Use of ginsenosides in the preparation of a composition for the treatment or prevention of neurodegeneration

Abstract

Provided is the use of ginsenoside in the preparation of a composition for treating or preventing neurodegeneration. Also provided is the use of ginsenoside in the preparation of a composition for improving ovarian reproductive function, or improving mechanical and / or olfactory sensitivity in a subject. The ginsenoside is Rg3 or Rg5.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of therapy, in particular to the use of rare ginsenoside Rg3 or Rg5 in the preparation of a composition for treating or preventing neurologic functional decline. BACKGROUND

[0002] Ginsenoside is a sterol compound, which is considered as an active ingredient in ginseng, mainly derived from Panax plants such as Panax ginseng, Panax quinquefolium and Panax notoginseng. Panax ginseng is the main source of ginsenoside, and the content and types of ginsenoside in Panax ginseng of different growth years and origins are different. Ginsenoside is a glycoside compound connected by aglycone and sugar. The aglycone is mainly tetracyclic triterpenoid or pentacyclic triterpenoid, and the sugar moiety includes glucose, galactose, rhamnose, etc. According to the structure of aglycone, ginsenoside can be divided into two types: dammarane type and oleanane type. The pharmacological functions of ginsenoside mainly include anti-fatigue, blood pressure regulation, anti-tumor, immune capacity improvement, and anti-oxidation, etc.

[0003] Therefore, it is still of great significance to develop ginsenoside composition with therapeutic or health care functions. SUMMARY

[0004] The present application uses Caenorhabditis elegans as a model to find that ginsenoside Rg3 or Rg5 can improve the mechanical sensation and / or olfaction of Caenorhabditis elegans and improve the ovary reproductive function. In addition, survival analysis shows that ginsenoside Rg3 or Rg5 has a significant effect of delaying the aging of Caenorhabditis elegans.

[0005] In one aspect, there is provided the use of ginsenoside in the preparation of a composition for assisting in improving memory in a subject.

[0006] In another aspect, there is provided the use of ginsenoside in the preparation of a composition for treating or preventing neurologic functional decline or a disease or condition caused by neurologic functional decline (or improving the degeneration of mechanoreceptor neurons or the degeneration of mechanoreceptor neurons) in a subject, or for improving ovary reproductive function or fertility or delaying ovary aging, or for treating or preventing a disease caused by ovary aging.

[0007] In one embodiment, the ginsenoside is one or more of ginsenoside Rg3 and Rg5.

[0008] In one embodiment, the improvement of ovary reproductive function or fertility is maintaining or improving the morphology of oocytes and / or increasing the number of oocytes.

[0009] In one embodiment, the composition is a composition for treating or preventing the decline of motor coordination and / or a composition for treating or preventing the decline of sensory cognitive function.

[0010] In one embodiment, the decline in neural function is caused by GABAergic neuron degeneration or mechanoreceptor neuron degeneration. In one embodiment, the disease or condition caused by the decline in neural function is one or more of neurodegenerative diseases, preferably one or more of dementia, movement disorder, post-infectious degenerative disease, degenerative changes after injury in young children, degenerative changes after injury in older children and adults, inherited degenerative diseases, and acquired neurodegenerative diseases.

[0011] In one embodiment, the dementia is one or more of Alzheimer’s disease, frontotemporal lobar degeneration (FTLD), frontotemporal dementia (FTD), dementia with Lewy bodies, posterior cortical atrophy, vascular or multi-infarct dementia, corticobasal degeneration, and prion disease.

[0012] In one embodiment, the movement disorder is one or more of Parkinson’s disease, Parkinson-plus syndrome, progressive supranuclear palsy, and amyotrophic lateral sclerosis.

[0013] In one embodiment, the Parkinson-plus syndrome is one or more of multiple system atrophy, olivopontocerebellar atrophy, and Shy-Drager syndrome.

[0014] In one embodiment, the inherited degenerative disease is one or more of a brain tissue iron deposition neurodegeneration, Wilson’s disease, Menkes syndrome, Fahr’s disease, Huntington’s disease, ataxia telangiectasia, Friedreich’s ataxia, and spinocerebellar ataxia.

[0015] In one embodiment, the disease caused by GABAergic neuron degeneration is one or more of Huntington’s disease, epilepsy, and dystonia, and the disease caused by mechanoreceptor neuron degeneration is one or more of sensory peripheral neuropathy, Friedreich’s ataxia, and age-related deterioration of touch or olfaction.

[0016] In one embodiment, the disease or condition caused by ovarian aging is one or more of endocrine disorder, menstrual cycle disorder or cessation, and infertility.

[0017] In one embodiment, the composition is a pharmaceutical composition or a food composition or a cosmetic composition.

[0018] In one embodiment, the food composition is one or more of a food, a health food, a special food, or a pet food.

[0019] In one embodiment, the composition is in one or more of a tablet, an ointment, a granule, a capsule, a powder, or an oral liquid.

[0020] In one embodiment, the subject is a mammal (including a female and / or a male). In a preferred embodiment, the subject is a human (e.g., a male and / or a female).

