Use of shionone in preparing Anti-frailty product

Abstract

Provided is use of shionone in preparing an anti-frailty product. New use of shionone is developed. Experiments demonstrate that shionone has an anti-frailty effect and can be used for preparing an anti-frailty product. A senescent cell model is established by inducing cells with adriamycin, and experiments demonstrate that shionone has the effects of eliminating senescent cells and promoting the apoptosis of senescent cells, has no influence on normal cells, and can bind to BCL-2 to enable targeted inhibition of BCL-2 activity. Shionone is injected into young mice and senescent mice intraperitoneally, and experiments demonstrate that shionone has the effects of enhancing muscle strength and exercise capacity in the senescent mice. The detection of P16 in the heart, kidney, and lung tissues of the mice demonstrates that shionone can significantly reduce the number of P16-positive senescent cells in these tissues. Safety evaluation indexes in the young mice show that shionone has no significant influence on liver and kidney function and the structures of major organs.

Description

Use of beta-eudesmol in preparing anti-frailty products

[0001] Cross-reference to Related Applications

[0002] This application claims priority to the Chinese patent application No. 202410444797.8, filed on April 12, 2024, and entitled "Use of beta-eudesmol in preparing anti-frailty products", the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present application relates to the use of beta-eudesmol, in particular to the use of beta-eudesmol in preparing anti-frailty products. BACKGROUND

[0004] Frailty is a complex syndrome associated with advanced age, characterized by increased vulnerability of individuals, decreased motor ability, and increased risk of severe illness. Frailty often coexists with chronic diseases associated with advanced age, such as obesity, diabetes, heart disease, cancer, stroke, and musculoskeletal diseases, and can affect the treatment effect of these diseases and increase the mortality rate of the diseases. However, there is a lack of effective intervention measures to improve the symptoms of frailty patients. Frailty and aging have similar pathophysiological changes, especially low-grade chronic inflammation associated with aging. Aging cells are becoming an important source of these inflammatory mediators. Recent studies have shown that the accumulation of aging cells in various tissues is a major factor leading to decreased organ function and body frailty. Cell senescence is a cellular stress response caused by DNA damage due to radiation, chemical agents or accumulation of reactive oxygen species (ROS). With age or long-term illness, increased tissue damage and decreased immune system function can lead to accumulation of senescent cells in the body, upregulation of cell cycle inhibitory molecules P16 and P21 expression and increased activity of anti-apoptotic molecule BCL2 in senescent cells, which are in a stable cell cycle arrest state and long-term survival in the body, while such cells produce senescence-associated secretory phenotype (SASP) factors, forming a harmful inflammatory environment. Removing these senescent cells can improve the pathological changes of various age-related diseases, enhance motor ability, resist frailty, and help to extend healthy life.

[0005] Beta-eudesmol is extracted from the roots and rhizomes of a common Asteraceae plant, Aster tataricus L.f. Traditional medicine believes that it is warm in nature, bitter and pungent in taste, and can moisten the lungs and lower qi, and has the effect of eliminating phlegm and relieving cough, and is often used to treat symptoms such as excessive phlegm and cough, new and prolonged cough, and labor cough with hemoptysis. However, the efficacy of beta-eudesmol in resisting frailty has not been reported.

[0006] SUMMARY

[0007] Therefore, the application provides an application of pinostrobin in preparation of an anti-aging product, solves the problem of lack of anti-aging products in the prior art, and proves that pinostrobin has an anti-aging effect through experiments, and can be used for preparation of an anti-aging product.

[0008] In a first aspect, the application provides an application of pinostrobin in preparation of an anti-aging product.

[0009] In some embodiments, the application of pinostrobin in preparation of an anti-aging product has a structure as follows:

[0010] Optionally, the anti-aging product is an anti-aging and / or physical fitness enhancing product.

[0011] In some embodiments, the anti-aging product is a product for eliminating senescent cells.

[0012] Optionally, the senescent cells include senescent 3T3 fibroblasts, senescent human epidermal cells and / or senescent skin fibroblasts.

[0013] In some embodiments, the anti-aging product is a product for promoting apoptosis of senescent cells.

[0014] Optionally, the senescent cells are 3T3 fibroblasts.

[0015] In some embodiments, the anti-aging product is a product for inhibiting the activity of BCL-2 protein.

[0016] In some embodiments, the anti-aging product is a product for preventing or treating senescence, an age-related disease or cancer caused by an increase in the activity of BCL-2 protein.

[0017] The age-related disease includes heart failure, pulmonary fibrosis and / or cirrhosis.

[0018] The senescence includes skin aging.

[0019] In some embodiments, the anti-aging product is a product for inhibiting the expression of P16 gene.

[0020] In some embodiments, the anti-aging product is a product for preventing or treating senescence or cancer caused by high expression of P16 gene.

[0021] In some embodiments, the anti-aging product is a product for enhancing muscle strength and exercise capacity.

[0022] In some embodiments, the application is applied to a subject including a human and an animal other than a human.

[0023] Optionally, the subject of the application includes the elderly, patients and other elderly animals other than humans.

[0024] The amount of the shionone used in the preparation of the anti-aging product for the elderly animals is 10 mg / Kg body weight, twice a week.

[0025] In some embodiments, the anti-aging product is a food, a skin care product, a cosmetic product or a medicine.

[0026] In some embodiments, when the anti-aging product is a medicine, it further comprises a pharmaceutically acceptable excipient.

[0027] Optionally, the excipient comprises one or more of a solvent, a solubilizer, a co-solvent, an emulsifier, a coloring agent, a binder, a disintegrant, a stabilizer, a flavoring agent, a preservative, a suspending agent, a coating material, an aromatic agent and a thickening agent.

