Stellaria yunnanensis extract as well as preparation method and application thereof

Abstract

The invention provides a stellaria yunnanensis extract as well as a preparation method and application thereof. The invention relates to the field of medicine, in particular to application of a stellaria yunnanensis Franch extract in preparation of a medicine composition for delaying senescence or preventing or treating senescence-related diseases, and particularly provides application of the stellaria yunnanensis Franch extract in preparation of the medicine composition for delaying senescence or preventing or treating senescence-related diseases.

Description

Technical Field

[0001] The present invention relates to the field of medicine and health care, and in particular to a Herba Lycopodii extract having an anti-aging effect, and a preparation method and application thereof. Background Art

[0002] Aging is a biological process common to all organisms. It is affected by environmental, nutritional and genetic factors and is a major risk factor for many chronic degenerative diseases and cancer. Anti-aging drugs are a technology that treats specific diseases by slowing down or reversing the basic aging process. In recent decades, researchers have used anti-aging drugs to delay aging and improve the health of the body. They are expected to be used to treat cancer, heart disease and other aging-related diseases.

[0003] Renal failure is a clinical syndrome accompanied by severe renal impairment. Its global incidence continues to rise and has become a public health problem that needs to be addressed urgently. The main treatments currently include dialysis and kidney transplantation, but they have limitations such as high cost and many postoperative complications, and cannot fully restore renal function. Therefore, it is of great significance to develop more effective anti-renal failure drugs.

[0004] Based on the screening of Chinese herbal medicine library, safe and reliable Chinese herbal medicines with good anti-aging activity are found. Further research and development of active component groups have broad prospects. The development of anti-aging drugs based on Chinese herbal medicines has great development potential. Most of the anti-aging drugs reported in recent years are chemical drugs, and anti-aging drugs developed based on Chinese herbal medicines are rare. And at present, there is still a lack of safe and reliable anti-aging drugs with good anti-aging activity in this field.

[0005] Therefore, there is a need in the art to develop new safe and effective anti-aging drugs for delaying aging or preventing or treating aging-related diseases. Summary of the invention

[0006] The purpose of the present invention is to provide a safe and effective anti-aging drug, and more specifically to provide a Herba Lycopodii extract with anti-aging effects, a preparation method thereof, and an application thereof as an anti-aging active ingredient.

[0007] In a first aspect, the present invention provides a use of a Herba Lycopodii Herba extract in preparing a pharmaceutical composition for delaying aging or preventing or treating aging or frailty-related diseases.

[0008] In another preferred embodiment, the extract is selected from the group consisting of: aqueous extract of Stellaria yunnanensis Franch., organic solvent extract of Stellaria yunnanensis Franch., water-organic mixed solvent extract of Stellaria yunnanensis Franch., or active components isolated from the extract of Stellaria yunnanensis Franch. (such as Stellaria yunnanensis Franch. polysaccharide, Stellaria yunnanensis Franch. alkaloid, Stellaria yunnanensis Franch. steroid compound, Stellaria yunnanensis Franch. phenolic acid compound, Stellaria yunnanensis Franch. flavonoid compound, Stellaria yunnanensis Franch. polypeptide compound, or a combination thereof) or a single component (phenols, cyclic peptides, steroids, indole alkaloids, flavonoid glycosides, phenyl ethanol glycosides, or a combination thereof), or a combination thereof.

[0009] In another preferred embodiment, the organic solvent is C1-C4 alkanol, ethyl acetate, acetone, ethane, or a combination thereof. Preferably, the organic solvent is C1-C4 alkanol, and is preferably selected from the group consisting of: methanol, ethanol, n-propanol, isopropanol, n-butanol, or a combination thereof. More preferably, it is ethanol.

[0010] In another preferred embodiment, the extract is a 0-100 v / v% alcohol-water extract of Stellaria yunnanensis Franch.

[0011] In another preferred embodiment, the extract is a 50-100 v / v% alcohol-water extract of Stellaria yunnanensis Franch. Preferably, the extract is a 60-90 v / v% alcohol-water extract of Stellaria yunnanensis Franch., more preferably a 65-85 v / v% alcohol-water extract or a 70-80 v / v% alcohol-water extract, and even more preferably a 75±2 v / v% alcohol-water extract.

[0012] In another preferred embodiment, the anti-aging includes one or more selected from the following group:

[0013] (i) improving the health level of cells and organisms, or improving healthy physiological parameters;

[0014] (ii) improving or enhancing the ability of locomotion (such as the ability of body sway);

[0015] (iii) enhancing the stress resistance ability;

[0016] (iv) delaying or reversing the aging of normal somatic cells;

[0017] (v) reducing the expression level of SA-β-Gal;

[0018] (vi) extending the lifespan.

[0019] In another preferred embodiment, the aging and / or frailty-related diseases are renal failure, heart failure, neurodegenerative diseases, musculoskeletal diseases, etc.

[0020] In another preferred embodiment, the above-mentioned renal failure includes chronic renal failure, renal fibrosis or acute renal failure.

[0021] In another preferred embodiment, preventing or treating renal failure includes improving one or more of the following indicators selected from the group consisting of:

[0022] (i) Reducing the serum level of blood urea nitrogen (BUN);

[0023] (ii) Reducing the serum level of creatinine (Cr);

[0024] (iii) Reducing the serum level of kidney injury molecule-1 (KIM-1);

[0025] (iv) Reducing the serum level of neutrophil gelatinase-associated lipocalin (NGAL);

[0026] (v) Reducing the serum level of the inflammatory factor interleukin-6 (IL-6);

[0027] (vi) Reducing the serum level of tumor necrosis factor α (TNF-α);

[0028] (vii) Reducing senescent kidney cells;

[0029] (viii) Reducing renal fibrosis;

[0030] (ix) Reducing the number of Ki67-positive cells in the kidney.

[0031] In a second aspect of the present invention, there is provided an extract of Silene fortunei, wherein the extract is a 0-100 v / v% alcohol-water extract of Silene fortunei.

[0032] In another preferred embodiment, the extract is a 50-100 v / v% alcohol-water extract of Silene fortunei. Preferably, the extract is a 60-90 v / v% alcohol-water extract of Silene fortunei, more preferably a 65-85 v / v% alcohol-water extract or a 70-80 v / v% alcohol-water extract, and even more preferably a 75±2 v / v% alcohol-water extract.