[0021] In another aspect, a method of activating lysosomal acidification, activating TOR signaling pathway, and / or activating vacuolar-type ATPase VHA-3 in a cell in vitro is provided, comprising contacting the cell with ginsenoside Rg3 or Rg5. In one embodiment, the effective concentration of ginsenoside Rg3 or Rg5 is 5-100 ug / mL.

[0022] In another aspect, the use of ginsenoside Rg3 or Rg5 in activating lysosomal acidification, activating TOR signaling pathway, and / or activating vacuolar-type ATPase VHA-3 is provided. In one embodiment, the effective concentration of ginsenoside Rg3 or Rg5 is 10 ug / mL.

[0023] The present application has the following beneficial effects:

[0024] 1. The present application uses C. elegans as a model and finds that ginsenoside Rg3 has a significant effect on improving the integrity of ALM, PLM, PVD mechanoreceptor neurons and GABA neurons, and improving touch avoidance response and olfactory sensitivity.

[0025] 2. By microscopically observing the morphology of oocyte cells and the number of offspring of C. elegans, it is found that Rg3 and Rg5 have the effect of improving the morphology of oocyte cells, and Rg5 can increase the number of offspring.

[0026] 3. The present application finds that ginsenoside Rg3 has a significant effect on delaying aging of C. elegans through survival analysis. The present application also finds that the anti-aging effect of ginsenoside Rg3 on C. elegans is achieved by activating vacuolar-type ATPase VHA-3 and ribosomal protein kinase RSKS-1; Rg5 prolongs the lifespan of C. elegans by activating vacuolar-type ATPase VHA-3.

[0027] 4. The present application provides a solid theoretical basis for the application of ginsenosides such as Rg3 and Rg5. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 The effects of ginsenosides Rg3 and Rg5 on the lifespan of C. elegans strain N2; wherein Figure A is a survival curve, and Figure B is the average lifespan. Figure 1 It can be seen that ginsenosides Rg3 and Rg5 have a significant effect on prolonging the lifespan of C. elegans.

[0029] Figures 2A-2ISurvival curves of the lifespan effect experiments of ginsenoside Rg3 and Rg5 on C. elegans strain daf-16(mu86) (Figure), Figure 2A Survival curves of the lifespan effect experiments of ginsenoside Rg3 and Rg5 on C. elegans strain eat-2(sd1116) (Figure), Figure 2B Survival curves of the lifespan effect experiments of ginsenoside Rg3 and Rg5 on C. elegans strain aak-2(gt33) (Figure), Figure 2C Survival curves of the lifespan effect experiments of ginsenoside Rg3 and Rg5 on C. elegans strain vha-3(ok1501) (Figure), Figure 2D Survival curves of the lifespan effect experiments of ginsenoside Rg3 and Rg5 on C. elegans strain glp-1(e2144) (Figure), Figure 2E Survival curves of the lifespan effect experiments of ginsenoside Rg3 and Rg5 on C. elegans strain rsks-1(ok1255) (Figure), Figure 2F Survival curves of the lifespan effect experiments of ginsenoside Rg3 and Rg5 on C. elegans strain clk-1(qm30) (Figure), Figure 2G Survival curves of the lifespan effect experiments of ginsenoside Rg3 and Rg5 on C. elegans strain atg-7(bp422) (Figure), Figure 2H Survival curves of the lifespan effect experiments of ginsenoside Rg3 and Rg5 on C. elegans strain daf-16(mu86) (Figure), Figure 2I Survival curves of the lifespan effect experiments of ginsenoside Rg3 and Rg5 on C. elegans strain eat-2(sd1116) (Figure), Figures 2A-2I It can be seen that the extension of C. elegans lifespan by ginsenoside Rg3 is mainly regulated by vha-3 and rsks-1 genes, that is, the extension of C. elegans lifespan by ginsenoside Rg3 depends on lysosomal acidification and TOR signaling pathway. The extension of C. elegans lifespan by ginsenoside Rg5 is mainly regulated by vha-3 gene, that is, the extension of C. elegans lifespan by ginsenoside Rg5 depends on lysosomal acidification pathway.

[0030] Figure 3 Effects of ginsenoside Rg3 and Rg5 on the ovary cell morphology and offspring number of C. elegans strain N2, wherein Figure A is the ovary cell morphology, and Figure B is the offspring number. From Figure A of Figure 3 It can be seen that ginsenoside Rg3 and Rg5 can significantly improve the ovary cell morphology on the 3rd day and the 5th day. From Figure B of Figure 3 It can be seen that ginsenoside Rg5 significantly improves the offspring number.

