Application of rare ginsenoside composition in preparation of products for delaying senescence

Abstract

The invention provides application of a rare ginsenoside composition in preparation of a product for delaying senescence, and belongs to the technical field of biological medicines. The invention provides a rare ginsenoside composition, the rare ginsenoside composition is prepared from Rk3, Rh4, Dehydroprotopanaxatrio I and Dehydroprotopanaxatriol II, and the mass ratio of the Rk3 to the Rh4 to the Dehydroprotopanaxatrio I to the Dehydroprotopanaxatriol II is (1 to 3) to (2 to 4) to 1 to (1 to 3). The ginsenoside composition can effectively delay senescence and has a wide application prospect in preparation of cosmetics or medicines for delaying senescence.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of rare ginsenoside compositions in the preparation of anti-aging products. Background Technology

[0002] Aging is a normal and irreversible physiological phenomenon. Along with aging, various functions decline, and the body's ability to resist environmental damage and restore homeostasis weakens, leading to increased disease incidence and mortality. Modern medicine shows that as people age, their metabolic rate slows down, and the body produces excessive reactive oxygen species (ROS). When ROS levels are excessive, they induce oxidative stress, causing significant damage to cell structure and biomolecular function, which in turn directly or indirectly leads to various diseases.

[0003] Currently, anti-aging products on the market are mainly divided into two categories: one is chemically synthesized anti-aging agents (such as vitamin E and coenzyme Q10), which can alleviate oxidative stress in the short term, but long-term use may have side effects and cannot specifically improve the multi-dimensional aging mechanisms; the other is natural extract products (such as grape seed extract and Ganoderma lucidum polysaccharides), which are safer, but generally suffer from unclear active ingredients and single targets, making it difficult to fundamentally delay the aging process. Therefore, developing anti-aging compositions based on highly active natural ingredients that can cover multiple aging targets has become an urgent need in the current technological field.

[0004] Existing research and products mostly focus on single rare ginsenosides (such as Rg3 and Rh2). For example, Chinese patent CN104644651A discloses the application of 20(R)-ginsenoside Rg3 in the preparation of anti-aging drugs. Experiments have shown that 20(R)-ginsenoside Rg3 has a rapid onset of anti-aging effects, few toxic side effects, and is a safe, efficient, stable, and simple-to-prepare anti-aging drug suitable for industrial production and easy to promote. However, it can only target one aspect of the aging mechanism, and its anti-aging effect is limited.

[0005] No studies have yet explored the combination of Rk3, Rh4 with Dehydroprotopanaxatrio I and Dehydroprotopanaxatriol II saponins. Therefore, developing a rare ginsenoside composition consisting of Rk3, Rh4, Dehydroprotopanaxatrio I, and Dehydroprotopanaxatriol II in a specific ratio has significant technological and application value: firstly, this combination can simultaneously target multiple core aging targets through the synergistic effect of the four components. The development of this composition not only fills a technological gap but also meets the market's urgent demand for safe, efficient, and multi-target natural anti-aging products, demonstrating broad application prospects. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides the application of rare ginsenoside compositions in the preparation of anti-aging products.

[0007] On one hand, the present invention provides a rare ginsenoside composition, wherein the rare ginsenoside composition is composed of Rk3, Rh4, Dehydroprotopanaxatrio Ⅰ and Dehydroprotopanaxatriol Ⅱ, and the mass ratio of Rk3, Rh4, Dehydroprotopanaxatrio Ⅰ and Dehydroprotopanaxatriol Ⅱ is (1-3):(2-4):1:(1-3).

[0008] According to some embodiments of the present invention, Rk3, Rh4, Dehydroprotopanaxatrio Ⅰ, and Dehydroprotopanaxatriol are mentioned. The mass ratio of II can be 1:2:1:1, 1:3:1:1, 1:4:1:1, 2:2:1:1, 2:3:1:1, 2:4:1:1, 3:2:1:1, 3:3:1:1, 3:4:1:1, 1:2:1:2, 1:3:1:2, 1:4:1:2, 2:2:1:2, 2:3:1:2, 2:4:1:2, 3:2:1:2, 3:3:1:2, 3:4:1:2, 1:2:1:3, 1:3:1:3, 1:4:1:3, 2:2:1:3, 2:3:1:3, 2:4:1:3, 3:2:1:3, 3:3:1:3, 3:4:1:3, and other point values ​​can be selected.