[0028] In some embodiments, the dosage form of the anti-aging product comprises any formulation acceptable in pharmacy.

[0029] Optionally, the dosage form of the anti-aging product is a tablet, a capsule, a powder, a drop, a lyophilizate, a granule, an ointment, a patch, a suspension, a syrup, an oral solution, an injection, a suppository or any combination of forms thereof.

[0030] The present application also provides an anti-aging method, characterized in that it comprises administering shionone to a subject in need, the structural formula of the shionone being as follows:

[0031] In some embodiments, the anti-aging is the removal of senescent cells.

[0032] Optionally, the senescent cells include senescent 3T3 fibroblasts, senescent human epidermal cells and / or senescent skin fibroblasts.

[0033] In some embodiments, the anti-aging is the promotion of apoptosis of senescent cells.

[0034] Optionally, the senescent cells are 3T3 fibroblasts.

[0035] In some embodiments, the anti-aging is the inhibition of BCL-2 protein activity.

[0036] In some embodiments, the anti-aging is the prevention or treatment of senescence, an age-related disease or cancer caused by an increase in BCL-2 protein activity.

[0037] The age-related disease includes heart failure, pulmonary fibrosis and / or liver cirrhosis.

[0038] The aging includes skin aging.

[0039] In some embodiments, the anti-aging is inhibiting P16 gene expression.

[0040] In some embodiments, the anti-aging is preventing or treating aging or cancer caused by high expression of P16 gene.

[0041] In some embodiments, the anti-aging is enhancing muscle strength and exercise capacity.

[0042] In some embodiments, the administration subject includes human and other animals except human.

[0043] In some embodiments, the administration dose is 5-20 mg / Kg body weight, 1-3 times per week.

[0044] In some embodiments, the administration dose is 5 mg / Kg body weight, 6 mg / Kg body weight, 7 mg / Kg body weight, 8 mg / Kg body weight, 9 mg / Kg body weight, 10 mg / Kg body weight, 11 mg / Kg body weight, 12 mg / Kg body weight, 13 mg / Kg body weight, 14 mg / Kg body weight, 15 mg / Kg body weight, 16 mg / Kg body weight, 17 mg / Kg body weight, 18 mg / Kg body weight, 19 mg / Kg body weight, 20 mg / Kg body weight; the administration frequency is once per week, twice per week or three times per week.

[0045] In some alternative embodiments, the administration dose is 10 mg / Kg body weight, twice per week. In some embodiments, when the administration subject is human, the administration dose is 1.1 mg / Kg body weight, twice per week.

[0046] In some embodiments, the administration route can be a systemic route. The following injection or administration routes can be particularly mentioned: intravenous, intrathecal, intraperitoneal, intranasal, subcutaneous, intramuscular, sublingual, oral and combinations thereof.

[0047] Compared with the prior art, the application has the following beneficial effects:

[0048] The application provides a use of camphorin in the preparation of an anti-aging product, and develops a new use of camphorin. The application proves by experiments that camphorin has an anti-aging effect and can be used for preparing an anti-aging product.

[0049] The application establishes a senescent cell model by inducing cells with doxorubicin (DOX), and proves by experiments that camphorin has the effects of clearing senescent cells and promoting apoptosis of senescent cells, and has no effect on normal cells, and camphorin can bind to BCL-2 and target to inhibit the activity of BCL-2.

[0050] The application proves that shionone has the effect of enhancing muscle strength and exercise capacity by intraperitoneal injection of shionone to young mice and aging mice, and proves that shionone can significantly reduce the number of P16 positive aging cells in each tissue by detecting P16 in the heart, kidney and lung tissues of mice.

[0051] The application proves the safety of shionone by intraperitoneal injection of shionone to 3-month-old male mice, and detecting the body weight and liver and kidney function indicators such as alanine aminotransferase, aspartate aminotransferase, creatinine and urea of the mice, and performing pathological analysis on the heart and liver tissues. BRIEF DESCRIPTION OF DRAWINGS

[0052] In order to more clearly illustrate the specific embodiments of the application or the technical solutions in the prior art, the following will briefly introduce the drawings needed to be used in the description of the specific embodiments or the prior art. Obviously, the drawings described below are some embodiments of the application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0053] Figure 1 is the effect of shionone with different concentrations (0, 0.5uM, 1uM, 5uM, 10uM, 20uM, 50uM, 100uM, 500uM) on normal and aging 3T3 fibroblasts in Example 1; Control is normal 3T3 fibroblasts, and Sens is aging 3T3 fibroblasts; wherein the abscissa is concentration, and the ordinate is cellular viability;

[0054] Figure 2A is the effect of shionone with different concentrations (0, 0.5, 1, 2.5, 5, 10, 20, 40uM) on aging human epidermal cells in Example 2; the ordinate is cell number per field;

[0055] Figure 2B is the effect of shionone with different concentrations (0, 0.5, 1, 2.5, 5, 10, 20, 40uM) on human skin fibroblasts in Example 2;

[0056] Figure 3A is the binding of shionone with different concentrations to BCL-2 detected by surface plasmon resonance technology in Example 3; the ordinate is response;

[0057] Figure 3B is a statistical graph of the binding of shionone to BCL-2 in Example 3;

[0058] Figure 3C is the effect of Shionone on BCL-2 activity in Example 3; DMSO is DMSO solution only; ABT-737 is ABT-737 solution (a reported BCL2 inhibitor); Shionone 5 is 5uM Shionone dilution; Shionone 10 is 10uM Shionone dilution;