[0031] Figure 4A Effects of Rg3 and Rg5 on the morphology of ALM and PLM neurons (ALM is anterior lateral microtubule cell, and PLM is posterior lateral microtubule cell) of nematode strain zdIs5 [mec-4p::GFP + lin-15(+)] observed by fluorescence microscope. Figure 4B Laser confocal observation of the effects of Rg3 on the morphology of ALM and PLM neurons. Figure 4C Scoring of the effects of Rg3 on the morphology of ALM and PLM neurons. Figure 4D Effects of Rg3 on the morphology of PVD neurons. Figure 4EThe impact of Rg3 on PVD neuronal morphology is scored. The scoring rules for neuronal morphology impact are as follows: Poor (lower part of the bar chart) indicates nematodes with all neurons broken; Middle (middle part of the bar chart) indicates nematodes with some neurons broken; and Great (upper part of the bar chart) indicates nematodes with all neurons intact. Figures 4A-4E It can be seen that Rg3 significantly improves the integrity of mechanoreceptor neurons in nematodes (maintaining high integrity on days 5 and 10, with a very significant effect). Rg5 can significantly improve the integrity of mechanoreceptor neurons in nematodes on day 5.

[0032] Figure 5A Fluorescence microscopy observation of the effects of Rg3 and Rg5 on the morphology of GABA neurons in the nematode strain oxls12 (unc-47::GFP). Figure 5B Laser confocal microscopy observation of the effect of Rg3 on the morphology of GABA neurons; Figure 5C The score represents the effect of Rg3 on the morphology of GABA neurons. (Based on...) Figures 5A-5C It can be seen that Rg3 is very effective in maintaining the integrity of GABA neurons in nematodes (significantly maintaining the integrity of GABA neurons on both day 5 and day 10); Rg5 has no significant improvement effect.

[0033] Figure 6 The scores for Rg3 and Rg5 in response to nematode touch avoidance and the olfactory tactile tropism index were assessed. Figure 6 Figure A shows that Rg3 significantly enhances the touch avoidance response in older nematodes (day 10); Figure 6 Figure B shows that Rg3 can significantly improve the olfactory sensitivity of middle-aged nematodes (day 5).

[0034] Figure 7 Figure A shows the odor preference experiment of nematodes. Figure 7 Figure B is a schematic diagram of the mechanoreceptor neurons ALM and PLM. Figure 7 Figure C is a schematic diagram of the PVD of a mechanoreceptor neuron. Figure 7 The diagram in Figure D is a schematic diagram of a GABA neuron. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0036] Ginsenosides are a class of sterol compounds that are considered to be active ingredients in ginseng, and are primarily derived from plants of the genus Panax in the family Araliaceae, such as Panax ginseng, Panax quinquefolius, and Panax notoginseng. The most common preparation process in the industry is biological transformation, including in vitro enzymatic degradation, microbial transformation, and biosynthesis. Microbial transformation uses specific microbial cells to modify the structure of exogenous substrates, and through the catalytic reaction of enzymes produced during the metabolic process, the content and medicinal properties of active ingredients are increased. In addition, pyrolysis and acid degradation are also common preparation methods. In this document, preferably, the ginsenoside is one or more of Rg3 and Rg5 ginsenosides.

[0037] Ginsenosides Rg3 and Rg5 are isolated or purified, and are rare ginsenosides known in the art, having molecular formulas of C 42 H 72 O 13 and C 42 H 70 O 12 , and structural formulas of:

[0038]

[0039] Ginsenoside Rg3

[0040]

[0041] Ginsenoside Rg5.

[0042] Ginsenosides Rg3 and Rg5 have been widely used in the art. In this document, ginsenoside Rg3 and / or Rg5 can be used to improve neurologic functional decline, treat diseases or conditions caused by neurologic functional decline, improve ovarian aging or diseases or conditions caused by ovarian aging, and can also be used to improve the anti-aging ability of biotics.

[0043] As used herein, “neurologic functional decline” refers to the progressive loss of neural function. Neurologic functional decline can cause a variety of diseases or conditions, including neurodegenerative diseases.

[0044] As used herein, "neurodegenerative disease" is a class of diseases that are characterized by the progressive degeneration, death, and loss of function of neurons. These diseases usually have an insidious onset and progress slowly, with symptoms that worsen progressively. The etiology is not fully understood and can be related to genetic, environmental, and age factors. The pathological changes are mainly manifested as neuronal degeneration and death, glial proliferation, and inflammatory response. Currently, there is no cure for neurodegenerative diseases, and clinical treatment mainly aims to relieve symptoms and delay disease progression, including drug treatment, rehabilitation treatment, and supportive treatment. Neurodegenerative diseases include, but are not limited to, one or more of dementia, movement disorder, post-infectious degenerative disease, degenerative changes after injury in young children, degenerative changes after injury in older children and adults, hereditary degenerative disease, and acquired neurodegenerative disease. In one embodiment, dementia is one or more of Alzheimer's disease, frontotemporal lobar degeneration (FTLD), frontotemporal dementia (FTD), Lewy body dementia, posterior cortical atrophy, vascular or multi-infarct dementia, corticobasal degeneration, and prion disease. In one embodiment, movement disorder is one or more of Parkinson's disease, Parkinson-plus syndrome, progressive supranuclear palsy, and amyotrophic lateral sclerosis. In one embodiment, Parkinson-plus syndrome is one or more of multiple system atrophy, olivopontocerebellar atrophy, and Shy-Drager syndrome. In one embodiment, hereditary degenerative disease is one or more of neurodegeneration with brain iron accumulation, Wilson's disease, Menkes syndrome, Fahr's disease, Huntington's disease, ataxia telangiectasia, Friedreich's ataxia, and spinocerebellar ataxia.