[0009] Furthermore, the mass ratios of Rk3, Rh4, Dehydroprotopanaxatrio Ⅰ, and Dehydroprotopanaxatriol Ⅱ are 1:2:1:1, 2:3:1:2, and 3:4:1:3.

[0010] Furthermore, the mass ratio of Rk3, Rh4, Dehydroprotopanaxatrio Ⅰ, and Dehydroprotopanaxatriol Ⅱ is 2:3:1:2.

[0011] On the other hand, the present invention provides a method for preparing the above-mentioned rare ginsenoside composition, comprising the following steps: (1) Dissolve the ginsenosides in a water-methanol system, add aspartic acid, cook, and remove the cooked hydrolysate. (2) After removing methanol from the hydrolysis product, the resulting suspension is first extracted with ethyl acetate 2-4 times. The ethyl acetate layers are combined, and the solvent in the ethyl acetate layer is removed to obtain the solid. (3) The solid was dissolved in methanol and separated by medium-pressure semi-preparative chromatography. C18 was selected as the stationary phase, the flow rate was 24-26 ml / min, the detection wavelength was 203 nm, the running time was 80-100 min, and the mobile phase conditions were acetonitrile (68%-100%) and water (32%-0%). Ginsenoside Rk3, ginsenoside Rh4, ginsenoside Dehydroprotopanaxatriol Ⅰ and ginsenoside Dehydroprotopanaxatriol Ⅱ were obtained. They were mixed according to the mass ratio to obtain the rare ginsenoside composition.

[0012] According to some embodiments of the present invention, in step (1), ginsenosides are dissolved in an 8-12 times water-methanol system; Furthermore, in step (1), the ginsenosides are dissolved in a 10-fold water-methanol system.

[0013] Specifically, in step (1), the volume ratio of water to methanol in the water-methanol system is 1:1.

[0014] According to some embodiments of the present invention, the amount of aspartic acid added in step (1) is 1 / 8 to 1 / 12 of the mass of the saponin; Furthermore, in step (1), the amount of aspartic acid added is 1 / 10 of the mass of the saponin.

[0015] According to some embodiments of the present invention, the cooking conditions in step (1) are cooking at 100°C under high pressure for 3-5 hours; Furthermore, the cooking conditions in step (1) are: cooking at 100°C under high pressure for 4 hours.

[0016] According to some embodiments of the present invention, in step (2), ethyl acetate of the same volume as the suspension is used for extraction.

[0017] According to some embodiments of the present invention, the extraction is performed three times in step (2).

[0018] According to some embodiments of the present invention, in step (3), methanol is used to dissolve the solution to 0.5 g / ml.

[0019] According to some embodiments of the present invention, the flow rate in step (3) is 25 ml / min.

[0020] According to some embodiments of the present invention, the running time in step (3) is 90 minutes.

[0021] Furthermore, this invention provides the application of the above-mentioned rare ginsenoside composition in the preparation of anti-aging products.

[0022] Specifically, the products include pharmaceuticals and cosmetics.

[0023] Specifically, the product contains a rare ginsenoside composition as its main active ingredient.

[0024] Specifically, the product has at least one of the following functions: (1) Reduce the level of lipofuscin in the body; (2) Reduce the level of ROS in the body.

[0025] Specifically, when the product is a drug, the drug also includes a pharmaceutically acceptable carrier.

[0026] Furthermore, the pharmaceutically acceptable carrier is selected from one or more of the following: excipients, buffers, emulsifiers, stabilizers, diluents, binders, preservatives, lubricants, pH adjusters, cryoprotectants, flavoring agents, and fillers. Specifically, the excipient is selected from at least one of microcrystalline cellulose, lactose, pregelatinized starch, cyclodextrin, carboxymethyl cellulose, mannitol, magnesium stearate, starch, calcium phosphate, ethyl cellulose, methyl cellulose, alginate, gelatin, gum arabic, glyceryl monostearate, sodium glycolate starch, guar gum, glycerol, and propylene glycol.

[0027] Specifically, the buffer is selected from at least one of sodium dihydrogen phosphate, sodium bicarbonate, ammonium bicarbonate, sodium acetate, citrate, histidine, and succinate.

[0028] Specifically, the emulsifier is selected from at least one of magnesium stearate, zinc stearate, calcium stearate, glyceryl stearate, sorbitan isostearate, sorbitan oleate, glyceryl oleate, and polyglycerol-3 polyricinoleate.