[0059] Figure 3D is the effect of Shionone treatment on apoptosis of normal 3T3 fibroblast cells and senescent 3T3 fibroblast cells by flow cytometry analysis in Example 3; wherein, PRO 3T3 is normal growing 3T3 fibroblast cells, SEN 3T3 is senescent 3T3 fibroblast cells; Viable cells are normal cells; Apoptosis cells are apoptosis cells of Annexin V + and PI + ;

[0060] Figure 4A is the administration dose, mode, time and sample detection time of old mice in Example 4;

[0061] Figure 4B is the detection results of grip strength, maximum exercise speed and tolerance time on the rotating rod of different mice in Example 4; young is young mice, aged-viechle is the control group of old mice, aged-SHI is the test group of old mice;

[0062] Figure 4C is the expression of P16 in the heart, kidney and lung tissues of different mice in Example 4; young is young mice, aged-viechle is the control group of old mice, aged-SHI is the test group of old mice;

[0063] Figure 5A is the body weight change of two groups of mice in Example 5;

[0064] Figure 5B is the detection results of alanine aminotransferase, aspartate aminotransferase, creatinine and urea of two groups of mice in Example 5;

[0065] Figure 5C is the histopathological analysis results of two groups of mice in Example 5. DETAILED DESCRIPTION

[0066] The following examples are provided to better enable those skilled in the art to further understand the application, and are not intended to limit the content and protection scope of the application, and do not limit the content and protection scope of the application, and any person who obtains any product same or similar to the application under the enlightenment of the application or combines the application with other prior art features falls within the protection scope of the application.

[0067] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the application.

[0068] In this application, the selection range related to "and / or", "or / and", "and / or" includes any one of two or more related listed items, and also includes any and all combinations of related listed items, which includes any two related listed items, any more related listed items, or all related listed items.

[0069] In this application, the terms "preferably", "more preferably", "most preferably", "suitably" only describe the better effect of the embodiments or examples, and it should be understood that it does not constitute a limitation on the scope of protection of the application.

[0070] In this application, the percentage content is used, and if not specified, it refers to the mass percentage for solid-liquid mixing and solid-solid mixing, and it refers to the volume percentage for liquid-liquid mixing.

[0071] In this application, the technical features described in an open manner include both the closed technical solution consisting of the listed features and the open technical solution containing the listed features.

[0072] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the application.

[0073] Frailty: refers to a clinical syndrome observed in the elderly, which has characteristics such as poor physical fitness, rapid onset and progression of disease, and decreased ability to cope with cellular and tissue stress.

[0074] Diseases with a positive correlation between incidence and age, most of which have an inflammatory pathogenesis. Frailty and aging have similar pathophysiological changes. Aging of cells will cause a series of changes in their internal structure and function, and may also affect other cells and microenvironments around them by secreting related substances, leading to a harmful inflammatory environment. Therefore, senescent cells may be one of the driving forces of aging in individuals, and the accumulation of senescent cells in various tissues is a major factor leading to decreased organ function and frailty. More and more studies have shown that removing senescent cells may be one of the most promising interventions for delaying aging and improving frailty.

[0075] In order to explore better anti-frailty products, according to the first aspect of the application, the application provides a use of a shionone in the preparation of an anti-frailty product, wherein the chemical structural formula of the shionone is 30 H 50O, and the specific structure is as follows:

[0076] Optionally, the anti-aging product is an anti-aging and / or fitness-enhancing product.

[0077] In some embodiments, the anti-aging product is a product that removes senescent cells.

[0078] Optionally, the senescent cells include senescent 3T3 fibroblasts, senescent human epidermal cells, and / or senescent skin fibroblasts.

[0079] The present application proves by experiments that Shionone can specifically kill senescent 3T3 fibroblasts at a low concentration, without affecting normal cells. Moreover, 5 uM of Shionone can significantly reduce the number of senescent human epidermal cells and fibroblasts, and has a killing effect on senescent cells.

[0080] In some embodiments, the anti-aging product is a product that promotes apoptosis of senescent cells.

[0081] Optionally, the senescent cells are 3T3 fibroblasts.

[0082] In some embodiments, the anti-aging product is a product that inhibits the activity of BCL-2 protein.

[0083] In some embodiments, the anti-aging product is a product that prevents or treats senescence, an age-related disease, or cancer caused by an increase in the activity of BCL-2 protein.

[0084] The age-related disease includes heart failure, pulmonary fibrosis, and / or cirrhosis.

[0085] B-cell lymphoma-2, BCL-2, can inhibit cell death caused by various cytotoxic factors. Senescent cells are more likely to survive after injury due to the increase in BCL2 expression and activity. A large number of reports have shown that drugs that can inhibit BCL2 activity have the effect of specifically removing senescent cells and improving various physiological indicators of elderly animals. In addition, targeted removal of senescent cells is also a potential intervention direction in other aging-related diseases, such as heart failure, pulmonary fibrosis, non-alcoholic cirrhosis, and cancer.

[0086] The present application proves by experiments that Shionone can bind to BCL-2, significantly inhibit the activity of BCL-2, and thus promote apoptosis, having the dual effects of anti-cancer and anti-aging, without affecting normal cells.

[0087] In some embodiments, the anti-aging product is a product that inhibits the expression of P16 gene.

[0088] In some embodiments, the anti-aging product is a product for preventing or treating aging or cancer caused by high expression of P16 gene.