[0045] As used herein, "GABAergic neuron degeneration" refers to the progressive degeneration, death, or loss of function of gamma-aminobutyric acid (GABA)ergic neurons in the brain or spinal cord, which in turn triggers a series of neurological dysfunctions. GABAergic neuron degeneration can include Huntington's disease.

[0046] As used herein, "motor coordination" refers to the effective coordination between the actions of different parts of the body, which is the effective coordination between perceptual ability and motor ability. "Decreased motor coordination" refers to the decreased ability of effective coordination between the actions of different parts of the body. In this context, decreased motor coordination is caused by neurologic decline.

[0047] As used herein, decreased sensory cognitive function can include cognitive impairment. Cognitive impairment is a pathological process caused by abnormalities in the function and structure of the cerebral cortex, resulting in abnormalities in functions related to the brain's high-level intelligent processing processes such as learning, memory, and thought judgment, which in turn triggers severe learning and memory impairment, and can be accompanied by changes such as aphasia, apraxia, agnosia, and ataxia.

[0048] As used herein, "Premature Ovarian Failure" refers to a condition in which a woman's ovaries stop functioning before the age of 40, resulting in irregular menstrual cycles or amenorrhea, infertility, and a range of associated symptoms. This condition not only impairs a woman's ability to reproduce, but can also lead to health problems such as osteoporosis, cardiovascular disease, and others.

[0049] As used herein, C. elegans is a bacterivorous, linear animal that is transparent, hermaphroditic, and feeds on bacteria. C. elegans has several advantages as a model organism. First, C. elegans has a short life cycle, from embryonic development to adulthood, which takes only about 3-4 days, and the entire life cycle is about 2-3 weeks. This allows researchers to observe the entire process of the worm from birth to death in a relatively short period of time, facilitating experimental research. Second, it has a clear genetic background, with a completely sequenced genome of about 19,000 genes. This clear genetic background provides great convenience for studying genes related to diseases or physiological processes, such as aging-related genes. Specific genes in C. elegans can be manipulated through gene editing techniques such as RNA interference, CRISPR / Cas9, etc., to explore the functions of these genes in the aging process. Third, it is easy to cultivate, and C. elegans can be cultured on simple agar plates with E. coli as a food source. The culture conditions are relatively simple, the cost is low, and a large number of cultures can be obtained to meet the needs of large-scale experiments. In addition, it has a simple but representative physiological structure, with a relatively simple body structure composed of about 1000 cells, with basic cell types and organ systems such as the nervous system, muscle system, and digestive system. Although the structure is simple, there are many similarities with higher organisms at the cellular and molecular levels, such as apoptosis, signal transduction, etc., making it possible to extend the results obtained from C. elegans research to other organisms, including humans. Finally and most importantly, it has obvious signs of aging. As age increases, C. elegans exhibits many obvious signs of aging, such as decreased motor ability, reduced reproductive capacity, and changes in body shape (such as muscle atrophy, intestinal degeneration, etc.). These characteristics are easy to observe and quantify, providing intuitive indicators for studying the aging of the nervous or reproductive system.

[0050] The method for verifying the anti-aging function and the anti-aging mechanism of ginsenoside Rg3 and Rg5 is based on Caenorhabditis elegans (for example, N2 strain of Caenorhabditis elegans). In this paper, the survival experiment of Caenorhabditis elegans after administration shows that both ginsenoside Rg3 and Rg5 can significantly prolong the life span of Caenorhabditis elegans. The existing technology has obtained gene edited nematode strains daf-16(mu86), eat-2(sd1116), aak-2(gt33), vha-3(ok1501), glp-1(e2144), rsks-1(ok1255), clk-1(qm30), atg-7(bp422) by gene engineering technology, such as CRISPR / Cas 9 and microinjection technology to point mutation to silence related genes. These genetically engineered nematodes are commercialized (see, for example, University of Minnesota, Caenorhabditis Genetics Center). The survival experiment of different nematode strains after administration shows that the prolongation of the life span of Caenorhabditis elegans by ginsenoside Rg3 is mainly regulated by vha-3 and rsks-1 genes, that is, the prolongation of the life span of Caenorhabditis elegans by ginsenoside Rg3 depends on lysosomal acidification and TOR signaling pathway; the prolongation of the life span of Caenorhabditis elegans by ginsenoside Rg5 is mainly regulated by vha-3 gene, that is, the prolongation of the life span of Caenorhabditis elegans by ginsenoside Rg5 depends on lysosomal acidification pathway. In this paper, the observation of ovary cells of N2 nematode strain and the counting of offspring confirm that both ginsenoside Rg3 and Rg5 can improve the morphology of ovary cells of nematodes on the 3rd day and the 5th day, and Rg5 can significantly increase the number of offspring. In this paper, the integrity of the mechanoreceptor neurons ALM, PLM and PVD of the nematode strain and the integrity of the GABA neurons are observed by fluorescence photography and laser confocal observation, and it is found that Rg3 can significantly improve the integrity of the mechanoreceptor neurons and the GABA neurons of the nematode, and also has improvement effect on the touch response and olfactory sensitivity phenotype.