[0029] Specifically, the stabilizer is selected from at least one of acacia gum, agar, alginate, cellulose ether, and carboxymethyl chitosan.

[0030] Specifically, the diluent is selected from at least one of erythritol, mannitol, sorbitol, xylitol, lactose, sucrose, corn starch, potato starch, calcium phosphate, calcium citrate, and crystalline cellulose.

[0031] Specifically, the adhesive is selected from at least one of ethanol, starch paste, pregelatinized starch, dextrin, syrup, hydroxypropyl methylcellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, sodium alginate, polyvinylpyrrolidone, gum arabic, gelatin, and alginic acid.

[0032] Specifically, the preservative is selected from at least one of methylparaben, propylparaben, methylparaben, ethylparaben, propylparaben, chlorobutanol, thimerosal, mercuric oxycyanide, phenoxyethanol, chlorhexidine, benzoic acid, sodium benzoate, chlorocresol, benzalkonium bromide, benzalkonium chloride, and ethylparaben.

[0033] Specifically, the lubricant is selected from at least one of magnesium stearate, zinc stearate, glyceryl monostearate, polyethylene glycol, stearic acid, talc, sodium chloride, sodium oleate, sodium lauryl sulfate, magnesium lauryl sulfate, sodium stearate fumarate, and poloxamer.

[0034] Specifically, the pH adjuster is selected from at least one of citric acid, fumaric acid, succinic acid, tartaric acid, malic acid, and ascorbic acid.

[0035] Specifically, the cryoprotectant is selected from at least one of sucrose, glucose, mannitol, fructose, trehalose, dextrose, lactose, glycerol, methanol, ethanol, ethylene glycol, propylene glycol, dimethyl sulfoxide (DMSO), acetamide, or formamide.

[0036] Specifically, the flavoring agent is selected from at least one of sweet orange flavoring, vanilla flavoring, strawberry flavoring, milk flavoring, banana flavoring, and cherry flavoring.

[0037] Specifically, the filler is selected from at least one of mannitol, xylitol, sorbitol, maltose, microcrystalline cellulose, glucose, lactose, sucrose, dextrin, starch, sodium alginate, and sodium bicarbonate.

[0038] Furthermore, the dosage form of the drug may be drops, tinctures, powders, tablets, capsules, granules, ointments, powders, emulsions, pills, lyophilized powder injections, gels, suppositories, or aerosols.

[0039] Specifically, when the product is a cosmetic, the cosmetic can be a conventional preparation such as lotion, toner, lotion, essence water, face cream, face mask, cleansing cream, facial cleanser, shampoo and conditioner, shaving cream, hair lotion, hair gel, lipstick, sunscreen, hand cream, etc.

[0040] Furthermore, the cosmetics may also include base materials and conventional excipients.

[0041] Furthermore, the matrix raw materials include, but are not limited to, gelatinous raw materials, powdery raw materials, oily raw materials, waxy raw materials, coagulants, and surfactants.

[0042] Furthermore, the aforementioned conventional excipients include, but are not limited to, antioxidants, adhesives, lubricants, humectants, preservatives, whitening agents, film-forming agents, emulsifiers, and cosmetic nutritional additives.

[0043] The beneficial effects of this invention are as follows: The rare ginsenoside composition of the present invention can significantly improve the lifespan, survival rate, body length, and head twitching frequency of naturally aging nematodes, and reduce lipofuscin and ROS levels. Attached Figure Description

[0044] Figure 1 Survival curves of nematodes treated with rare ginsenoside composition drugs.

[0045] Figure 2 The effect of rare ginsenoside compositions on the heat stress resistance of nematodes, p <0.01, *** p <0.001.

[0046] Figure 3 The effects of rare ginsenoside compositions on the motility of nematodes, *** p <0.001.

[0047] Figure 4 The effect of rare ginsenoside compositions on nematode body length, *** p <0.001.

[0048] Figures 5-6 The effect of rare ginsenoside compositions on lipofuscin levels in nematodes, *** p <0.001.

[0049] Figures 7-8 To investigate the effect of rare ginsenoside compositions on ROS levels in nematodes, ***p <0.001. Detailed Implementation

[0050] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0051] Example 1: Intervention effect of rare ginsenoside composition on naturally aging Caenorhabditis elegans The mass ratio of the rare ginsenoside composition is: The mass ratio of Rk3, Rh4, Dehydroprotopanaxatrio Ⅰ and Dehydroprotopanaxatriol Ⅱ is 2:3:1:2.

