsp22 in Drosophila promotes longevity; mutant mice with a common chaperone (CHIP) exhibit a faster aging process, while certain HSP genes are upregulated in long-lived mutant mice.

[0070] This invention uses the HSF-1 mutant nematode hsf-1(sy441) as a model and conducts experiments according to the method in Example 2. The results show that, compared with the control group (DMSO), the glycyrrhizin treatment group with willow leaf water does not prolong the lifespan of the hsf-1(sy441) nematode. Figure 5 AB).

[0071] Example 5: Effects of glycyrrhizin in willow leaf extract on the expression of heat shock protein genes

[0072] In nematodes, genes associated with oxidative stress mainly include hsp12.6 , hsp16.2, hsp17, hsp25 HSP70 The required primers are shown in Table 1.

[0073] Table 1 Primers for genes related to oxidative stress

[0074]

[0075] The experimental steps are as follows:

[0076] 1) First, pick 20-25 day 1 adult nematodes and place them on a sterile plate. After 2 hours of oviposition, pick out the adults and kill them to obtain synchronized nematodes. Then, place 200 day 1 nematodes into NGM medium coated with inactivated E. coli OP50 and add 1 μM glycyrrhizic acid (DMSO as a control). Incubate at 20°C for 12 h.

[0077] 2) Transfer the cultured nematodes to a 1.5 mL EP tube and wash three to five times with M9 buffer;

[0078] 3) Whole-genome RNA was extracted from 200 adult insects using TRI reagent (Life Technologies);

[0079] 4) Using a high-capacity cDNA reverse transcription kit (thermoscientific), reverse transcription PCR was performed using the extracted whole-genome RNA as a template;

[0080] 5) Using cDNA obtained from reverse transcription PCR as a template, real-time quantitative PCR analysis was performed on an ABI real-time quantitative PCR detection system using CYBR Green reagent (Vazyme);

[0081] 6) In the processing of experimental data, this invention selects... act-1 (actin) was used as an internal reference, and the results of real-time quantitative PCR were processed and analyzed using the ΔΔCt analysis method. All real-time quantitative PCR experiments were repeated three times. The experimental results were statistically analyzed using the ANOVA method.

[0082] The results showed that glycyrrhizin in willow leaf water could induce the growth of wild-type nematodes N2. hsp12.6 , hsp16.2, hsp17, hsp25 HSP70 The expression levels of oxidative stress-related genes were significantly upregulated, at 1.4, 3, 2, 1.2, and 2.5 times that of the control group, respectively, which was statistically significant. (See [link to relevant documentation]). Figure 6 .

[0083] The preferred embodiments of this application have been described in detail above with reference to the accompanying drawings. However, this application is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this application, various simple modifications can be made to the technical solution of this application, and these simple modifications all fall within the protection scope of this application.

[0084] It should also be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this application will not describe the various possible combinations separately.

[0085] Furthermore, various different implementations of this application can be combined in any way, as long as they do not violate the spirit of this application, they should also be regarded as the content disclosed in this application. sequence list <110> Henan University <120> Application of glycyrrhizin in the preparation of products with extended shelf life <141> 2022-03-09 <160> 12 <170> SIPOSequenceListing 1.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <400> 1 tccttaccga gcgtggttac 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 gtttccgacg gtgatgactt 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 gtgatggctg acgaaggaac 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 gggaggaagt tatgggcttc 20 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <400> 5 tccatctgag tcttctgaga ttgtta 26 <210> 6 <211> twenty three <212> DNA <213> Artificial Sequence <400> 6 tggtttaaac tgtgagacgt tga 23 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <400> 7 taaccatggc cgcagatt 18 <210> 8 <211> twenty three <212> DNA <213> Artificial Sequence <400> 8 ttcacaacat caatagcatc tcc 23 <210> 9 <211> twenty one <212> DNA <213> Artificial Sequence <400> 9 acaaccgtct tcttgtccac g 21 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <400> 10 cacgccatca gtcgaaagag 20 <210> 11 <211> twenty one <212> DNA <213> Artificial Sequence <400> 11 gtctacatgc aaagcgattg g 21 <210> 12 <211> twenty one <212> DNA <213> Artificial Sequence <400> 12 tgcatcacca acgagtctct c 21

Claims

1. Application of glycyrrhizin as the sole active ingredient in the preparation of anti-aging drugs; The glycyrrhizin in the willow leaf extract exerts its anti-aging effect by phosphorylating the S326 site of HSF1 and increasing the expression level of oxidative stress-related genes. The oxidative stress-related genes include HSP27, hsp12.6, hsp16.2, hsp17, hsp25, and hsp70; Anti-aging refers to reducing or delaying the increase of biological age.

2. The application as described in claim 1, wherein the medicament further comprises a pharmaceutically acceptable buffer solution, excipient, or carrier.

3. The application as described in claim 2, wherein the dosage form of the drug includes solution, suspension, emulsion, extract, elixir, powder, granule, tablet, and capsule.

Images