Application of Ectoin in preparation of medicine for prolonging service life

Abstract

The invention discloses application of Ectoin in preparation of a medicine for prolonging the service life, and belongs to the technical field of biology. Life experiments prove that the life of caenorhabditis elegans can be obviously prolonged by 25.8% by 5mM of Ectoine; 10 mM of Ectoine can obviously prolong the service life of caenorhabditis elegans, and the service life is prolonged by 43.4%; 15mM Ectoine can obviously prolong the service life of caenorhabditis elegans, and the service life is prolonged by 21.1%. The invention proves that Ectoin has an excellent effect of prolonging the service life, so that related products can be effectively developed on the basis of the medicine, and the Ectoin has wide application prospects and popularization values.

Description

Technical Field

[0001] This invention specifically relates to the application of ectoine in the preparation of life-extending drugs, and belongs to the field of biotechnology. Background Technology

[0002] As society's demand for extended healthy lifespan increases, the research and development of life-extending drugs becomes particularly important. Extending lifespan not only concerns individual health and well-being but also has profound implications for socioeconomic development and the allocation of medical resources. With the increasing aging of the global population, extending healthy lifespan can reduce the burden of age-related diseases, improve the quality of life for older adults, and potentially alleviate the pressure on social healthcare systems.

[0003] Existing technologies suggest a potential link between ectoine and anti-aging: for example, Chinese invention patent application CN120837410A mentions that a combination of ectoine with safflower seed oil, extracts of golden button flower / leaf / stem, and extract of Astragalus membranaceus root has anti-aging effects. Although existing technologies indicate a possible link between ectoine and anti-aging, it should be clear that anti-aging is not equivalent to extending lifespan. Aging is a slow, systemic, multifaceted, highly complex, and gradual degenerative process that occurs in the later stages of an organism's life. This degenerative process leads to a gradual decline in the body's adaptive and reserve capabilities, specifically manifested as a decrease or loss of function in various systems, organs, and tissues. Anti-aging refers to a proactive health care system based on an understanding of the mechanisms of aging, focusing on delaying the aging process of cells and tissues, and employing various programs, health products, and drugs to maintain youthful physical function and appearance, reduce the risk of age-related chronic diseases, and slow down the aging process. Lifespan refers to the time from birth to death. The measure of lifespan is age, and the length of lifespan depends on both innate genetic factors and acquired environmental factors.

[0004] Therefore, the development of lifespan-extending drugs differs from anti-aging drugs. Lifespan-extending drugs focus more directly on increasing an organism's maximum lifespan, that is, extending the total time from birth to death. This typically involves gene regulation, cell signaling pathways, and adjustments to environmental factors. In short, anti-aging primarily focuses on slowing the aging process and maintaining a healthy state, while lifespan extension emphasizes actually increasing the length of an individual's lifespan. Current technology does not mention the effects of ectoine on lifespan extension, nor is there evidence to prove that ectoine has lifespan-extending efficacy.

[0005] In conclusion, the development of life-extending drugs is of great significance for addressing the challenges of an aging population, improving quality of life, and preventing diseases. The development of these drugs will not only help meet people's pursuit of healthy longevity but also provide new ideas and methods for medical research and clinical practice. Summary of the Invention

[0006] Ectoine is a compound produced by halophilic microorganisms (such as halophytes). Halomonas elongata Small molecule natural compounds synthesized in extreme environments such as high salinity, high temperature, or strong radiation, with the chemical formula C6H. 10 N2O2. To address the shortcomings of existing technologies, the purpose of this invention is to provide the application of ectoine in extending lifespan.

[0007] The first objective of this invention is to provide the use of ectoine in the preparation of products that extend lifespan.

[0008] In one embodiment of the invention, the extended lifespan refers to increasing the number of days that *Caenorhabditis elegans* survives.

[0009] In one embodiment of the invention, the drug is ectoine as the sole active ingredient or as one of the active ingredients.

[0010] In one embodiment of the present invention, the dose of ectoine in the drug is not less than 5 mM.

[0011] In one embodiment of the present invention, the product includes, but is not limited to, drugs, feed, feed additives, cosmetics, and health products.

[0012] In one embodiment of the invention, the drug contains ectoine and pharmaceutically acceptable excipients.