[0033] In another preferred embodiment, the alcohol is a C1-C4 alkanol. Preferably, the alcohol is selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, n-butanol, or a combination thereof.

[0034] In another preferred embodiment, the Silene fortunei can be the whole plant, above-ground part, underground part, root, stem, leaf, flower of Silene fortunei, or a combination thereof, and is preferably the root.

[0035] In another preferred embodiment, the extract is prepared by the following method:

[0036] Silene fortunei is heated under reflux in 0-100 v / v% alcohol-water for 1-10 times, the extraction solutions are combined, and the solvent is removed to obtain an extract paste or a dried extract.

[0037] In another preferred embodiment, the extraction comprises one or more of the following features:

[0038] (a) The temperature of the extraction is 70 - 120 °C, preferably 80 - 100 °C, more preferably 90 - 100 °C;

[0039] (b) The number of extractions is 2 - 10 times, preferably 2 - 3 times;

[0040] (c) The time of each extraction is 1 - 4 h / time, preferably 2 - 3 h / time;

[0041] (d) The mass - volume ratio of Silene gallica L. to alcohol - water is 1 g: 1 - 20 mL, preferably 1 g: 2 - 10 mL.

[0042] In another preferred embodiment, the extract contains β - ecdysterone.

[0043] In another preferred embodiment, the extract can be further separated. Preferably, it includes the steps of:

[0044] After adsorbing the above 50 - 100 v / v% alcohol - water extract with macroporous adsorption resin (preferably non - polar macroporous resin, such as D101 resin), eluting with water, 30% ethanol - water, 40% ethanol - water and 100% ethanol - water respectively, the obtained fractions can be used as the active ingredients of the present invention or a combination of 2, 3 or 4 of them can be used as the active ingredients of the present invention.

[0045] In another preferred embodiment, the extract contains β - ecdysterone. Preferably, the content of β - ecdysterone > 0.01 wt%, preferably > 0.02 wt%, > 0.05 wt%, > 0.1 wt%, > 0.2 wt% or > 0.5 wt%, more preferably 0.01 - 10 wt%, preferably 0.1 - 5 wt%, still more preferably 0.3 - 0.8 wt%, such as 0.2 wt%, 0.2 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.8 wt% or 1 wt%, calculated based on the dry weight of the extract.

[0046] In another preferred embodiment, the extract contains steroidones, cyclic peptides and phenols. Preferably, the sum of the contents of these substances ≥ 0.1 wt%, calculated based on the dry weight of the extract; more preferably ≥ 0.2 wt%, ≥ 0.3 wt%, ≥ 0.4 wt% or ≥ 0.5 wt%; still more preferably, the sum of the contents of these substances is 0.1 - 20 wt%, such as 0.2 - 10 wt%, 0.3 - 5 wt% or 0.3 - 1 wt%, calculated based on the dry weight of the extract.

[0047] In another preferred embodiment, the extract contains sterones, cyclic peptides, phenols, indole alkaloids, flavonoid glycosides and phenyl ethanol glycosides. Preferably, the sum of the contents of the above substances ≥ 0.1 wt%, calculated based on the dry weight of the extract; more preferably ≥ 1 wt%, ≥ 2 wt%, ≥ 3 wt% or ≥ 4 wt%; more preferably still, the sum of the contents of the above substances is 0.1 - 20 wt%, such as 0.5 - 10 wt%, 1 - 6 wt% or 2 - 5 wt%, calculated based on the dry weight of the extract.

[0048] In the third aspect of the present invention, there is provided the use of the extract of Silene fortunei Vis. in the preparation of a pharmaceutical composition for preventing or treating renal failure. Preferably, the extract is a 0 - 100 v / v% alcohol - water extract of Silene fortunei Vis.

[0049] In another preferred embodiment, the extract contains β - ecdysterone.

[0050] In another preferred embodiment, the above - mentioned renal failure includes chronic renal failure, renal fibrosis or acute renal failure.

[0051] In another preferred embodiment, preventing or treating renal failure includes improving one or more of the following indicators:

[0052] (i) Reducing the serum level of blood urea nitrogen (BUN);

[0053] (ii) Reducing the serum level of creatinine (Cr);

[0054] (iii) Reducing the serum level of kidney injury molecule - 1 (KIM - 1);

[0055] (iv) Reducing the serum level of neutrophil gelatinase - associated lipocalin (NGAL);

[0056] (v) Reducing the serum level of inflammatory factor interleukin - 6 (IL - 6);

[0057] (vi) Reducing the serum level of tumor necrosis factor α (TNF - α);

[0058] (vii) Reducing senescent cells in the kidney;

[0059] (viii) Reducing renal fibrosis;

[0060] (ix) Reducing the number of Ki67 - positive cells in the kidney.

[0061] In the fourth aspect of the present invention, there is provided the use of the extract of Silene fortunei Vis. in the preparation of a cosmetic composition for anti - aging. Preferably, the extract is a 0 - 100 v / v% alcohol - water extract of Silene fortunei Vis.

[0062] In another preferred embodiment, the anti-aging effect includes delaying skin aging.

[0063] In the fifth aspect of the present invention, there is provided a composition comprising the extract of Silene fortunei as described in the second aspect of the present invention, and a pharmaceutically or cosmetically acceptable carrier.

[0064] In another preferred embodiment, the pharmaceutical composition or preparation further comprises an additional anti-aging active ingredient selected from the group consisting of metformin, resveratrol, rapamycin, acarbose, dasatinib, quercetin, curcumin, astaxanthin, and arbutin.

[0065] In another preferred embodiment, in the pharmaceutical composition, the content of the extract is 0.001-99 wt%, preferably 0.01-90 wt% or 0.1-15 wt%, such as 0.1 wt%, 0.2 wt%, 0.5 wt%, 1 wt%, 2 wt%, 5 wt%, 8 wt%, 10 wt%, based on the total weight of the composition.

[0066] In another preferred embodiment, the composition is a solid preparation or a liquid preparation.

[0067] In another preferred embodiment, the form of the composition is selected from the group consisting of tablets, capsules, granules, suspensions, pills, solutions, syrups, injections, ointments, gels, or patches.