[0089] p16 gene is directly involved in the regulation of cell cycle, negatively regulates cell proliferation and division, and homozygous deletion is found in 50% of human tumor cell lines, and mutation, which is considered to be a new anticancer gene more important than p53. Some people compare it to the brake device in the cell cycle, which will cause malignant proliferation of cells and lead to the occurrence of malignant tumors if it fails. Cell aging needs to be transmitted by signal pathways, of which the most important two are P16Ink4a / Rb (retinoblastoma protein) pathway and P19Arf / P53 / P21Cip1 pathway. These two pathways interact with each other but independently regulate the progress of cell cycle. The tumor suppressor gene p16 is a key gene for regulating cell aging. Studies have shown that when cells age, the transcription and protein expression level of p16 gene increases, inhibits cell mitosis, stimulates the phosphorylation of RB protein to produce a response, thereby maintaining the irreversible growth arrest state of senescent cells. Inhibiting the transcription and protein expression of p16 gene can inhibit cell aging and also has anticancer efficacy.

[0090] In some embodiments, the anti-aging product is a product for enhancing muscle strength and exercise capacity.

[0091] The present application proves by experiments that the grip strength, maximum exercise speed and tolerance time of old mice on a rotating rod are significantly lower than those of young mice, but the grip strength, maximum exercise speed and tolerance time of old mice treated with shionone are significantly increased

[0092] In some embodiments, the administration subject of the application includes humans and other animals except humans.

[0093] Optionally, the administration subject of the application includes the elderly, patients and other elderly animals except humans.

[0094] In some embodiments, the amount of shionone used for preparing an animal anti-aging product is 10 mg / Kg body weight, twice a week.

[0095] In some embodiments, the anti-aging product is food, skin care product, cosmetic or drug.

[0096] In some embodiments, when the anti-aging product is a drug, it further includes a pharmaceutically acceptable excipient.

[0097] Optionally, the adjuvant includes one or more of a solvent, a solubilizing agent, a co-solvent, an emulsifier, a coloring agent, a binding agent, a disintegrating agent, a stabilizing agent, a flavoring agent, a preservative, a suspending agent, a coating material, an aromatic agent, and a thickening agent.

[0098] In some embodiments, the dosage form of the anti-aging product includes any formulation pharmaceutically acceptable;

[0099] Optionally, the dosage form of the anti-aging product is a tablet, a capsule, a powder, a drop, a lyophilizate, a granule, an ointment, a patch, a suspension, a syrup, an oral solution, an injection, a suppository, or any combination of forms thereof.

[0100] The present application is further described in detail by the following specific examples, which should not be interpreted as limiting the scope of the present application.

[0101] The specific experimental procedures or conditions not mentioned in the examples can be performed according to the conventional experimental procedures or conditions described in the literature in the art. The reagents or instruments used are not specified by the manufacturer, and are all conventional reagent products that can be obtained by purchase.

[0102] 1. The following drugs and reagents were used in the following tests:

[0103] Shionone was purchased from MedChemExpress with the product number HY-N0829. The chemical structure is C30H50O, and the specific chemical structure is shown below.

[0104] 2. Cells

[0105] 3T3 mouse embryonic fibroblast cell line, HaCat human epidermal cell line, and BJ human skin fibroblast cell line were purchased from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences.

[0106] Passage culture was performed using complete medium of DMEM high-sugar medium + 10% fetal bovine serum + 1% double antibody.

[0107] 3. The following is the method for establishing the senescent cell model in each example:

[0108] Cells were seeded in the culture wells of 96-well plates, cultured with 100ul of complete culture medium (DMEM high-sugar culture medium + 10% fetal bovine serum + 1% double-antibiotic), and when the density reached about 70%, 1ul of 1mol / L doxorubicin (DOX, final concentration 10nmol / L) was added to each culture well to stimulate cells for 48 hours, then all the culture medium was removed, 100ul of new culture medium (DMEM high-sugar culture medium + 10% fetal bovine serum + 1% double-antibiotic complete culture medium) was added, and then placed in the incubator for 72 hours to induce cell aging.

[0109] 4. Test animals:

[0110] 3-month-old and 20-month-old C57bl6 / J male mice were purchased from Sirebioscience Co., Ltd. and bred and reproduced in the Experimental Animal Center of Beijing Anzhen Hospital, Capital Medical University, at a constant temperature of (22±2)℃, with 12 hours of light per day, and free access to food and water. All experimental procedures comply with the relevant laws and regulations of national experimental animal management and are approved by the Ethics Committee of Beijing Anzhen Hospital, Capital Medical University.

[0111] Example 1

[0112] 1. Purpose of the experiment

[0113] To investigate the effect of Shionone on 3T3 fibroblasts of aging mice.

[0114] 2. Test instruments: CCK8 kit (for detecting cell viability) purchased from Beijing Solabio Technology Co., Ltd., item number CA1210.

[0115] 3. Test method

[0116] 3T3 mouse embryonic fibroblasts were seeded in the culture wells of 96-well plates at a density of 5×10 3 cells per well, randomly divided into two groups, the model group and the control group (control), the model group (Sens) was induced by DOX to establish an aging cell model. The control group was normal growing mouse 3T3 fibroblasts (control). Blank wells (no cells, no drugs, only culture medium) were also set up.

[0117] After removing the culture medium of normal growing mouse 3T3 fibroblasts and aging mouse 3T3 fibroblasts, equal amounts of 100uL of different concentrations (0, 0.5uM, 1uM, 5uM, 10uM, 20uM, 50uM, 100uM, 500uM) of Shionone diluent were added (4 replicates for each dose).

[0118] Preparation of Shionone dilution: Shionone powder was dissolved in ethanol to a concentration of 5 mmol / L. Then the medium was used to dilute to the required working concentration according to the required concentration.