[0052] The preparation method of the rare ginsenoside composition is as follows: (1) Dissolve the ginsenosides in a 10-fold water-methanol (1:1) system, add 1 / 10 of the ginsenoside mass of aspartic acid, cook under high pressure at 100°C for 4 hours, and take out the cooked hydrolysate.

[0053] (2) After removing methanol from the product, the resulting suspension is first extracted with an equal volume of ethyl acetate three times. Each time, the ethyl acetate layers are combined, and then the solvent of the ethyl acetate layer is removed to obtain a solid.

[0054] (3) The hydrolysis product solid was dissolved in an appropriate amount of methanol to 0.5 g / ml and then separated by medium-pressure semi-preparative chromatography. C18 was selected as the stationary phase, the flow rate was 25 ml / min, the detection wavelength was 203 nm, the running time was 90 min, and the mobile phase conditions were acetonitrile (68%-100%) and water (32%-0%). More than 90% of ginsenosides Rk3, ginsenoside Rh4, ginsenoside Dehydroprotopanaxatriol Ⅰ and ginsenoside Dehydroprotopanaxatriol Ⅱ were obtained. The rare ginsenoside composition was obtained by mixing them according to the mass ratio.

[0055] 1. Experimental Materials 1.1 Materials Wild-type Caenorhabditis elegans (N2) was preserved in the laboratory.

[0056] Uracil-deficient Escherichia coli OP50 was preserved in the laboratory.

[0057] 1.2 Instruments Table 1 Main Experimental Instruments

[0058] 1.3 Reagents Table 2 Drugs and Reagents

[0059] 1.4 Software Software such as Upoview, Image J, GraphPad Prism 8.0, etc.

[0060] 2 Experimental Methods 2.1 Preparation of main solvents and culture media Cholesterol solution: Add 50 mg of cholesterol to 10 mL of 95% ethanol, mix well, and then filter through a 0.22 μm filter.

[0061] 5 M KOH: Add 28 g KOH to ultrapure water and bring the volume to 100 ml.

[0062] 1 M CaCl2: Add 22.2 g CaCl2 to ultrapure water, bring the volume to 200 mL, and autoclave at 121℃ for 30 min.

[0063] 1 M MgSO4: Add 24 g MgSO4 to ultrapure water, bring the volume to 200 mL, and autoclave at 121 °C for 30 min.

[0064] 1 M potassium hydrogen phosphate (pH 6.0): 129.25 g KH2PO4, 67.75 g K2HPO4, dissolved in an appropriate amount of ultrapure water, adjusted to pH 6.0, then added ultrapure water, brought to a final volume of 200 mL, and autoclaved at 121℃ for 30 min.

[0065] M9 buffer: KH2PO4 3.0 g, Na2HPO4 5.8 g, NH4Cl 1.0 g, NaCl 0.5 g, add ultrapure water, bring to a final volume of 1 L, and autoclave at 121℃ for 30 min.

[0066] FUDR: Add 40 mg FUDR to 4 ml of ultrapure water, vortex to mix, and then aliquot into centrifuge tubes. Store at -20°C.

[0067] 10 mM levamisole: Add 0.024 g levamisole powder to 10 ml of ultrapure water, shake to mix, and dispense into centrifuge tubes.

[0068] LB liquid medium: 25 g of LB liquid medium, ultrapure water, bring to a final volume of 1 L, autoclave at 121°C for 30 min.

[0069] LB solid medium: 25 g of LB solid medium, ultrapure water, bring to a final volume of 1 L, autoclave at 121°C for 30 min.

[0070] Solid cryopreservation solution: 1.65 ml phosphate buffer, 17.5 ml H2O, 30 ml 50% glycerol, 0.29 mg NaCl, 0.2 g agar. Sterilize and incubate at 37 ℃ for later use. Store at 4 ℃.

[0071] Lysis buffer: 1g NaOH, 5ml NaClO added to 95ml water.

[0072] NGM medium: 6.8g agar powder, 1.2g NaCl, 1.0g peptone. First, add about 390mL of distilled water, then autoclave at 121℃ for 30 minutes, incubate at 60℃ for 30 minutes, and then add 400μL 5mg / mL cholesterol, 10mL 1M potassium hydrogen phosphate buffer (pH 6.0), 400μL 1M MgSO4, and 400μL 1M CaCl2 in a clean bench.