[0068] In the sixth aspect of the present invention, there is provided a method for anti-aging, comprising the step of administering to a subject in need the extract of Silene fortunei or a composition containing the same as described in the present invention.

[0069] In another preferred embodiment, the subject is a human or non-human mammal (such as a rat, a mouse) or a nematode.

[0070] In the seventh aspect of the present invention, there is provided a method for prolonging the lifespan or delaying the aging of nematodes in vitro, comprising the step of adding the active ingredient or composition of the present invention to a nematode culture medium and culturing the nematodes, thereby prolonging the lifespan or delaying the aging of the nematodes.

[0071] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 Shows the protective effects of the water extract and 75% ethanol extract of Silene fortunei in a renal cell injury model.

[0073] Figure 2Shows the chemical composition analysis of Stellaria yunnanensis Franch. extract (JM11002). (A) Schematic diagram of solvent stepwise separation, silica gel column chromatography separation and macroporous resin separation; (B) Liquid phase data of JM11002 (254 nm); (C) Schematic diagram of the structure of the abundant components; (D-J): UV data of the active components.

[0074] Figure 3 Shows the liquid phase data (254 nm) of 4 active components isolated from Stellaria yunnanensis Franch..

[0075] Figure 4 Shows the liquid phase data (254 nm) of each segment after separation by D101 macroporous resin.

[0076] Figure 5 Shows the anti-aging efficacy of JM11002 at the level of nematodes and mammalian cells. (A) Nematode lifespan experiment; (B) Nematode healthspan experiment; (C) Nematode stress resistance experiment; (D) SA-β-Gal staining experiment on human embryonic lung cells.

[0077] Figure 6 Shows the in vivo efficacy of the crude extract JM11002 under the UIRI model. (A) Schematic diagram of the experiment; (B-G) Changes in the levels of serum urea nitrogen (BUN), creatinine (Cr), kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), inflammatory factor interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α); (H) Representative images of X-gal staining, HE staining, Sirius red staining and Ki67 immunofluorescence of mouse kidney sections; (I) Quantitative map of X-gal staining; (J) Quantitative map of Sirius red staining; (K) Quantitative map of Ki67-positive cells; The dose of metformin (Met) is 200 mg / kg; The doses of JM11002 are 1 g / kg and 2 g / kg respectively. P<0.001 vs Sham; *P<0.05, **P<0.01, ***P<0.001 vs UIRI.

[0078] Figure 7 Shows the in vivo efficacy of the crude extract JM11002, the abundant component JM11201, and the macroporous resin fractions under the UUO model. (A) Schematic diagram of the experiment; (B-D) Changes in the levels of serum creatinine (Cr), urea nitrogen (BUN), and cystatin C (Cys-C); (E-F) Representative images and quantitative maps of Sirius red staining. ##P<0.01, P<0.001 vs Sham; *P<0.05, **P<0.01, ***P<0.001 vs UUO.

[0079] Figure 8Shows the in vivo pharmacodynamic effects of the crude extract JM11002, the abundant component JM11201, and the macroporous resin fractions under the UIRIx model. (A) Schematic diagram of the experiment; (B) Representative image of the kidney appearance; (C) Kidney-body ratio; (D) Serum level of kidney injury molecule-1 (KIM-1); (E) Serum level of neutrophil gelatinase-associated lipocalin (NGAL). ##P<0.01 vs Sham; *P<0.05, ***P<0.001 vs Model.

[0080] Figure 9 Shows the results of the 2-week subacute toxicity experiment of JM11002 at a dose of 10 g / kg. (A) Body weight curve; (B) Food intake per mouse per day; (C) Water intake per mouse per day; (D) Representative pictures of various organs; (E) Organ-body ratio.

[0081] Figure 10 Shows the serum and HE section results of the 2-week subacute toxicity experiment of JM11002 at a dose of 10 g / kg. (A-D) Changes in the levels of serum alanine aminotransferase (ALT); aspartate aminotransferase (AST); urea (UREA); creatinine (Cr); (E) Representative pictures of HE sections of each organ. Detailed implementation mode

[0082] Through extensive and in-depth research, and through a large number of screenings and tests, the present inventors have provided a Stellaria yunnanensis Franch. extract, its preparation method and application. Through the screening and testing of hundreds of traditional Chinese medicines, the present inventors have first discovered that a specific extract of Stellaria yunnanensis Franch. and its active ingredients have excellent anti-aging-related activities. On this basis, the present invention has been completed.

[0083] Terms

[0084] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains.

[0085] As used herein, when referring to a specifically recited numerical value, the term "about" means that the value can vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0086] As used herein, the term "comprising" or "including" can be open-ended, semi-closed, and closed. In other words, the term also includes "consisting essentially of..." or "consisting of...".

[0087] As used herein, the term "room temperature" or "normal temperature" refers to a temperature of 4 - 40°C, preferably, 25 ± 5°C.

[0088] Stellaria yunnanensis Franch.

[0089] Stellaria yunnanensis Franch. is a plant of the genus Stellaria in the Caryophyllaceae family. It is mainly distributed in Dali, Lijiang, Diqing, Chuxiong, Kunming and other places in China; it grows at an altitude of 1,800 - 3,200 meters.

[0090] Stellaria yunnanensis Franch. is sweet in taste and warm in nature, and has the effects of replenishing qi and nourishing blood, strengthening the spleen and tonifying the kidney, etc.; it is used for symptoms such as qi and blood weakness, listlessness, dizziness and palpitation, soreness and weakness of the waist and knees, irregular menstruation, etc.; it is an important medicine for treating women's deficiency. The main medicinal part is the root. At present, there is no report on its anti - aging effect.

[0091] The present invention first discovers the use of a specific extract of Stellaria yunnanensis Franch. in anti - aging / anti - debility.

[0092] Active ingredient

[0093] The active ingredient of the present invention is the extract of Stellaria yunnanensis Franch., which can be the water extract of Stellaria yunnanensis Franch., the organic solvent extract of Stellaria yunnanensis Franch., the water - organic mixed solvent extract of Stellaria yunnanensis Franch., or the active components separated from the extract of Stellaria yunnanensis Franch. (such as Stellaria yunnanensis Franch. polysaccharide, Stellaria yunnanensis Franch. alkaloid, Stellaria yunnanensis Franch. steroid compounds, Stellaria yunnanensis Franch. phenolic acid compounds, Stellaria yunnanensis Franch. flavonoid compounds, Stellaria yunnanensis Franch. polypeptide compounds, or their combinations) or single components (phenols, cyclic peptides, steroids, indole alkaloids, flavonoid glycosides, phenyl ethanol glycosides, or their combinations), or their combinations.