[0051] In this paper, ginsenoside Rg3 and Rg5 are natural sterol compounds, which are safe, have no adverse reactions and toxic side effects, can effectively prolong the life span of Caenorhabditis elegans, and have significant anti-aging activity. The anti-aging mechanism of ginsenoside Rg3 and Rg5 is studied in depth, and the specific anti-aging pathway is found. The beneficial effect of ginsenoside Rg3 and Rg5 on reproduction (ovary cell morphology) and offspring number of nematodes is studied, and it is found that Rg5 significantly increases the number of offspring of nematodes. The effect of ginsenoside Rg3 and Rg5 on the integrity of ALM, PLM, PVD and GABA neurons of nematodes, touch and olfactory sensitivity phenotype response is studied, and it is found that Rg3 has improvement effect on the integrity of neurons, touch response and olfactory sensitivity.

[0052] EMBODIMENT

[0053] The presently disclosed subject matter will be better understood by reference to the following examples, which are presented as exemplary of the application and not in limitation of the present application. The experimental methods and detection methods described in the various examples are routine methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified. C. elegans strain N2 and E. coli OP50 are commercially available.

[0054] 1. Materials

[0055] Ginsenoside Rg3 or Rg5 solution: dissolved in DMSO and diluted with distilled water to 10 ug / mL.

[0056] Nematode growth medium (NGM) plates were prepared by weighing 3.5 g NaCl, 2.0 g peptone, 25 g agar powder into 800 mL deionized water, and dissolving thoroughly, followed by autoclaving (121 °C, 20 min). When cooled to 55 °C, 1 mL of 1 mol / L CaCl2, 1 mL of 1 mol / L MgSO4, 1 mL of 5 mg / L cholesterol, and 25 mL of 1 mol / L potassium phosphate solution (pH = 6.0) were added in a clean bench, and ddH2O was added to 1 L. After mixing, it was quickly dispensed into culture dishes to prepare.

[0057] FUDR plates were prepared by weighing 3.5 g NaCl, 2.0 g peptone, 25 g agar powder into 800 mL deionized water, and dissolving thoroughly, followed by autoclaving (121 °C, 20 min). When cooled to 55 °C, 1 mL of 1 mol / L CaCl2, 1 mL of 1 mol / L MgSO4, 1 mL of 5 mg / L cholesterol, 5 mL of 10 mg / mL 5-fluoro-2'-deoxyguanosine (FUdR), and 25 mL of 1 mol / L potassium phosphate solution (pH = 6.0) were added in a clean bench, and ddH2O was added to 1 L. After mixing, it was quickly dispensed into culture dishes to prepare. NGM plates and FUdR plates were packaged with sealing film and stored at 4 °C.

[0058] M9 buffer was prepared by weighing 0.25 g MgSO4·7H2O, 3.0 g KH2PO4, 6.0 g Na2HPO4, and 5.0 g NaCl, respectively, and adding ddH2O to 1 L. Filtration was performed with a 0.45 μm filter membrane in a clean bench, and the filtrate was dispensed into sterile containers and stored at room temperature for use.

[0059] Rinse buffer was prepared by mixing 10 mL NaClO (4%), 10 mL NaOH (1 M), and 20 mL ddH2O, and then transferring to a brown centrifuge tube and stored at room temperature in the dark.

[0060] Incubator was a 20 °C constant temperature incubator.

[0061] In this example, the solvent control group (or "vehicle") was a 0.1% DMSO treatment group.

[0062] 2. Methods

[0063] 2.1 Culture of C. elegans

[0064] In this example, ginsenoside Rg3 or Rg5 (both purchased from Source Leaf Biologies) was ingested by mixing with biological feed. The mixing process was as follows: ginsenoside Rg3 or Rg5 was dissolved in an organic solvent and diluted with distilled water to 10 ug / mL before mixing with biological feed in equal proportions. The biological feed was E. coli OP50. The organic solvent was dimethyl sulfoxide (DMSO). The distilled water was sterilized distilled water (ddH2O).

[0065] Step 1: Prepare plates of nematode growth medium (NGM) coated with Rg3 or Rg5 solution mixed with OP50 E. coli and plates of NGM with OP50 E. coli only, and plates of limiting nematode egg laying medium (FUdR).

[0066] Step 2: Synchronization of nematodes: Prepare C. elegans strain N2. Culture C. elegans on NGM plates for 2-3 days, and obtain plates rich in eggs before the OP50 E. coli is exhausted. Rinse the plates repeatedly with 1-2 mL of M9 buffer using a pipette to ensure that the eggs are not retained on the plates, and transfer the liquid containing the eggs to a 1.5 mL centrifuge tube. Centrifuge at 4,000 rcf for 1 min, and discard the supernatant. Add 500 μL of rinse buffer to the remaining 100 μL of liquid, tighten the cap, and shake for 3-5 min, ensuring that the entire nematode is not visible under a microscope, but the eggs are not over-soaked and inactivated. Centrifuge at 4,000 rcf for 0.5 min, and discard the supernatant. Resuspend the pellet by adding 1 mL of M9 buffer, centrifuge at 4,000 rcf for 1 min, and discard the supernatant. Repeat this step once more. Transfer the final 100 μL of liquid to a new NGM plate near the bacterial lawn, and after drying, place the plate upside down in an incubator.