[0073] 2.2 OP50 culture and plate spreading 1) Activation of E. coil OP50 After removing the E. coli OP50 culture from the -80℃ freezer, it was vigorously shaken in a 37℃ constant temperature water bath until thawed. 100 μl of the OP50 culture was then pipetted into a pre-prepared sterile LB liquid medium and incubated at 37℃ with shaking for 10 h.

[0074] 2) Preparation of E. coil OP50 monoclonal colonies Dip a sterile inoculation loop into the bacterial suspension prepared in step 1) and streak it onto the surface of the culture medium. Refer to "Molecular Cloning: A Laboratory Manual" and "A Concise Guide to Molecular Biology Experiments" for specific methods. Incubate at 37°C for 12 hours until single colonies appear.

[0075] 3) E. coli OP50 inoculation Dip a sterile inoculation loop into 200 mL of sterile LB broth and add the E. coil OP50 monoclonal colony prepared in step 2). Incubate overnight at 37°C with shaking. An OD600 value between 0.4 and 0.6 is acceptable for subsequent experiments.

[0076] 2.3 Cultivation and Subculturing of Nematodes All nematodes were cultured on standard nematode culture medium (NGM) and maintained according to standard procedures. Unless otherwise specified, all nematodes were cultured at 20°C and fed with OP50.

[0077] When the nematodes reach adulthood or food becomes scarce, they are passaged. In a clean bench, a small piece of old agar plate is cut with a sterile scalpel and placed upside down on freshly prepared NGM medium containing OP50. The nematodes are then encouraged to crawl onto the fresh medium.

[0078] 2.4 Synchronization of Nematodes 2.4.1 Spawning Method 1) Select nematodes in the oviposition period and place them on the surface of freshly prepared NGM medium with OP50; 2) Incubate at a constant temperature of 20℃ for 3-4 hours until egg-laying occurs; 3) Remove the adult insects, leaving the eggs; 4) Continue culturing the eggs for three more days to obtain synchronous nematodes.

[0079] 2.4.2 Pyrolysis method 1) Collect and flush nematodes at their peak oviposition period using M9 buffer; 2) Centrifuge at 2000 rpm for 2 min and discard the supernatant; 3) Add the lysis buffer and shake vigorously for 40 seconds; 4) Centrifuge at 3000 rpm for 1 min to precipitate the eggs, and discard the supernatant; 5) Rinse three times with M9 buffer solution; 6) Centrifuge at 2000 rpm for 2 min, then discard the supernatant; 7) Transfer the precipitated eggs to the surface of freshly prepared NGM medium with OP50; 8) Continue culturing the eggs for three more days to obtain synchronized nematodes.

[0080] 2.5 Administration methods for rare ginsenoside compositions Before the experiment, a working solution was prepared by adding a rare ginsenoside composition to Escherichia coli OP50 at a concentration of 25 μg / mL.

[0081] The administration method involves coating the working solution of the rare ginsenoside composition onto NGM medium, drying it, and then using it to culture nematodes for bioassays.

[0082] 2.6 Effects of Rare Ginsenoside Combinations on Nematode Lifespan 1) Nematodes were cultured on NGM plates to the L4 stage; 2) Use a sterile nematode picker to pick up the nematodes onto NGM medium containing a rare ginsenoside composition; 3) One board per group, with at least 100 pieces per board; 4) Incubate at a constant temperature of 20℃; 5) The day the nematodes were picked is recorded as DAY 0; 6) Each day, transfer nematodes to fresh culture medium and record the number of deaths, survivals, and losses. Nematodes that burrow into the culture medium or dry out on the side of the petri dish are recorded as lost. Nematodes that do not react to the platinum wire are recorded as dead. Deaths of nematodes due to eggs hatching inside their bodies are not recorded.

[0083] 2.7 Effects of rare ginsenoside compositions on the heat stress resistance of nematodes 1) Cultured nematodes on NGM plates to the L4 stage; 2) Use a sterile nematode picker to pick up the nematodes onto NGM medium containing a rare ginsenoside composition; 3) One tray per group, with at least 30 pieces per tray; 4) Incubate at a constant temperature of 20℃ for three days, changing the culture medium daily; 5) Three days after administration, the nematodes were placed in a 37°C incubator and subjected to heat shock for 3 hours; 6) Recover in an incubator at 20℃ for 12 hours; 7) Statistical analysis of nematode survival and mortality.