[0094] In particular, the active ingredient of the present invention is a specific extract of Stellaria yunnanensis Franch. Preferably, the active ingredient is the 0 - 100 v / v% alcohol - water extract of Stellaria yunnanensis Franch. (the percentage is the alcohol concentration), more preferably the 20 - 100 v / v% alcohol - water extract of Stellaria yunnanensis Franch., more preferably the 50 - 100 v / v% alcohol - water extract of Stellaria yunnanensis Franch.; more preferably the extract is the 60 - 90 v / v% alcohol - water extract of Stellaria yunnanensis Franch., more preferably the 65 - 85 v / v% alcohol - water extract or the 70 - 80 v / v% alcohol - water extract, even more preferably such as the 75 ± 2 v / v% alcohol - water extract. Preferably, it is an ethanol - water extract.

[0095] According to the analysis of the extract of the present invention, its main components include: steroid compounds, cyclic peptides, phenols, indole alkaloids, flavonoid glycosides and phenyl ethanol glycosides. Among them, β - ecdysterone is the component with the largest amount and has excellent activity. Preferably, in the extract, the content of β - ecdysterone is 1 - 10 mg / g; more preferably 4 - 6 mg / g.

[0096] Experimental results have shown that extracts of Stellaria yunnanensis Franch., especially the ethanol-water extract of the present invention and the components obtained by further separating and purifying the ethanol-water extract, all have certain activities. However, the directly extracted ethanol-water extract generally has higher activity than other components, indicating that there is a synergistic effect among the components in terms of anti-aging.

[0097] The inventors of the present invention have found through research that the active ingredients of the present invention have the ability to significantly extend lifespan, delay aging, increase antioxidant stress and anti-osmotic stress resistance, and are expected to be developed into anti-aging drugs and can be applied to extending lifespan, delaying aging, and preparing anti-aging cosmetics, etc.

[0098] Aging and related diseases

[0099] As used interchangeably herein, the terms "delay aging" or "anti-aging" refer to "preventing", "delaying", "resisting" or "improving" the process of organism aging, such as local aging (e.g., skin, muscle, organ, bone, joint, brain, etc.) and systemic aging of the whole body. Biologically speaking, aging is an inevitable spontaneous process of organisms over time. It is a complex natural phenomenon, manifested as degenerative changes in structure and decline in function, as well as reduced adaptability and resistance. In the present invention, the aging may be physiological aging or pathological aging, such as natural aging, chemotherapy drug-induced aging, oncogene-induced aging, irradiation-induced aging, replicative aging, oxidative stress-induced aging, DNA damage-induced aging, or a combination thereof. One aspect of the anti-aging of the present invention is manifested as extending lifespan.

[0100] Cellular senescence is a phenomenon that causes individual cells to be unable to continue dividing and to stop after several divisions. To detect cellular senescence, a cell staining test is generally used, which detects senescence-related markers (such as β-galactosidase activity). Senescent cells can interfere with important functions of the entire organism and thus cause certain disorders. Aging of the entire organism is accompanied by an increased risk of certain disorders (such as diseases, complications, and symptoms).

[0101] Some representative senescent cell markers or signs include (but are not limited to): SA-β-galactosidase (an increase in its expression level indicates an increase in the degree of senescence), cell proliferation ability (a decrease in which indicates an increase in the degree of senescence), and collagen (a decrease in its expression level indicates an increase in senescence).

[0102] Delaying aging includes (but is not limited to) delaying the aging of skin, muscle, organ, bone, joint, and brain.

[0103] Typically, the aging and / or frailty-related diseases include (but are not limited to): renal failure, heart failure, neurodegenerative diseases, and musculoskeletal diseases (such as osteoarthritis, sarcopenia, etc.).

[0104] As used herein, the terms "prevent", "delay", "resist" or "ameliorate" aging, etc. include delaying or alleviating to some extent one or more indicators or aspects related to the aging of an organism, but do not necessarily require 100% delay of aging. In some embodiments, compared with the absence of the active ingredient of the present invention, the active ingredient of the present invention or a composition containing the same delays or alleviates one or more indicators related to the aging of the organism of the subject by, for example, at least about 5%, at least about 10%, at least about 15%, at least about 20%, or at least about 30%; or the lifespan of the subject is extended by at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, or at least about 30%.

[0105] Renal failure

[0106] Renal failure is a pathological state in which the renal function is partially or completely lost due to the progression of various chronic kidney diseases to the late stage. Renal failure can be divided into acute renal failure and chronic renal failure. The condition of acute renal failure progresses rapidly, and the main causes are insufficient blood supply to the kidneys (such as trauma or burns), damage to the kidneys due to obstruction by certain factors, or poisoning; while the main cause of chronic renal failure is long-term kidney disease. Chronic renal failure is one of the common diseases in the urinary system of the elderly.

[0107] Studies have shown that using anti-aging drugs to improve or eliminate cellular senescence in the kidneys can effectively relieve renal fibrosis and inflammation, enhance the regenerative ability of the kidneys, improve renal function, and thus delay the progression of chronic renal failure.

[0108] Preparation method

[0109] In the present invention, the extract of Silene fortunei can be obtained by extracting the Silene fortunei medicinal material with water, an organic solvent (such as an alcohol, ester, or ether solvent), or a mixed solvent thereof. Further, the solvent extract can be further extracted / extracted or separated and purified to obtain an enriched active component or a single active component.

[0110] A preferred method for preparing an extract includes the steps of:

[0111] (i) Heating and refluxing Silene fortunei in 0 - 100 v / v% alcohol-water for 1 - 10 times, combining the extracts, and removing the solvent to obtain an extract paste or a dried extract.

[0112] In another preferred example, the extract is a 50 - 100 v / v% alcohol-water extract or a 60 - 90 v / v% alcohol-water extract of Silene fortunei, preferably a 65 - 85 v / v% alcohol-water extract or a 70 - 80 v / v% alcohol-water extract, and more preferably a 75 ± 2 v / v% alcohol-water extract.