[0013] In one embodiment of the present invention, the excipients include any one or a combination of at least two of the following: carrier, diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

[0014] In one embodiment of the present invention, the carrier includes, but is not limited to, liposomes and nanoparticles.

[0015] In one embodiment of the present invention, the excipient is one or more of mannitol, lactose, fatty acids, and polyethylene glycol.

[0016] In one embodiment of the present invention, the dosage form of the drug includes any one of suspension, granules, capsules, powders, tablets, emulsions, solutions, pellets, injections, suppositories, enemas, aerosols, patches, or drops.

[0017] In one embodiment of the present invention, the route of administration of the drug includes oral administration, sublingual administration, skin and mucous membrane administration, inhalation administration, or injection administration.

[0018] In one embodiment of the present invention, the dose of ectoine in the drug is not less than 5 mM.

[0019] In one embodiment of the present invention, the product has at least the function of extending lifespan.

[0020] A second object of the present invention is to provide a medicine having a life-extending function, the medicine containing ectoderma as the sole active ingredient or as one of the active ingredients, and pharmaceutically acceptable excipients.

[0021] In one embodiment of the present invention, the excipients include any one or a combination of at least two of the following: carrier, diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

[0022] In one embodiment of the present invention, the carrier includes, but is not limited to, liposomes and nanoparticles.

[0023] In one embodiment of the present invention, the excipient is one or more of mannitol, lactose, fatty acids, and polyethylene glycol.

[0024] In one embodiment of the present invention, the dosage form of the drug includes any one of suspension, granules, capsules, powders, tablets, emulsions, solutions, pellets, injections, suppositories, enemas, aerosols, patches, or drops.

[0025] In one embodiment of the present invention, the route of administration of the drug includes oral administration, sublingual administration, skin and mucous membrane administration, inhalation administration, or injection administration.

[0026] In one embodiment of the present invention, the dose of ectoine in the drug is not less than 5 mM.

[0027] In one embodiment of the present invention, the drug has at least the function of prolonging life.

[0028] Beneficial effects: This invention confirms that ectoine has a life-extending effect, specifically manifested in: (1) Lifespan experiments confirmed that 5 mM ectoin can significantly prolong the lifespan of Caenorhabditis elegans by 25.8%; (2) 10 mM ectoine can significantly prolong the lifespan of Caenorhabditis elegans by 43.4%; (3) 15 mM ectoine significantly prolonged the lifespan of Caenorhabditis elegans by 21.1%; Therefore, ectoine has broad application prospects and promotional value in the preparation of life-extending drugs. Attached Figure Description

[0029] Figure 1 This is a diagram showing the effect of ectoin on the lifespan of Caenorhabditis elegans. Detailed Implementation

[0030] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0031] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0032] The reagents and materials involved in the following examples are as follows: Ectoin (item number: 011517696) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; cholesterol (item number: 69008216), anhydrous ethanol (item number: 40021279), sodium hypochlorite (item number: 40021279), sodium chloride (item number: 10019360), agar powder (item number: 10000582), tryptone (item number: 69024138), anhydrous disodium hydrogen phosphate (item number: XW755879402), anhydrous potassium dihydrogen phosphate (item number: 100176193), anhydrous dipotassium hydrogen phosphate (item number: 1042920), anhydrous magnesium sulfate (item number: T40864), and anhydrous calcium chloride (item number: 53219961) were purchased from Sinopharm Chemical Reagent Co., Ltd. N2 wild-type Caenorhabditis elegans was provided by the CGC Center for Caenorhabditis elegans (University of Minnesota, MN, USA).

[0033] The solutions involved in the following examples are as follows: Stock solution preparation: 1 M phosphate buffer (35.6 g K2HPO4, 108.3 g KH2PO4, pH 6.0, adjusted to 1 L); 1 M CaCl2 solution; 1 M MgSO4 solution; 5 mg / mL cholesterol solution (prepared with anhydrous ethanol).

[0034] M9 buffer: 3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl and 1 mL 1 M MgSO4 solution, 1 L deionized water, mix well, autoclave at 121 ℃ for 20 min.

[0035] Lysis buffer: Prepared with 5 mL NaClO (5%), 1.25 mL NaOH (10 mol / L), and 18.75 mL sterile water. Prepare fresh before use.