[0067] Step 3: 50 worms were picked from each of the different feeding groups (FUdR + Rg3 group, FUdR + Rg5 group, FUdR + vehicle group and vehicle group) at L4 stage and placed on the corresponding fresh FUdR plates, 3 replicates for each group.

[0068] Step 4: The number of dead worms was counted every day. The worms were considered dead if they did not respond after being touched. The counting was continued until all the worms on the plate were dead.

[0069] The calculation of percent survival and average lifespan was as follows:

[0070] Percent survival = (number of surviving worms / total number of worms) x 100%

[0071] Average lifespan = total lifespan / total number of worms

[0072] 2.2 Experimental study on the mechanism of ginsenoside Rg3 or Rg5 improving the lifespan of nematodes

[0073] This experiment involves the specific mechanism of ginsenoside Rg3 improving biological anti-aging or improving the lifespan of nematodes. The process of studying this mechanism uses the gene knockdown strains of daf-16, eat-2, aak-2, vha-3, glp-1, rsks-1, clk-1, atg-7, daf-16(mu86), eat-2(sd1116), aak-2(gt33), vha-3(ok1501), glp-1(e2144), rsks-1(ok1255), clk-1(qm30), atg-7(bp422) (provided by Professor Zheng Lingjun's team at Shanghai Jiaotong University, or can also be replaced by commercially available corresponding gene knockdown strains). Gene knockdown strains can also be obtained by CRISPR / Cas9 technology for gene editing of C. elegans. By feeding ordinary C. elegans N2 with a mixture of ginsenoside Rg3 or Rg5 and heat-killed E. coli, the mechanism of action of ginsenoside Rg3 or Rg5 is determined.

[0074] Step 1: Prepare the nematode strains daf-16(mu86), eat-2(sd1116), aak-2(gt33), vha-3(ok1501), glp-1(e2144), rsks-1(ok1255), clk-1(qm30), atg-7(bp422) and heat-killed E. coli (HK E. coli). Prepare NGM and FUdR plates coated with 5 μL of heat-killed E. coli.

[0075] The daf-16(mu86) strain represents the insulin / IGF-1 signaling pathway. The eat-2(sd1116) strain represents the caloric restriction pathway. The aak-2(gt33) strain represents the AMPK signaling pathway. The vha-3(ok1501) strain represents the lysosomal acidification and autophagy pathway. The glp-1(e2144) strain represents the germline stem cell Notch signaling pathway. The rsks-1(ok1255) strain represents the TOR signaling pathway. The clk-1(qm30) strain represents the mitochondrial function pathway. The atg-7(bp422) strain represents the autophagy pathway. The heat-killed E. coli coated plate represents the microbial host interaction pathway.

[0076] Step 2: Prepare a large number of synchronized nematode eggs to be cultured on NGM plates coated with heat-killed E. coli to the L4 stage as in Method 2.1 above, and then pick 50 each to FUdR plates coated with heat-killed E. coli for incubation in an incubator. Different feeding groups (FUdR + Rg3, FUdR + Rg5, and FUdR + vehicle) of nematodes, each with three replicates.

[0077] Step 3: Count the number of dead nematodes every day, and death is determined by lack of response after touching. Until all nematodes in the plate are dead.

[0078] Experiments were entrusted to Professor Zheng Lingjun's team at Shanghai Jiaotong University for processing.

[0079] 2.3 Neuronal morphology impact score

[0080] The example uses the scoring method to count the proportion of intact neuronal nematodes after administration. The main observation objects include ALM neurons and PLM neurons, PVD neurons, and GABA neurons. The general scoring rules are as follows: poor: the proportion of nematodes with all neurons broken, Middle: the proportion of nematodes with some neurons broken, and great: the proportion of nematodes with all neurons intact. The nematode body microscope bright field observation experiment and the fluorescence microscope observation experiment can be performed by using microscopes and conventional methods known in the art. The body microscope bright field and fluorescence microscope observation experiments of the present application were entrusted to Professor Zheng Lingjun's team at Shanghai Jiaotong University for processing. Figure 7 Figure 1B is a schematic diagram of the mechanoreceptor neurons ALM and PLM, Figure 7 Figure 1C is a schematic diagram of the mechanoreceptor neuron PVD, Figure 1 Figure 1D is a schematic diagram of the GABA neuron.