[0119] After the cells were treated with Shionone diluent for 3 days, 10 uL of CCK8 solution was added to the cell culture hole of the 96-well plate, and then incubated in the cell incubator for 2 hours. The absorbance at 450 nm was detected using a microplate reader. The cell viability in each culture hole was calculated using the following formula.

[0120] Cell viability (%) = [A (with drug) - A (blank)] / [A (0 with drug) - A (blank)] x 100;

[0121] A (with drug): OD value of the hole with cells, CCK-8 solution and drug solution;

[0122] A (0 with drug): OD value of the hole with cells, CCK-8 solution and no drug solution;

[0123] A (blank): OD value of the hole without cells.

[0124] 4. Test results

[0125] The test results are shown in Figure 1. The results of calculating the IC50 value of the drug show that the IC50 value of shionone for senescent cells is 5.152 uM, which is much lower than the IC50 value for normal cells (400 uM). The results show that Shionone can specifically kill senescent mouse fibroblasts (Senc) at low concentrations, and has no effect on normal cells (control).

[0126] Example 2

[0127] 1. Purpose of the experiment

[0128] To investigate the effect of Shionone on senescent human epidermal cells (HaCat cell line) and skin fibroblasts (BJ cell line).

[0129] 2. Test method

[0130] The specific steps are as follows:

[0131] (1) Human epidermal cells (HaCat cell line) and skin fibroblasts (BJ cell line) were inoculated in 96-well plates at a density of 5 x 10 3 cells per well, and DOX was used to induce cell aging to establish a senescent cell model.

[0132] (2) After 3 days of incubation, the residual cell number was analyzed after adding equal amount of 100 uL of Shionone dilution solution with different concentrations (0, 0.5 uM, 1 uM, 2.5 uM, 5 uM, 10 uM, 20 uM, 40 uM) respectively.

[0133] (3) Preparation of Shionone dilution solution (same as Example 1): Shionone powder was dissolved in ethanol to a concentration of 5 mmol / L. Then the culture medium was used to dilute to the required working solution concentration according to the required concentration.

[0134] 3. Test results

[0135] The test results are shown in Figure 2, and the results show that Shionone dilution solution of 5 uM can significantly reduce the number of senescent human epidermal cells (A) and fibroblasts (B), and has a killing effect on senescent cells.

[0136] Example 3

[0137] 1. Purpose of the experiment

[0138] To investigate the effect of Shionone on BCL-2 protein.

[0139] Test 1, Surface Plasmon Resonance (SPR) detection

[0140] Test instrument: The surface plasmon resonance detection instrument is Biacore T200, and the detection chip is S series CM5 chip (29149603).

[0141] Test method:

[0142] The specific steps are as follows:

[0143] (1) Running buffer configuration: The buffer used in the experiment is PBS-P + .

[0144] (2) Ligand capture: Dilute BCL-2 protein to 20 ug / mL with sodium acetate at pH 4.0, activate the chip with EDC (1-(3-dimethylaminopropyl)-3-ethyl carbodiimide) and NHS (N-hydroxysuccinimide) mixture (volume ratio 1:1) on the experimental channel Fc2 for 420 s, immobilize about 15000 RU, then flow EA through the chip surface to block the excess sites. The reference channel Fc1 was activated and then directly blocked.

[0145] (3) Analyte injection: Dilute small molecule Shionone to 1.5625 μΜ, 3.125 μΜ, 6.25 μΜ, 12.5 μΜ, 25 μΜ respectively using Running buffer, and then perform 2-fold gradient dilution in sequence. The analyte flows through the experimental channel and the reference channel at the same time, the binding time is 60 s, the flow rate is 50 μL / min, and the dissociation time is 60 s.

[0146] (4) Regeneration: The regeneration buffer is PBS-P+, and the regeneration time is 30 s, and the flow rate is 30 μL / min.

[0147] Test results:

[0148] The results are shown in FIGS. 3A and 3B. As the concentration increases, the binding signal of Shionone and BCL-2 is enhanced, and the calculated KD=4.529 x 10 -6 M.

[0149] Test two BCL-2 activity detection

[0150] Test instrument: Use BCL-2TR-FRET Assay Kit (item number 50222) of BPS Bioscience Company to detect the inhibition of Shionone on the binding of BCL-2 and peptide ligand according to the operation instruction, and use PerkinElmer EnSpire multifunctional enzyme label instrument to detect the fluorescence intensity.

[0151] Test method:

[0152] The specific steps are as follows:

[0153] (1) Reagent preparation: Dilute BCL TR-FRET assay buffer with ultrapure water at a ratio of 1:3. Dilute Anti-His terbium-labeled donors and dye-labeled acceptors with the diluted BCL TR-FRET assay buffer diluent at a volume ratio of 1:100. Dilute BCL-2 polypeptide ligand with BCL TR-FRET assay buffer diluent at a ratio of 1:40. Dilute BCL-2 protein to a working concentration of 1 ng / μl with BCL TR-FRET assay buffer diluent.

[0154] Dissolve Shionone powder to a concentration of 5 mmol / L using an ethanol solution, then further dilute Shionone to 5 uM and 10 uM using PBS, and then prepare the test substance. Use PBS solution as a blank control, and use ABT-737 solution provided in the kit as a negative control.

[0155] (2) Prepare the reaction system: Add 5 μl of diluted donors, 5 μl of acceptors, 2 μl of the molecule to be detected, 5 μl of the peptide ligand and 3 μl of BCL-2 protein to each well of a white flat-bottom 384-well plate and mix well.

[0156] (3) After reacting at room temperature for 3 hours, the fluorescence intensity was detected using a microplate reader. The detection wavelength for TR was 665 nm, and the detection wavelength for FRET was 620 nm.