[0036] The culture media involved in the following examples are as follows: LB liquid medium: 10 g tryptone, 5 g yeast extract, 10 g NaCl, add deionized water to a final volume of 1 L, adjust pH to 7.0, and autoclave at 121°C for 20 min.

[0037] NGM medium preparation (1 L): 3 g NaCl, 2.5 g peptone, 20 g agar, 25 mL 1 M phosphate buffer, 1 mL 1 M CaCl2 solution, 1 mL 1 M MgSO4 solution, 973 mL H2O. Autoclave at 121 °C for 20 min. Cool to room temperature, add 1 mL 5 mg / mL cholesterol solution, mix well, and pour into petri dishes.

[0038] The experimental methods involved in the following embodiments are as follows: 1. Preparation of bacterial suspension: (1) Activation of strain: Escherichia coli ( Escherichia coli OP50 glycerol preservation tubes were inoculated into LB liquid medium and cultured in a 37 ℃ shaker incubator (180 rpm) for 18 h.

[0039] (2) Subculture of strains: Escherichia coli ( Escherichia coli The OP50 activated culture medium was inoculated into LB liquid medium in the same manner as above and cultured in a 37 ℃ shaker incubator (180 rpm).

[0040] (3) Calculation of viable bacteria count: The OP50 bacterial suspension was diluted to 10⁻⁶ using the plate count method. 5 10 6 and 10 7 The diluted OP50 bacterial solution was spread onto the surface of LB solid medium and incubated for 48 h before being counted.

[0041] (4) Preparation of bacterial suspension: 1×10⁻⁶ live bacteria were respectively prepared. 8The OP50 bacterial suspension of CFU / mL was centrifuged at 5000 rpm for 5 min to remove the culture medium. After washing with sterile water, it was centrifuged again (5000 rpm for 5 min) to obtain bacterial precipitate. The precipitate was then resuspended in sterile water to prepare OP50 bacterial suspensions containing 5 mM, 10 mM and 15 mM ectoine.

[0042] 2. Preparation of nematodes: (1) Nematode culture This invention uses N2 wild-type Caenorhabditis elegans containing 1×10 8 OP50 bacterial suspensions at CFU / mL were cultured in NGM culture dishes, and all were maintained and tested in a 20 ℃ constant temperature incubator using standard methods.

[0043] (2) Nematode propagation Under laboratory conditions, nematodes reproduce rapidly. To prevent the nematodes from entering a state of starvation and affecting the experiment, subculturing is necessary. Use a blade to cut a piece of old culture medium containing nematodes of appropriate size and transfer it to a new NGM culture dish.

[0044] (3) Nematode synchronization When there are enough eggs and reproductive nematodes on the NGM culture dish, wash them with M9 buffer into a 15 mL centrifuge tube. Repeat the washing twice, discard the supernatant and transfer it to a 1.5 mL centrifuge tube. Add 1 mL of lysis buffer, rotate the mixture for 10 min, centrifuge at 5000×g for 1 min, wash three more times with M9 buffer, add M9 buffer and incubate overnight at 20 ℃. Once the eggs hatch, L1 stage larvae with consistent development stages can be obtained.

[0045] Example 1: Application of ectoine in the preparation of life-extending drugs 1. To investigate the effects of 5 mM, 10 mM and 15 mM ectoine on the lifespan of Caenorhabditis elegans.

[0046] The specific experiment is as follows: OP50 bacterial suspensions containing 5 mM, 10 mM, and 15 mM ectoine, OP50 bacterial suspensions without ectoine, and L1 stage larvae were taken respectively to detect the effect of ectoine on nematodes. The steps are as follows: OP50 bacterial suspensions containing 5 mM, 10 mM, and 15 mM ectoine (bacterial concentration 1×10⁻⁶) were respectively prepared. 8 CFU / mL), OP50 bacterial suspension without ectoine (bacterial concentration of 1×10⁻⁶ CFU / mL), 8The CFU / mL nematodes were spread in 60 mm NGM culture dishes. Equal amounts of synchronized L1-stage nematodes were transferred to the above-mentioned OP50 bacterial suspensions containing 5 mM, 10 mM, and 15 mM ectoine or OP50 bacterial suspensions without ectoine, which were designated as the control group (without ectoine), the 5 mM group, the 10 mM group, and the 15 mM group, respectively. The dishes were placed in a constant temperature incubator at 20 ℃ and cultured until the nematodes died.