[0084] 2.7.1 Body length measurement 1) Nematodes were cultured on NGM plates to the L4 stage; 2) Use a sterile nematode picker to pick up the nematodes onto NGM medium containing a rare ginsenoside composition; one plate per group, 10 nematodes per plate; 3) After administering the medication for eight days, transfer the nematodes to freshly prepared culture medium daily to ensure an adequate food supply; 4) Preparation of agar pads: Add 2% agar to a clean glass slide, place a coverslip vertically on top, press the agar flat, and remove any air bubbles. After the agar has cooled and solidified, remove the coverslip and it is ready for use. 5) On the eighth day after administration, collect the nematodes in sterile centrifuge tubes and add levamisole to anesthetize them; 6) Remove the anesthetized nematodes and arrange them neatly on the surface of an agar plate; 7) Take photos under a microscope; 8) Use Upoview software to measure the relative body length of the nematodes.

[0085] 2.7.2 Measurement of head oscillation frequency 1) Nematodes were cultured on NGM plates to the L4 stage; 2) Use a sterile nematode picker to pick up the nematodes onto NGM medium containing a rare ginsenoside composition; one plate per group, 10 nematodes per plate; 3) After administering the medication for eight days, transfer the nematodes to freshly prepared culture medium daily to ensure an adequate food supply; 4) On the eighth day after administration, the nematodes were collected in M9 droplets; 5) The number of times the nematode's head swung from left to right within 10 seconds was recorded.

[0086] 2.8 Effects of rare ginsenoside compositions on lipofuscin levels in nematodes 1) Nematodes were cultured on NGM plates to the L4 stage; 2) Use a sterile nematode picker to pick up the nematodes onto NGM medium containing a rare ginsenoside composition; one plate per group, 10 nematodes per plate; 3) After administering the medication for eight days, transfer the nematodes to freshly prepared culture medium daily to ensure an adequate food supply; 4) On the eighth day after administration, collect the nematodes in sterile centrifuge tubes and add levamisole to anesthetize the nematodes; 5) Remove the anesthetized nematodes and arrange them neatly on the surface of an agar plate; 6) Observe the fluorescence value under a fluorescence microscope and take pictures.

[0087] 2.9 Effects of rare ginsenoside compositions on ROS levels in nematodes 1) Nematodes were cultured on NGM plates to the L4 stage; 2) Use a sterile nematode picker to pick up the nematodes onto NGM medium containing a rare ginsenoside composition; 3) One board per group, 10 pieces per board; 4) After administering the medication for eight days, transfer the nematodes to freshly prepared culture medium daily to ensure an adequate food supply; 5) On the eighth day after administration, collect the nematodes in sterile centrifuge tubes and wash with M9 buffer to remove OP50; 6) Add 10 μl of H2DCF-DA molecular probe to each tube, gently tap the bottom of the centrifuge tube to mix, and incubate at 37°C for 1 h; 7) Wash the nematodes repeatedly with M9 buffer to remove excess probes; 8) Remove the nematodes and arrange them neatly on the surface of the agar plate; 9) Observe the fluorescence value under a fluorescence microscope and take pictures.

[0088] 2.10 Data Processing and Analysis All fluorescence images were captured at 10x magnification. Relative fluorescence intensity of the nematodes was measured using ImageJ software (NIH, Bethesda, MD, USA). Student's t-test was used to calculate comparisons between the two groups. Statistical analysis was performed using GraphPadPrism 8.0 software. Data are expressed as mean ± standard deviation of three independent experiments. P < 0.05 was considered statistically significant.

[0089] Example 2 The only difference from Example 1 is that the mass ratio of the rare ginsenoside composition is: Rk3: Rh4: Dehydroprotopanaxatriol Ⅰ: Dehydroprotopanaxatriol Ⅱ=1:2:1:1.

[0090] Example 3 The only difference from Example 1 is that the mass ratio of the rare ginsenoside composition is Rk3:Rh4:Dehydroprotopanaxatrio Ⅰ:Dehydroprotopanaxatriol Ⅱ=3:4:1:3.