[0081] Example 1: Ginsenoside Rg3 or Rg5 improves nematode lifespan and its mechanism

[0082] Figure 1The results of the experiment showing that ginsenoside Rg3 or Rg5 prolongs the lifespan of nematodes are shown. The results show the effect of ginsenoside Rg3 or Rg5 on the lifespan of the common Caenorhabditis elegans strain N2. In Figure 1 Figure A is a survival curve, and Figure B is the average lifespan. As can be seen from Figures 2A-2I ginsenoside Rg3 or Rg5 has a significant effect of prolonging the lifespan of Caenorhabditis elegans.

[0083] Figures 2A-2I The results of the experiment showing the effect of ginsenoside Rg3 and Rg5 on the lifespan of Caenorhabditis elegans strains daf-16(mu86), eat-2(sd1116), aak-2(gt33), vha-3(ok1501), glp-1(e2144), rsks-1(ok1255), clk-1(qm30), and atg-7(bp422) are shown. The survival curves of the lifespan effect experiment and the lifespan effect curve of feeding with heat-killed Escherichia coli are shown. As can be seen from Figure 3 ginsenoside Rg3 prolongs the lifespan of Caenorhabditis elegans mainly by the regulation of the vha-3 and rsks-1 genes, that is, the prolongation of the lifespan of Caenorhabditis elegans by ginsenoside Rg3 depends on the lysosomal acidification and TOR signaling pathway. Ginsenoside Rg5 prolongs the lifespan of Caenorhabditis elegans mainly by the regulation of the vha-3 gene, that is, the prolongation of the lifespan of Caenorhabditis elegans by ginsenoside Rg5 depends on the lysosomal acidification pathway.

[0084] Example 2: Ginsenoside Rg3 or Rg5 improves oocyte morphology of nematodes

[0085] As described above in Method 2.1, after the Caenorhabditis elegans strain N2 nematodes were synchronized and cultured to the L4 stage, they were transferred to FudR plates (coated with Rg3, Rg5, or solvent control mixed with Escherichia coli OP50, respectively). The oocyte morphology was observed and the number of offspring hatched from the nematodes was counted using a body microscope bright field at 1 day, 3 days, 5 days, and 10 days, respectively.

[0086] Figure 3 The results of the body microscope bright field photographing experiment showing the effect of ginsenoside Rg3 and Rg5 on the oocyte morphology and offspring number of Caenorhabditis elegans strain N2 are shown. Figure 3 Figure A is the oocyte morphology, and Figure 3 Figure B is the offspring number. As can be seen from Figure 3 Figure A, ginsenoside Rg3 and Rg5 can significantly improve the oocyte morphology at 3 days and 5 days. As can be seen from Figures 4A-4B Figure B, ginsenoside Rg5 significantly increases the offspring number. This example proves that ginsenoside Rg5 can improve the ovarian reproductive function or delay the early ovarian aging or aging.

[0087] Example 3: Effects of ginsenoside Rg3 or Rg5 on the integrity of the mechanosensory neuron of nematode

[0088] This example is performed using zdIs5 [mec-4p::GFP + lin-15(+)] nematodes. The zdIs5 [mec-4p::GFP + lin-15(+)] nematodes can be used to establish a model of degeneration of the mechanosensory neuron of nematode. The nematodes are provided by Professor Zheng Lingjun of Shanghai Jiaotong University, and the nematodes can be prepared by conventional methods. Alternatively, the nematodes can also be replaced by commercially available nematodes, such as the Caenorhabditis Genetics Center (CGC) website.

[0089] As described above in Method 2.1, after the nematodes are synchronized and cultured to the L4 stage, they are transferred to FudR plates coated with E. coli mixed solution containing Rg3 or Rg5 or solvent control. Fluorescence photographs are taken using a body fluorescence microscope or laser confocal at 3 days, 5 days, and 10 days, respectively, to observe the integrity of the neurons. The intact mechanosensory neurons have the characteristics of long strip, smooth and flat without breakpoints. During the aging process, the neurons will have breakpoints and loss, and the aging of the mechanosensory neurons will affect the response of the nematode to touch avoidance and external stimuli.

[0090] Figure 4C The effects of Rg3 or Rg5 on the morphology of ALM and PLM neurons of nematode strain zdIs5 [mec-4p::GFP + lin-15(+)] are shown by fluorescence microscope observation. As Figure 4E and Figure 4C It can be seen that the percentage of ALM, PLM and PVD neurons with poor morphology score increases over time in the presence of solvent without ginsenoside Rg3, indicating that the nematode strain zdIs5 [mec-4p::GFP + lin-15(+)] can simulate the degeneration of the mechanosensory neuron. In contrast, Figure 4E and Figures 4A-4E It can be seen that the percentage of ALM, PLM and PVD neurons with good morphology score increases over time in the presence of ginsenoside Rg3. From Figure 5A It can be seen that Rg3 significantly improves the integrity of the mechanosensory neurons (ALM, PLM and PVD) of nematode, and still maintains high integrity at 5 days and 10 days, with very significant effect. Rg5 can significantly improve the integrity of the mechanosensory neurons of nematode at 5 days.

[0091] According to this example, ginsenoside, particularly ginsenoside Rg3, can significantly improve the degeneration of the mechanosensory neuron and treat or prevent diseases caused by the degeneration of the mechanosensory neuron.