[0157] (4) Calculate the TR-FRET ratio (665 nm / 620 nm) of all reactions, taking the negative control as 1, and calculate the relative activity percentage.

[0158] Test results:

[0159] Both 5uM and 10uM Shionone dilutions significantly inhibited BCL-2 activity, similar to the inhibitory effect of the reported inhibitor ABT-737. The effects of 1uM ABT-737 and 5uM and 10uM Shionone dilutions on BCL-2 are shown in Figure 3C. In the figure, DMSO refers to the use of only DMSO solution as the solution of the molecule to be detected; ABT-737 refers to the use of 1uM ABT-737 solution (a reported BCL2 inhibitor) as the solution of the molecule to be detected; Shionone 5 refers to the use of 5uM Shionone dilution as the solution of the molecule to be detected; and Shionone 10 refers to the use of 10uM Shionone dilution as the solution of the molecule to be detected.

[0160] Experiment 3: Detection of cell apoptosis using Annexin V-PI flow cytometry

[0161] Test instruments: BD Bioscience's FITC Annexin V apoptosis detection kit (Cat. No. 556547) was used to detect cell apoptosis using a BD Fortessa flow cytometer.

[0162] Test method:

[0163] The specific steps are as follows:

[0164] 1) 3T3 cells were seeded in 12-well plates and divided into two groups, one group to establish a senescent cell model; the other group to be normal cells. The medium was used to dilute the dissolved 5 mmol / L Shionone solution to 10 mol / L Shionone diluent, and the medium of normal cells and senescent 3T3 fibroblasts was replaced with 10 mol / L Shionone diluent. After 48 hours of culture, all cells were digested using 0.25% trypsin, centrifuged at 1500 rpm for 5 minutes, and the supernatant was removed.

[0165] 2) After diluting the 10x binding buffer in the kit with deionized water to 1x binding buffer, 100ul of 1x binding buffer was used to resuspend the cells.

[0166] 3) 5ul of FITC Annexin V and 5ul of PI reagent were added to each sample, and incubated at room temperature for 15 minutes in the dark.

[0167] 4) After adding 400ul of 1x binding buffer to each sample to terminate staining, the flow cytometer was used to detect within 1 hour.

[0168] The results showed that 10uM Shionone diluent treatment had no effect on normal 3T3 fibroblasts, but significantly increased the proportion of apoptotic senescent fibroblasts, as shown in Figure 3D, where Viable cells are normal 3T3 fibroblasts; Apoptosis cells are senescent 3T3 fibroblasts.

[0169] Example 4

[0170] Test instruments: KW-ZL mouse grip strength tester to detect the grip strength of the forelimbs of mice; Nanodrop 2000 spectrophotometer to measure extracted RNA; KW-6C mouse rotarod fatigue instrument to detect mouse exercise endurance.

[0171] Preparation of Shionone diluent: Shionone was dissolved using a mixture of 10 vol% ethanol + 90 vol% sulfobutyl-β-cyclodextrin (SBE-β-CD) salt solution to form a Shionone diluent; the SBE-β-CD salt solution is a 20% SBE-β-CD physiological saline solution.

[0172] Method of administration: 4 young mice of 3 months old; 10 old mice of 22 weeks old, the old mice were randomly divided into two groups, the first group was the test group (5), according to the body weight, the dissolved Shionone diluent was given intraperitoneally at a dose of 10 mg / Kg, once every 3 days, the second group was the control group (5), the same volume of 20% SBE-β-CD saline solution was given, and the continuous administration lasted for four weeks as shown in Figure 4A.

[0173] Main detection method:

[0174] 1. The grip strength of the forelimbs of the mice was detected using a KW-ZL type mouse grip strength tester, and the detection started after 4 weeks of administration. Each mouse was measured continuously 8 times a day, and the average value of the grip strength of two days was taken.

[0175] 2. The exercise tolerance of the mice was detected using a KW-6C mouse rotarod fatigue instrument. The detection started after 4 weeks of administration, and each mouse was detected continuously twice a day, and the average value of the maximum exercise speed and tolerance time obtained in two days was taken.

[0176] 3. Mouse tissue RNA extraction method:

[0177] (1) The heart, kidney and lung tissues of the size of a soybean were cut into small pieces and placed in an RNAase-free 1.5ml centrifuge tube, 2 RNAase-inactivated beads and 1mL TRIzol reagent were added, and the homogenizer was used for shock digestion for 2-3 times, and the homogenization temperature was kept below 10℃;

[0178] (2) The homogenized tissue suspension (as long as no obvious tissue blocks are observed) was taken, 200μL of chloroform was added to each tube, the tube was tightly capped and shaken vigorously for about 10 seconds, then placed on ice for stratification and repeated shaking for 2-3 times;

[0179] (3) Put into a 4℃ centrifuge, centrifuge at 12000rpm for 15 minutes, and then transfer the upper transparent liquid to a new 1.5mL centrifuge tube;

[0180] (4) Add about 800μL of isopropyl alcohol to each tube, mix well, and then place in a -20℃ refrigerator overnight to promote the precipitation of RNA; the next day, place in a 4℃ centrifuge, centrifuge at 12000rpm for 15 minutes;

[0181] (5) At the same time, 75% ethanol was prepared using anhydrous ethanol and RNase-free H2O, after centrifugation and discarding the supernatant, 1mL of 75% ethanol was added to each tube, and then placed in a 4℃ centrifuge, centrifuged at 12000rpm for 10 minutes; repeat once;

[0182] (6)Discard the supernatant and centrifuge again, use 200 μL pipette to suck the residual alcohol, blow dry the residual liquid in the clean bench, and then add 20-30 μL RNase-free H2O to dissolve the RNA to obtain the RNA solution;

[0183] (7) The quality and concentration of the extracted RNA were measured using a Nanodrop 2000 spectrophotometer.