[0113] In another preferred embodiment, the alcohol is a C1-C4 alkanol. Preferably, the alcohol is selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, n-butanol, or a combination thereof.

[0114] In another preferred embodiment, the Herba Dianthi Superba can be the whole plant, above-ground part, underground part, root, stem, leaf, flower of Herba Dianthi Superba, or a combination thereof, preferably the root.

[0115] In another preferred embodiment, the extraction includes one or more of the following features:

[0116] (a) The temperature of the extraction is 70-120 °C, preferably 80-100 °C, more preferably 90-100 °C;

[0117] (b) The number of extractions is 2-10 times, preferably 2-5 times, 2-3 times;

[0118] (c) The time of each extraction is 1-4 h / time, preferably 2-3 h / time;

[0119] (d) The mass-volume ratio of Herba Dianthi Superba to the alcohol-water is 1 g: 1-20 mL, preferably 1 g: 2-10 mL.

[0120] In another preferred embodiment, the (crude) extract obtained in the above step can be further subjected to solvent extraction to obtain an enriched component. For example, the crude extract obtained by solvent extraction is dissolved in water, and the aqueous phase is successively extracted with petroleum ether, dichloromethane, or ethyl acetate to obtain multiple organic phases and a final aqueous phase. Preferably, the ethyl acetate extract is collected.

[0121] Upon analysis, the ethyl acetate extract mainly contains sterones (including β-ecdysterone), cyclic peptides, and phenolic components in Herba Dianthi Superba, and can be separated and enriched by silica gel column chromatography or macroporous adsorption resin as needed. The aqueous phase after ethyl acetate extraction mainly contains indole alkaloids, flavonoid glycosides, and phenethyl alcohol glycoside components in Herba Dianthi Superba, and can be separated and enriched by silica gel column chromatography or macroporous adsorption resin as needed.

[0122] Preferably, the ethyl acetate extract is further passed through a macroporous adsorption resin (such as D101), and gradient elution is performed with water-ethanol (0→100 v / v% ethanol) to obtain an enriched active component; preferably, the components obtained at 0-5 v / v% (preferably 0-2 v / v%) ethanol, 30±5 v / v% (preferably 30±2 v / v%) ethanol, 40±5 v / v% (preferably 40±2 v / v%) ethanol, 95-100 v / v% (preferably 98-100 v / v%) ethanol.

[0123] Composition and administration method

[0124] The present invention also provides a composition, preparation or product containing the active ingredient of the present invention, and the composition, preparation or product can be used for anti-aging. Representative compositions, preparations or products include anti-aging drugs and cosmetics.

[0125] The composition of the present invention comprises:

[0126] A) The active ingredient of the present invention or the extract of the present invention, as an anti-aging active ingredient; and

[0127] B) A pharmaceutically or cosmetically acceptable carrier.

[0128] In another preferred embodiment, the pharmaceutical composition or preparation of the present invention further comprises A2) an additional anti-aging active ingredient selected from the group consisting of metformin, resveratrol, rapamycin, acarbose, dasatinib, quercetin, curcumin, astaxanthin and arbutin.

[0129] As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity (i.e., anti-aging function) in humans and / or animals and is acceptable to humans and / or animals.

[0130] As used herein, the component of the term "pharmaceutically acceptable" is applicable to humans and / or mammals without excessive adverse side effects (such as toxicity, irritation and allergic reaction), that is, a substance with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent, including various excipients and diluents.

[0131] The composition of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): water, NaCl, physiological saline solution, lactated Ringer's solution, conventional sucrose, conventional glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavoring agents, salt solutions (such as Ringer's solution), alcohols, oils, gelatin, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxypropyl methylcellulose, polyvinylpyrrolidone and pigments, etc. Such preparations can be sterilized and, if necessary, mixed with adjuvants that do not react harmfully with the compounds provided herein or interfere with the activity of the compounds provided herein, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts affecting osmotic pressure, buffers, coloring agents and / or aromatic substances, etc. Those of ordinary skill in the art will recognize that other pharmaceutical carriers and excipients are suitable for the active ingredient of the present invention. The choice of carrier should match the mode of administration, which is well known to those of ordinary skill in the art.

[0132] Typically, the dosage forms of the compositions of the present invention can be injection preparations, oral preparations, transdermal preparations, sustained-release preparations. The compositions suitable for the present invention will typically be discrete units in solid form, such as in the form of tablets, capsules, cachets, powders, granules, lozenges, patches, suppositories, pills, or in liquid form, such as liquid preparations, injectable or infusible solutions or suspensions.

[0133] The effective amount of the active ingredient described in the present invention can vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by those of ordinary skill in the art based on various factors (such as through clinical trials). Such factors include, but are not limited to: the pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated in the patient, the patient's body weight, the patient's immune status, the route of administration, etc. Generally, when the active ingredient of the present invention is administered at a dose of about 0.00001 mg - 50 mg / kg of animal body weight per day (preferably 0.0001 mg - 10 mg / kg of animal body weight), satisfactory effects can be obtained. For example, depending on the urgency of the treatment situation, several separate doses can be administered per day, or the dose can be proportionally reduced.

[0134] Typically, when the pharmaceutical composition of the present invention is administered orally, for a subject (human) with a body weight of 60 kg, the average daily dose is usually 10 - 500 mg, preferably 20 - 300 mg, more preferably 50 - 250 mg. The daily dose can be taken once, twice, or multiple times.

[0135] The present invention also provides a method for prolonging the lifespan or delaying the aging of nematodes in vitro, including the steps of adding the active ingredient or composition of the present invention to a nematode culture medium and culturing the nematodes, thereby prolonging the lifespan or delaying the aging of the nematodes.

[0136] Cosmetic composition

[0137] The present invention also provides a cosmetic composition, and the cosmetic composition contains the active ingredient as described in the present invention as an effective ingredient for anti-aging.