[0091] Comparative Example 1 The only difference from Example 1 is that the rare ginsenoside composition in Comparative Example 1 contains only Rk3 and Dehydroprotopanaxatrio I, with a mass ratio of Rk3 to Dehydroprotopanaxatrio I of 2:1.

[0092] Comparative Example 2 The only difference from Example 1 is that the rare ginsenoside composition in Comparative Example 1 contains only Rh4 and Dehydroprotopanaxatriol II, with a mass ratio of Rh4 to Dehydroprotopanaxatriol II of 3:2.

[0093] Comparative Example 3 The only difference from Example 1 is that the mass ratio of the rare ginsenoside composition is Rk3:Rh4:Dehydroprotopanaxatrio Ⅰ:Dehydroprotopanaxatriol Ⅱ=5:1:1:4.

[0094] Comparative Example 4 The only difference from Example 1 is that the mass ratio of the rare ginsenoside composition is Rg3:Rh4:Dehydroprotopanaxatrio Ⅰ:Dehydroprotopanaxatriol Ⅱ=2:3:1:2.

[0095] Experimental Example 1: Intervention Effect of Rare Ginsenoside Combinations on Naturally Aging Caenorhabditis elegans The experimental method is the same as in Example 1.

[0096] The experimental results are as follows: 3.1 Effects of rare ginsenoside compositions on nematode lifespan The results showed that rare ginsenoside compositions could prolong the lifespan of nematodes. Figure 1Under suitable growth conditions, the lifespan of nematodes in the control group was 14.67 days. The maximum lifespans of Examples 1, 2, and 3 were 25 days, 25 days, and 25.33 days, respectively. The maximum lifespans of Comparative Examples 1, 2, 3, and 4 were 18.33 days, 17.67 days, 18 days, and 17 days, respectively. The lifespans of the Examples were significantly longer than the control group and superior to the Comparative Examples. These results indicate that the rare ginsenoside composition can significantly prolong the lifespan of nematodes. p <0.001, p <0.001, p <0.001).

[0097] 3.2 Effects of rare ginsenoside compositions on the heat stress resistance of nematodes A key characteristic of aging is a decline in the ability to withstand environmental stress. The following experiment investigated the heat stress resistance of nematodes treated with a rare ginsenoside composition. The results are as follows... Figure 2 As shown, after heat stress at 37℃, the survival rate of nematodes in the control group was 49.4%, while the survival rates in Examples 1, 2, and 3 were 75.67%, 70.67%, and 71.62%, respectively, significantly higher than that in the control group. p <0.001, p <0.001, p <0.001), the survival rates of Comparative Examples 1, 2, 3, and 4 were 59.3%, 49.67%, 54.3%, and 50%, respectively. In conclusion, the heat stress resistance of nematodes was significantly improved after treatment with the rare ginsenoside composition.

[0098] 3.3 Effects of rare ginsenoside compositions on physiological indicators of nematodes The head-waving frequency of nematodes treated with a rare ginsenoside composition for 8 days was measured. The average head-waving frequency of the control group nematodes was 21.93 times per 10 seconds. The head-waving frequencies of Examples 1, 2, and 3 were 28.70 times, 28.25 times, and 28.50 times per 10 seconds, respectively, significantly higher than that of the control group. p <0.001, p <0.001, p <0.001), the head-wobbling frequencies of Comparative Examples 1, 2, 3, and 4 were 24.40, 24.80, 24.90, and 24.90 times, respectively. The results indicate that the head-wobbling frequencies of nematodes treated with the rare ginsenoside composition were all higher than those of the control group (…). Figure 3 ).

[0099] Aging is often accompanied by a decline in other physiological indicators. Therefore, the body length of nematodes treated with a rare ginsenoside composition was evaluated on the eighth day. With the average relative body length of the control group set at 1, the relative body lengths of Examples 1, 2, and 3 were 1.73, 1.72, and 1.70, respectively, significantly higher than that of the control group. p <0.001, p <0.001, p <0.001), the relative body lengths of Comparative Examples 1, 2, 3, and 4 were 1.06, 1.09, 1.15, and 1.11, respectively. The results indicate that the relative body length of nematodes treated with the rare ginsenoside composition was significantly increased. Figure 4 ).