[0184] 4. Reverse transcription of RNA to cDNA

[0185] (1) Calculate the volume of 5ug RNA according to the concentration, and then perform reverse transcription. Perform the operation according to the instructions of the reverse transcription kit (Promega);

[0186] (2) On ice, mix 1 μL Random Primer and 5 μg mRNA (calculate the volume of RNA needed according to the RNA concentration detection result), and use RNase-free H2O to make up the total volume to 10 μL;

[0187] (3) After mixing and centrifuging, use a PCR instrument to perform the first step of reverse transcription, and follow the following procedure: ① 72℃, 10 minutes; ② 0℃, 10 minutes; mix the working solution of various reagents in the kit according to the instructions, and after the first step is completed, divide 10 μL of the mixed working solution into each sample tube, mix well and centrifuge, and the final volume of each tube is 20 μL;

[0188] (4) Use a PCR instrument to perform the second step of reverse transcription, and the reaction conditions are: ① 25℃, 5 minutes; ② 42℃, 60 minutes; ③ 75℃, 15 minutes; ④ 4℃, forever.

[0189] 5. Real-time PCR method for detecting the expression of various genes in aortic tissue:

[0190] Use 2xSYBR Green II (Takara) kit and prepare the reaction system on ice according to the instructions;

[0191] Use CFX Real-time quantitative PCR instrument (Bio-Rad) to perform real-time fluorescent quantitative PCR reaction, reaction conditions: ① 95℃, 3 minutes; ② 95℃, 15 seconds; ③ 60℃, 45 seconds; 45 cycles. Take GAPDH as the internal reference, and the expression level of the gene is calculated by 2-Ct method using the corresponding Ct value obtained.

[0192] The primer sequences used in the experiment are as follows:

[0193] Cdkn2a (P16) upstream: 5'-CGCAGGTTCTTGGTCACTGT-3';

[0194] Cdkn2a (P16) upstream: 5'-TGTTCACGAAAGCCAGAGCG-3';

[0195] Gapdh upstream: 5'-AGGTCGGTGTGAACGGATTTG-3';

[0196] Gapdh downstream: 5'-TGTAGACCATGTAGTTGAGGTCA-3';

[0197] Detection results:

[0198] At the 5th week, the exercise capacity was evaluated, and the grip strength, maximum exercise speed and tolerance time of the old mice on the rotating rod were detected. The results are shown in Figure 4B. The grip strength, maximum exercise speed and tolerance time of the old mice on the rotating rod were significantly decreased compared with the young mice, but the grip strength, maximum exercise speed and tolerance time of the old mice treated with shionone were significantly increased.

[0199] The expression of the aging marker molecule P16 in the heart, kidney and lung tissues of the old mice was significantly increased compared with the young mice, but the expression level of the aging marker molecule P16 in each tissue was significantly reduced by shionone treatment. The results are shown in Figure 4C (young for young mice, aged-viechle for old mice control group, aged-SHI for old mice test group).

[0200] The above results show that the treatment of shionone in old mice can improve exercise capacity, reduce the expression of P16 gene in each organ, and reduce the number of P16 positive aging cells.

[0201] Example 5

[0202] Purpose of the test:

[0203] To investigate the effect of shionone on body weight, liver and kidney function, and the structure of major organs.

[0204] Test method: 8 three-month-old male mice were randomly divided into two groups. One group was the shionone group, and the other group was the control group. The shionone group was given intraperitoneal injection of the shionone diluent dissolved according to the method of Example 4 at a dose of 10 mg / Kg once every 3 days, and the control group was given the same volume of 20% SBE-β-CD physiological saline solution. The body weight was measured every week, and the serum was collected after 4 weeks for detection of serum liver and kidney function indicators and histopathological analysis.

[0205] Test method and results:

[0206] 1. Body weight detection results: There was no significant difference in body weight between the two groups of mice during the 4-week administration period, and both groups increased normally. The results are shown in Figure 5A.

[0207] 2. Serum liver and kidney function index detection results: After 4 weeks, the serum was collected, and the alanine aminotransferase, aspartate aminotransferase, creatinine, urea, and other liver and kidney function indexes were detected using the Mindray BS200 automatic biochemical analyzer.

[0208] The results showed that there was no significant difference in the detection values of alanine aminotransferase, aspartate aminotransferase, creatinine, and urea between the two groups of mice. The results are shown in Figure 5B.

[0209] 3. Histopathological analysis results:

[0210] After the collected heart, liver, lung, and kidney tissues were prepared into paraffin sections, HE staining was performed to observe the tissue structure, and Sirius red staining was performed to observe whether there was residual repair collagen deposition after tissue damage.

[0211] HE staining was performed according to the steps of the HE staining kit (company: Solarbio; kit number G1120) instructions, as follows:

[0212] 1) The sections were deparaffinized in xylene for 2 times, each for 5-10 min. Gradient ethanol (100%, 95%, 85%, 75%) was used for rehydration, each for 3 min. Distilled water was used for soaking for 2 min.

[0213] 2) Stain with hematoxylin staining solution for 5 min, and wash off the excess color with distilled water.

[0214] 3) Differentiate for 10-60 s, and add tap water dropwise or soak for 2 times, each for 3-5 min.

[0215] 4) Place in the eosin staining solution for 30 s-2 min, and quickly dehydrate after pouring off the excess staining solution.