[0138] Within the scope that does not interfere with the effects of the present invention, other ingredients commonly used in cosmetics can be added to the cosmetics of the present invention, such as film-forming agents, oil-soluble gelling agents, organically modified clay minerals, resins, moisturizers, preservatives, antibacterial agents, fragrances, salts, antioxidants, pH regulators, chelating agents, cooling agents, anti-inflammatory agents, skin beautifying ingredients (whitening agents, cell activators, skin roughness improvers, blood circulation promoters, skin astringents, anti-seborrheic agents, etc.), vitamins, amino acids, nucleic acids, hormones, inclusion compounds, etc.

[0139] The cosmetic composition of the present invention can be used for skin anti-aging.

[0140] The main advantages of the present invention include:

[0141] 1) The present invention discovers and verifies for the first time the anti-aging use of the traditional Chinese medicine Stellaria yunnanensis Franch., which has application prospects in the preparation of anti-aging drugs, cosmetics, etc.

[0142] 2) The present invention isolates and identifies for the first time the extract of Stellaria yunnanensis Franch. and its main active ingredients, as well as their uses in anti-aging and the treatment of aging-related diseases (such as renal failure), significantly expanding the application of the traditional important Stellaria yunnanensis Franch. and providing a material and theoretical basis for its industrial application.

[0143] 3) Experiments prove that the extract of Stellaria yunnanensis Franch. of the present invention has better anti-aging and activity in the treatment of aging-related diseases (such as renal failure) than existing drugs.

[0144] 4) As a traditional Chinese medicinal material, Stellaria yunnanensis Franch. has rich clinical medication experience for those skilled in the art. Moreover, experiments of the present invention prove that it has no obvious toxic and side effects at effective doses, has high safety, and has excellent application prospects.

[0145] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0146] Example 1 Stellaria yunnanensis Franch. Renal Cell Protection Experiment

[0147] In this experiment, a renal cell injury model was established to investigate the effect of the drug on cell viability, thereby evaluating the protective effect of the drug on renal cells. The cells were divided into a normal control group, a model group, a positive drug control group (empagliflozin EMPA, 100 mg / L), an aqueous extract group (10 mg / L), and a 75% ethanol extract group (10 mg / L). When the growth density of HK-2 (human renal epithelial cells) exceeded 90%, cell plating was performed. In a 96-well plate, plating was carried out at a density of 10,000 cells per milliliter, and 100 μL of culture medium was added to each well. To prevent the solvent evaporation effect from affecting the experimental results, only PBS was added to the wells in the outer circle of the 96-well plate. The 96-well plate was placed in an incubator and left standing for more than 12 h to ensure stable cell adhesion. Then, pre-drug treatment was carried out. According to the stock solution concentration of the sample to be tested, the corresponding final concentration was prepared with culture medium. The original culture medium was removed and co-incubated with the cells for 12 h before modeling. Except for the cells in the normal control group, the cells in the other groups were co-incubated with the culture medium containing the modeling agent (200 mg / L cobalt chloride CoCl2) for 12 h. After modeling, the supernatant was discarded, and the cell viability was detected by the Cell Counting Kit-8 (CCK8) method. The data were summarized, and a bar chart was drawn using Graph Pad, and the significant difference (t-test) was calculated.

[0148] Results: The data of the renal cell protection experiment are as Figure 1 shown. With the help of the cell injury model of HK-2 (human renal epithelial cells), we preliminarily screened the activities of different solvent extracts (water, 75% ethanol) of Stellaria yunnanensis Franch. In the cell injury model induced by cobalt chloride, compared with the model group, both the aqueous extract group and the 75% ethanol extract group had protective effects, both were superior to the positive drug EMPA, and the 75% ethanol extract group was superior to the aqueous extract group. * indicates P < 0.05, and ** indicates P < 0.01. The above experimental results show that JM11002 (75% ethanol extract) has a better protective effect on renal cells in the cobalt chloride-induced renal cell injury model.

[0149] Example 2 Preparation of the crude extract of Stellaria yunnanensis Franch.

[0150] Using Stellaria yunnanensis Franch. (recorded in the "Yunnan Chinese Medicinal Materials Standard (2005 Edition)" (Volume II·Yi Medicine)), a medicinal plant collected locally in Dali Bai Autonomous Prefecture, Yunnan Province, as the raw material, the naturally air-dried dry material (root) was taken, crushed, and refluxed with the extraction solvent at 95 °C for 3 times, 2 h each time. The extraction solutions were combined, and the extraction solution was concentrated under reduced pressure to obtain an extract, and finally freeze-dried to obtain the crude extract dry powder. Among them, the extraction solvent used for the 75% ethanol extract (JM11002) of Stellaria yunnanensis Franch. was 75% ethanol, and a total of 1.06 kg of yellow extract was obtained after extracting 5.95 kg of dry material of Stellaria yunnanensis Franch.

[0151] Chemical Constituent Analysis of 75% Ethanol Extract of Silene fortunei

[0152] As Figure 2 shown, 400 g of the 75% ethanol extract was dissolved in water and successively extracted with petroleum ether, dichloromethane, and ethyl acetate to obtain 0.4 g of petroleum ether extract, 1.413 g of dichloromethane extract, and 9.743 g of ethyl acetate extract.

[0153] The dried ethyl acetate extract was subjected to column chromatography on a silica gel column of 200 - 300 mesh, eluted with a mixed solvent of dichloromethane / methanol, and finally three types of structures were obtained. The structures and purities were confirmed by spectroscopic and chromatographic methods such as nuclear magnetic resonance (proton NMR, carbon NMR), mass spectrometry (ESI-high resolution), and HPLC. It was determined that JM11201 (β-ecdysterone, the component with high content) and JM11203 are both steroid ketone structures. Combining ultraviolet and nuclear magnetic data, JM11202 was preliminarily determined to be a cyclic peptide, and JM11204 was a phenolic compound.

[0154] The aqueous phase after ethyl acetate extraction was separated using D101 macroporous adsorption resin and further separated by a rapid preparative liquid chromatograph. Combining ultraviolet and nuclear magnetic data, JM11205 was preliminarily determined to be an indole alkaloid, JM11206 was a flavonoid glycoside, and JM11207 was a phenylethanol glycoside.

[0155] As Figure 4 Using D101 macroporous adsorption resin, the main peak of JM11002 was enriched. After static adsorption, it was eluted with water, 30% ethanol, 40% ethanol, and 100% ethanol respectively to obtain four components: JM11101 (washed part with water), JM11102 (eluted part with 30% ethanol), JM11103 (eluted part with 40% ethanol), and JM11104 (eluted part with 100% ethanol).