[0092] Example 4: Effect of Ginsenoside Rg3 or Rg5 on GABA neuron integrity

[0093] This example was performed using oxls12 (unc-47::GFP) nematodes. oxls12 (unc-47::GFP) nematodes can be used to establish a model of GABA neuron degeneration in nematodes. The nematodes were provided by Professor Zheng Lingjun of Shanghai Jiaotong University, and the nematodes can be prepared by conventional methods. Alternatively, the nematodes can also be replaced by commercially available nematodes.

[0094] As described above in Method 2.1, after the oxls12 (unc-47::GFP) nematodes were synchronized and cultured to the L4 stage, they were transferred to FudR plates for drug administration. Rg3, Rg5 or solvent control was mixed with E. coli OP50 and used to coat the plates. At the 3rd, 5th, and 10th stages, fluorescence photographs were taken using a body fluorescence microscope or laser confocal as shown in Figure 5A and 5B The integrity of GABA neurons was observed. Complete GABA neurons have short stripes, smooth and flat features, and as the aging process progresses, some will appear broken with spots and even completely broken.

[0095] Figure 5C Fluorescence microscope observation of the effect of Rg3 and Rg5 on the morphology of GABA neurons of the nematode strain oxls12 (unc-47::GFP). Figure 5C Scoring of the effect of Rg3 on GABA neuron morphology.

[0096] As shown in Figure 5A It can be seen that in the presence of solvent without ginsenoside Rg3, the percentage of GABA neurons with poor morphology scores increases over time, indicating that the nematode strain oxls12 (unc-47::GFP) can simulate GABA neuron degeneration. Conversely, in the presence of ginsenoside Rg3, the percentage of GABA neurons with good morphology scores increases over time. As shown in Figure 7 , 5B , 5C, compared with solvent and Rg5, Rg3 has a very obvious effect on maintaining the integrity of nematode GABA neurons (significantly maintaining GABA neuron integrity at Day 5 and Day 10).

[0097] As can be seen from this example, ginsenosides, particularly ginsenoside Rg3, can significantly improve GABA neuron degeneration. It has been reported that learning and memory decline is associated with GABA neuron loss. Therefore, ginsenoside Rg3 can improve memory by improving GABA neuron degeneration. In addition, ginsenoside Rg3 can also treat or prevent diseases or conditions caused by GABA neuron degeneration.

[0098] Example 5: Effects of ginsenoside Rg3 or Rg5 on touch avoidance response and olfactory sensitivity of nematodes

[0099] After the C. elegans strain N2 nematodes were synchronized and cultivated to the L4 stage, they were transferred to FudR plates for administration according to the above method 2.1. The mixture of Rg3, Rg5 or solvent control and E. coli OP50 was used to coat the plates. On the 3rd day, the 5th day and the 10th day, touch response experiments were performed. Nematodes naturally seek benefits and avoid harm. The head and tail of the nematodes were touched with an eyebrow, and the nematodes would flee away from the touched part. This feature was used to study the speed and loss of touch response. The touch response sample n = 15, the head and tail were touched three times respectively, and the reaction was counted as 1, and no reaction was 0.

[0100] After the C. elegans strain N2 nematodes were synchronized and cultivated to the L4 stage, they were administered according to the above method 2.1. Ethanol was used as a control, and the olfactory test was performed using the preference of nematodes for benzaldehyde. Three plates of nematodes were prepared, about 50-80 per plate, and the odor preference experiment was performed on the 3rd day and the 5th day as shown in FIG. A. Figure 6 Specifically, a 1% benzaldehyde ethanol solution was prepared, 10% benzaldehyde solution and ethanol solution were respectively dropped 5 uL at points A and B, and after drying, 5 uL NaN3 solution (500 mM) was added. The purpose was to anesthetize the nematodes. 200-400 nematodes were transferred to the starting point of the test plate (Origin) with a pipette or a glass dropper, and incubated at room temperature for 1 h. The number of nematodes in the benzaldehyde and EtOH circles was counted respectively. Nematodes naturally choose to approach the sample, benzaldehyde is counted as 1, and ethanol control is counted as 2. The Chemotaxis Index (CI) was calculated according to the formula: CI = (n1-n2) / (n1+n2), where n1 is the number of nematodes that tend to benzaldehyde, and n2 is the number of nematodes that tend to ethanol.

[0101] Figure 6 The touch avoidance response score and olfactory tendency index of Rg3 and Rg5 were evaluated. Figure 6 As shown in FIG. A, Rg3 can significantly improve the touch avoidance response of old nematodes (10th day). ​ As shown in FIG. B, Rg3 can significantly improve the olfactory sensitivity of middle-aged nematodes (5th day).

[0102] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement methods and shall be within the scope of protection of the present application.

Claims

1. Use of ginsenoside for the preparation of a composition for the treatment or prevention of age-related deterioration of touch or olfactory sensation, wherein the ginsenoside is ginsenoside Rg3.

2. Use according to claim 1, wherein the composition is in one or more of the following dosage forms: tablet, ointment, granule, capsule, powder or oral liquid.

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