[0216] 5) Start with 75% gradient ethanol dehydration, xylene transparency, and neutral gum sealing.

[0217] Sirius red staining was performed according to the steps of the modified Sirius red staining kit (company: Solarbio; kit number G1472) instructions, as follows:

[0218] 1) The sections were deparaffinized in xylene and gradient ethanol to water.

[0219] 2) Add hematoxylin staining solution dropwise and stain for 5-10 min, and wash off the excess staining solution with distilled water for 10-20 s.

[0220] 3) Soak in tap water for 5-10 min, and wash with distilled water for 3 times, each for 5-10 s.

[0221] 4) Stain the sections with Sirius red for 15-30 min, and rinse with running water to remove the surface dye.

[0222] 5) Dehydrate in graded ethanol, clear in xylene, and mount in neutral gum.

[0223] Test results:

[0224] The results show that there is no obvious collagen deposition in the heart and liver tissues, and there is no obvious abnormality in the alveolar structure of the lung and the glomerular structure of the kidney (C), as shown in FIG. 5C.

[0225] The above tests show that shionone has no damage to organs after 4 weeks of administration, and has a certain safety.

[0226] In summary, the present application establishes a senescent cell model by doxorubicin (DOX) induction, and tests prove that shionone has the effect of specifically killing senescent cells and promoting the apoptosis of senescent cells, and has no effect on normal cells. It is further confirmed that shionone can bind to the anti-apoptotic molecule BCL-2, target and inhibit the activity of BCL-2, thereby promoting the apoptosis of senescent cells.

[0227] The present application intraperitoneally injects shionone into young mice and senescent mice, and tests prove that shionone has the effect of significantly improving the muscle strength and exercise capacity originally decreased in senescent mice. At the same time, by detecting P16 in the heart, kidney and lung tissues of mice, it is proved that shionone can significantly reduce the expression level of P16 in various tissues of senescent mice, and also indicates that shionone can remove the senescent cells accumulated in the body of senescent mice.

[0228] The present application intraperitoneally injects shionone into 3-month-old male mice, and detects the body weight and liver and kidney function indexes such as glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase, creatinine and urea, and performs pathological analysis on the heart, liver, lung and kidney tissues, thereby proving the safety of shionone.

[0229] Obviously, the above examples are only examples for clearly illustrating, but not limitation to the embodiments. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, it is not necessary and impossible to exhaust all the embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. The application of asterone in the preparation of anti-debilitating products, characterized in that: The structural formula of the asterone is as follows:

2. The use according to claim 1, characterized in that The anti-aging product is a product that removes senescent cells; Preferably, the senescent cells include senescent 3T3 fibroblasts, senescent human epidermal cells and / or senescent skin fibroblasts.

3. The use according to claim 1, characterized in that The anti-aging product is a product that promotes apoptosis of aging cells; Preferably, the senescent cells are 3T3 fibroblasts.

4. The use according to claim 1, characterized in that The anti-asthenia product is a product that inhibits the activity of BCL-2 protein.

5. The use according to claim 4, characterized in that The anti-frailty product is a product that prevents or treats aging, age-related diseases or cancer caused by increased BCL-2 protein activity; The age-related diseases include heart failure, pulmonary fibrosis and / or liver cirrhosis; The aging includes skin aging.

6. The use according to claim 1, characterized in that The anti-frailty product is a product that inhibits the expression of the P16 gene.

7. The use according to claim 6, characterized in that The anti-aging product is a product for preventing or treating aging or cancer caused by high expression of the P16 gene.

8. The use according to claim 1, characterized in that The anti-frailty product is a product that enhances muscle strength and athletic ability.

9. The use according to any one of claims 1 to 8, characterized in that: The subjects of application include humans and animals other than humans.

10. The use according to any one of claims 1 to 9, characterized in that: The anti-aging product is food, skin care product, cosmetic or medicine.

11. An anti-frailty method, characterized in that: The method comprises administering asterone to a subject in need thereof, wherein the structural formula of the asterone is as follows:

12. The method according to claim 11, characterized in that The anti-aging method is to remove senescent cells; Preferably, the senescent cells include senescent 3T3 fibroblasts, senescent human epidermal cells and / or senescent skin fibroblasts.

13. The method according to claim 11, characterized in that The anti-aging method is to promote the apoptosis of senescent cells; Preferably, the senescent cells are 3T3 fibroblasts.

14. The method according to claim 11, characterized in that The anti-asthenia effect is to inhibit the activity of BCL-2 protein.

15. The method according to claim 14, characterized in that The anti-frailty is to prevent or treat aging, age-related diseases or cancer caused by increased BCL-2 protein activity; The age-related diseases include heart failure, pulmonary fibrosis and / or liver cirrhosis; The aging includes skin aging.

16. The method according to claim 11, characterized in that The anti-asthenia effect is to inhibit the expression of P16 gene.

17. The method according to claim 16, characterized in that The anti-frailty method is to prevent or treat aging or cancer caused by high expression of P16 gene.

18. The method according to claim 11, characterized in that The anti-asthenia effect is to enhance muscle strength and exercise ability.

19. The method according to any one of claims 11 to 18, characterized in that: The subjects of administration include humans and animals other than humans.

20. The anti-frailty method according to claim 11, characterized in that: The dosage is 5-20 mg / kg body weight, 1-3 times per week.

21. The anti-frailty method according to claim 20, characterized in that: The dosage was 10 mg / kg body weight, twice a week.

22. The anti-frailty method according to claim 11, characterized in that: When the subject is a human, the dosage is 1.1 mg / kg body weight, twice a week.

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