[0156] Example 4 Silene fortunei Prolongs the Lifespan of Caenorhabditis elegans

[0157] In this experiment, the lifespan of nematodes was measured to investigate whether the drug has an anti-aging effect on nematodes. Synchronized L1-stage nematodes were transferred to the culture medium for cultivation until they developed to the L4 stage. At this time, nematodes in the same growth stage were selected and transferred to the blank control group pretreated with bacteria and the culture medium of the drug-containing experimental group using a nematode pick, with 15 to 20 nematodes placed in each petri dish. Then, every 2 to 3 days, the nematodes were transferred to a new culture medium. After the nematodes grew on the drug-containing culture medium for 10 days, they were transferred to the drug-free culture medium to complete a total of 10 days of drug treatment. During this period, the number of surviving nematodes was recorded daily, and the dead nematodes were removed from the petri dish. At the same time, nematodes that died due to vulva bursting or crawling to the edge and drying out were excluded. After the experiment, a survival curve was plotted using GraphPad software, and a significant difference analysis was performed using The log-rank (Mantel-Cox) test.

[0158] The experimental data are shown in Table 1, and the survival curve is as Figure 5 (A). The extract of Silene tatarinowii Regel (JM11002) significantly prolonged the lifespan of nematodes at 100 mg / L and 200 mg / L. Compared with the control group, the lifespan of nematodes was prolonged by 5.99% and 7.76% respectively. ** indicates P < 0.01, and *** indicates P < 0.001.

[0159] Table 1. Experimental data of the lifespan of nematodes treated with the extract of Silene tatarinowii Regel

[0160]

[0161] Example 5 Silene tatarinowii Regel improves the body sway ability of nematodes

[0162] In this experiment, the body sway ability of nematodes was tested to investigate the effect of the drug on improving the body health parameters. The life cycle of nematodes is short. Under natural conditions, nematodes enter the senescence stage on the 8th day of adulthood, and their body sway ability significantly decreases. Nematodes were cultured according to the method of the above nematode lifespan experiment. On the 3rd and 8th days of nematode adulthood, 20 to 30 nematodes were selected from the control group and Silene tatarinowii Regel (200 mg / L) respectively, and the number of body sways of each nematode in the buffer solution (M9) for 30 seconds was recorded. After the experiment, the data were statistically analyzed, and the significant difference was calculated using GraphPad (Two-way ANOVA, Sidak multiple comparisons test). The error bar indicates the standard error of the mean (SEM).

[0163] Results: As Figure 5(As shown in (B), the extract of *Stellaria yunnanensis* Franch. (200 mg / L) can improve the body swaying ability of nematodes, with significant differences on the 3rd and 8th days, indicating that *Stellaria yunnanensis* Franch. has good anti-aging effects and can improve the body health parameters. * indicates P < 0.05.)

[0164] Example 6: *Stellaria yunnanensis* Franch. Improves the Osmotic Stress Resistance Ability of Nematodes

[0165] The osmotic stress resistance ability is one of the indicators for evaluating the healthy lifespan of nematodes. In this experiment, the osmotic stress resistance ability of nematodes was measured to investigate the enhancing effect of drugs on the osmotic stress resistance ability of nematodes. In the experiment, nematodes in the blank control group and the experimental group were cultured according to the standards of the lifespan experiment. On the 4th day after drug administration, nematodes in each group were transferred to NGM plates containing *E. coli* OP50 and then placed on NGM culture plates added with 500 mM sodium chloride. The excess M9 buffer was sucked away with filter paper, and the timing started from the moment when the NGM plates began to dry. Under hypertonic conditions, starting from 3 minutes, the number of crawling nematodes was recorded every 2 minutes until all nematodes could no longer move. When a nematode showed no movement response when touched with a platinum wire, it was recorded as dead. After the experiment was completed, survival curves were plotted using GraphPad software, and the significant differences between the survival curves were analyzed using The log-rank (Mantel-Cox) test.)

[0166] Results: As Figure 5 (shown in (C), the extract of *Stellaria yunnanensis* Franch. (200 mg / L) improves the osmotic stress resistance ability of nematodes, with a significant difference on the 4th day, indicating that *Stellaria yunnanensis* Franch. can enhance the osmotic stress resistance ability of nematodes. *** indicates P < 0.001. This shows that the extract of *Stellaria yunnanensis* Franch. can improve the health parameters of nematodes, increase their healthy lifespan, and thus delay aging.)

[0167] Example 7: *Stellaria yunnanensis* Franch. Reduces SA-β-Gal in Human Embryonic Lung Cells

[0168] SA-β-Gal is a senescent cell marker that can be used to evaluate the degree of cell senescence. In this experiment, by measuring SA-β-Gal in cells, the alleviating effect of the drug on cell senescence was investigated. The vast majority of normal cells are considered to have only limited division ability and enter the senescent state after they can no longer divide. Senescent cells are usually larger in volume and have highly active β-galactosidase. The β-gal can be stained with a β-galactosidase staining kit (Beyotime) to evaluate the degree of cell senescence. In this example, SA-β-Gal was used as the senescent cell marker and stained with the chemical reagent X-Gal to make it blue-green. Human embryonic lung cells (MRC-5, purchased from the Cell Bank of the Chinese Academy of Sciences) were cultured in MEM medium (containing 10% FBS, 1% sodium pyruvate, 1% non-essential amino acids, 1% double antibiotics). After culturing to the 39th passage (P39), the degree of cell senescence was detected by preliminary experiments. The experiment could be carried out when the cell senescence rate was between 50% and 60%. The experimental groups were set as the control group, extracts of Stellaria yunnanensis Franch. (5, 25 mg / L), and metformin group (100 μM, positive drug group). The drugs were co-incubated with the cells for 3 days. The cells could be stained with a β-galactosidase staining kit, and the cell nuclei also needed to be stained with DAPI (Sigma). The number of senescent cells and the total number of cells were counted, and the cell senescence rate was calculated. GraphPad was used to calculate the significant difference (Unpaired t-test), and the error bars indicated the standard error of the mean (SEM). Three valid repeated experiments were carried out.