[0100] 3.4 Effects of rare ginsenoside compositions on lipofuscin levels in nematodes Previous studies have shown that lipofuscin, a product of oxidative damage, accumulates in nematodes with increasing age, reflecting their aging status. Therefore, the lipofuscin levels in nematodes treated with a rare ginsenoside composition for eight days were evaluated. The results showed that, with the relative lipofuscin level in the control group set at 1, the relative lipofuscin levels in Examples 1, 2, and 3 were 0.80, 0.81, and 0.89, respectively, significantly lower than those in the control group. p <0.001, p <0.001, p <0.001), the relative levels of lipofuscin in Comparative Examples 1, 2, 3, and 4 were 0.96, 0.97, 0.97, and 0.95, respectively. The results indicate that the relative level of lipofuscin in nematodes significantly decreased after intervention with the rare ginsenoside composition. Figures 5-6 ).

[0101] 3.5 Effects of rare ginsenoside compositions on ROS levels in nematodes In the following experiments, the effect of rare ginsenoside compositions on intracellular ROS levels in nematode cells was investigated. The reactive oxygen species (ROS) level in N2 nematodes was determined using the reactive oxygen species indicator fluorescein dichlorodiacetate (H2DCFH-DA). The non-fluorescent H2DCFH-DA could only be oxidized to 2',7'-dichlorofluorescein (DCF) in the presence of intracellular ROS, thus determining the intracellular ROS level. After 8 days of pretreatment, all groups treated with rare ginsenoside compositions showed lower relative ROS levels. With the relative ROS level of the control group set at 1, the relative ROS levels in Examples 1, 2, and 3 were 0.48, 0.49, and 0.49, respectively, significantly lower than that of the control group (…). p <0.001, p <0.001, p<0.001), and the relative ROS levels of Comparative Examples 1, 2, 3, and 4 were 0.73, 0.82, 0.77, and 0.76, respectively. The results indicate that the relative ROS level of nematodes treated with the rare ginsenoside composition was significantly reduced (<0.001). Figures 7-8 ).

[0102] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A rare ginsenoside composition, characterized in that, The rare ginsenoside composition is composed of Rk3, Rh4, Dehydroprotopanaxatrio Ⅰ and Dehydroprotopanaxatriol Ⅱ, with a mass ratio of (1-3):(2-4):1:(1-3).

2. The rare ginsenoside composition according to claim 1, characterized in that, The mass ratios of Rk3, Rh4, Dehydroprotopanaxatrio Ⅰ, and Dehydroprotopanaxatriol Ⅱ are 1:2:1:1, 2:3:1:2, and 3:4:1:

3.

3. The rare ginsenoside composition according to claim 1, characterized in that, The mass ratio of Rk3, Rh4, Dehydroprotopanaxatrio Ⅰ, and Dehydroprotopanaxatriol Ⅱ is 2:3:1:

2.

4. The method for preparing the rare ginsenoside composition according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Dissolve the ginsenosides in a water-methanol system, add aspartic acid, cook, and remove the cooked hydrolysate. (2) After removing methanol from the hydrolysis product, the resulting suspension is first extracted with ethyl acetate 2-4 times. The ethyl acetate layers are combined, and the solvent in the ethyl acetate layer is removed to obtain the solid. (3) The solid was dissolved in methanol and separated by medium-pressure semi-preparative chromatography. C18 was selected as the stationary phase, the flow rate was 24-26 ml / min, the detection wavelength was 203 nm, the running time was 80-100 min, and the mobile phase conditions were acetonitrile / 68%-100% and water / 32%-0%. Ginsenoside Rk3, ginsenoside Rh4, ginsenoside Dehydroprotopanaxatriol Ⅰ and ginsenoside Dehydroprotopanaxatriol Ⅱ were obtained. They were mixed according to the mass ratio to obtain the rare ginsenoside composition.

5. The use of the rare ginsenoside composition according to any one of claims 1-3 in the preparation of anti-aging products.

6. The application according to claim 5, characterized in that, The products mentioned include pharmaceuticals and cosmetics.

7. The application according to claim 5, characterized in that, The product contains a rare ginsenoside composition as its main active ingredient.

8. The application according to claim 5, characterized in that, The product has at least one of the following functions: (1) Reduce the level of lipofuscin in the body; (2) Reduce the level of ROS in the body.

9. The application according to claim 6, characterized in that, When the product is a drug, the drug may also include a pharmaceutically acceptable carrier.

10. The application according to claim 6, characterized in that, The cosmetics mentioned also include base materials and conventional excipients.

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