[0047] 2. Experimental Results The lifespan of nematodes is counted in days: (1) Analysis of nematode lifespan Unless otherwise specified, all experiments used L4 stage (day 3) as t=0. 150 nematodes were tested in 5 culture dishes per group (30 nematodes / dish). Nematode survival was checked and recorded daily. Every 2 days, the nematodes were transferred to fresh culture dishes until all nematodes died. Nematodes were considered dead when pharyngeal aspiration ceased or when they did not respond to gentle mechanical stimulation of the platinum wire of the nematode pick. Nematodes that were accidentally lost, died from internal hatching, suffered organ damage from compression, or were injured during transfer were not included in the lifespan statistics.

[0048] Calculate the mean lifetime (MLS, days) using the following formula:

[0049] Where, d j For the time period (x) j x j+1 The number of dead nematodes, where N is the total number of dead nematodes. The standard error of mean lifespan (SEM) is estimated using the following formula:

[0050] Lifespan survival curve as follows Figure 1 As shown.

[0051] The results showed that compared with the control group (mean lifespan 17.16±0.68 days, longest lifespan 28 days), intervention with 5 mM ectoine significantly prolonged the lifespan of nematodes (P<0.0001), with a mean lifespan of 21.60±1.06 days and a longest lifespan of 36 days, representing a 25.8% increase in mean lifespan compared with the control group. Intervention with 10 mM ectoine significantly prolonged the lifespan of nematodes (P<0.0001), with a mean lifespan of 24.62±1.12 days and a longest lifespan of 38 days, representing a 43.4% increase in mean lifespan compared with the control group. Intervention with 15 mM ectoine significantly prolonged the lifespan of nematodes (P<0.0001), with a mean lifespan of 20.79±1.06 days and a longest lifespan of 36 days, representing a 21.1% increase in mean lifespan compared with the control group.

[0052] In summary, ectoine in this invention has the effect of prolonging life and can be used in the preparation of life-extending drugs. Those skilled in the art can refer to the content of this article and appropriately modify the process parameters, all of which are within the protection scope of this invention.

[0053] It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The products of this invention have been described through preferred embodiments, and those skilled in the art will readily be able to modify or appropriately alter and combine the products described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

Claims

1. Application of ectoine in the preparation of products with extended lifespan.

2. The application according to claim 1, characterized in that, The product uses Ectodextrin as its sole active ingredient or as one of its active ingredients.

3. The application according to claim 1, characterized in that, The products include, but are not limited to, drugs, feed, feed additives, cosmetics, and health products.

4. The application according to claim 3, characterized in that, The drug is made with ectoderma as the sole active ingredient or as one of the active ingredients, and pharmaceutically acceptable excipients. Preferably, the dosage form of the drug includes any one of suspension, granules, capsules, powders, tablets, emulsions, solutions, pellets, injections, suppositories, enemas, aerosols, patches, or drops; Preferably, the route of administration of the drug includes oral administration, sublingual administration, skin and mucous membrane administration, inhalation administration, or injection administration.

5. The application according to claim 3 or 4, characterized in that, The dosage of ectoine in the product is not less than 5 mM.

6. A drug with life-extending function, characterized in that, The drug contains ectoderma as its sole active ingredient or as one of its active ingredients, as well as pharmaceutically acceptable excipients.

7. The drug according to claim 6, characterized in that, The excipients include any one or a combination of at least two of the following: carrier, diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

8. The medicament according to claim 7, characterized in that, The carriers include, but are not limited to, liposomes and nanoparticles; The excipient is one or more of mannitol, lactose, fatty acids, and polyethylene glycol; The dosage form of the drug includes any one of the following: suspension, granules, capsules, powders, tablets, emulsions, solutions, pellets, injections, suppositories, enemas, aerosols, patches, or drops.

9. The drug according to claim 6, characterized in that, The routes of administration of the drug include oral administration, sublingual administration, skin and mucous membrane administration, inhalation administration, or injection administration.

10. The medicament according to claim 6, characterized in that, The dosage of ectoine in the drug is not less than 5 mM.

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