[0169] Results: As Figure 5 (D) shows, compared with the control group, the SA-β-Gal of the extracts of Stellaria yunnanensis Franch. (5, 25 mg / L) and the treatment groups were significantly reduced, indicating that the extracts of Stellaria yunnanensis Franch. could effectively reduce the cell senescence rate. The activity of reducing the senescence rate at a concentration of 5 mg / L was equivalent to that of the positive drug metformin (100 μM), and the effect was better at a concentration of 25 mg / L. The significant differences relative to the control group: * indicates P < 0.05, *** indicates P < 0.001.

[0170] Example 8 Treatment of chronic renal failure in mice induced by unilateral renal ischemia-reperfusion (UIRI) with Stellaria yunnanensis Franch.

[0171] In this experiment, a mouse model of chronic renal failure induced by unilateral renal ischemia-reperfusion (UIRI) was established to investigate the effects of the drug on renal function, pathological indexes, senescence indexes and other indexes, so as to judge the effect of the drug on chronic renal failure.

[0172] UIRI-induced Chronic Renal Failure Mouse Model: After adaptively raising 8-week-old male C57BL / 6J mice, the mice were continuously anesthetized with isoflurane. After the corneal reflex of the mice disappeared, the mice were placed in the supine position, and their four limbs were fixed to the operating table. The hair in the abdominal surgical area was shaved off. An electric heating plate was used to prevent the body temperature of the mice from dropping, and the body temperature of the mice was maintained at about 37 °C. After disinfecting twice with iodophor, the muscle was then incised, the intestinal canal was gently separated with a cotton swab, the left renal pedicle was bluntly dissected, and the renal pedicle was clamped with a non-invasive arterial clamp for 35 minutes. The success of renal pedicle clamping was judged by the naked eye as the color of the kidney gradually deepened from pink to finally purple-black; the surgical incision was covered with a gauze soaked in warm saline to prevent dehydration of the renal tissue. After 35 minutes, the arterial clamp was removed, and the surrounding organs were gently separated with a cotton swab and then the condition of the kidney was observed. If the kidney returned from purple-black to the red color before clamping within 30 seconds, it indicated successful reperfusion of the kidney. Finally, the abdominal organs such as the kidney were repositioned, the abdominal cavity was closed, and the wound was sutured. From the 7th day after the operation, the mice in the drug administration group were given the corresponding concentration of the medicinal liquid by gavage every day, and the drug administration continued for 30 days. The positive drug was the marketed drug metformin (200 mg / kg).

[0173] After the experiment, serum biochemical indexes such as blood urea nitrogen (BUN), creatinine (Cr), kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), inflammatory factor interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α) were detected to evaluate renal function.

[0174] Pathological Detection of Kidney Tissue: After embedding a part of the kidney tissue with OCT, frozen sections were made, and another part was immersed in 4% paraformaldehyde solution for fixation, paraffin-embedded, and cut into thin slices with a thickness of about 4 μm.

[0175] a. 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) staining was used to evaluate the senescence of kidney cells: After the frozen sections were placed at room temperature, they were washed with PBS and then immersed in the fixative (2% formaldehyde + 0.2% glutaraldehyde). After washing twice with PBS at room temperature, the preheated X-gal staining solution was used for overnight staining. Then, dehydration was carried out successively with gradients, made transparent with xylene, sealed, and photographed for observation.

[0176] a. Hematoxylin-eosin (HE) staining was used to detect the morphological changes of kidney tissue: After dewaxing and hydrating the sections, they were stained with hematoxylin for 10 minutes, rinsed with running water for 5 minutes, then the sections were placed in 1% hydrochloric acid ethanol for differentiation, blued, rinsed with running water, and then the sections were stained with eosin solution for 5 - 10 minutes, rinsed with running water, and sealed with neutral gum for photographing and observation.

[0177] b. Sirius red staining was used to observe kidney fibrosis: After dewaxing and hydrating the sections, they were stained with Sirius red for 1 hour, dehydrated successively with gradients, made transparent with xylene, sealed, and observed under a microscope.

[0178] c. Immunofluorescence labeling experiment of tissue sections was used to evaluate the changes of the renal injury marker Ki67. The sections were dewaxed and hydrated, and the antigens were repaired by water bath heating using EDTA buffer. After being placed at room temperature, they were washed 3 times with PBS. After blocking with 1.5% horse serum at room temperature for 1 hour, they were washed 3 times with PBS and incubated with the primary antibody overnight at 4°C. After being washed 3 times with PBS, they were incubated with the secondary antibody at room temperature for 2 hours, then washed 3 times with PBS, stained with Hoechst at room temperature for 5 minutes, and mounted with 50% glycerol-PBS after washing with PBS and photographed.

[0179] The results are as Figure 6 shown. JM11002 significantly decreased the serum indexes related to renal function induced by UIRI in a concentration-dependent manner, and significantly alleviated renal senescent cells, tissue lesions, fibrosis, and the number of Ki67-positive cells. The serum indexes of the high-concentration group of the crude extract JM11002 (2 g / kg) were better than those of the positive drug, and the histopathological results were comparable to those of the positive drug.

[0180] Example 9 Treatment of unilateral ureteral ligation (UUO)-induced renal fibrosis in mice with Herba Sileneae Conoideae

[0181] In this experiment, a mouse renal fibrosis model was established by unilateral ureteral ligation (UUO) to induce obstruction, and the effects of the drug on renal function, pathological indexes, fibrosis and other indexes were investigated to judge the effect of the drug on chronic renal failure.

[0182] UUO-induced mouse renal fibrosis model: After 8-week-old C57BL / 6J male mice were adaptively fed, the mice were continuously anesthetized with isoflurane. After the corneal reflex of the mice disappeared, the mice were placed in the supine position and their limbs were fixed on the operating table, and the hair in the abdominal surgical area was shaved off. The electric heating plate was used to heat to prevent the body temperature of the mice from dropping, and the body temperature of the mice was maintained at about 37°C. After disinfection with iodophor twice, the muscle was cut open, and the intestinal canal was gently separated with a cotton swab. The renal hilum of the kidney was exposed using an ophthalmic forceps. After separating the left ureter, double silk ligations were performed at the upper 1 / 3 and lower 1 / 3 of the ureter and then cut off to completely obstruct the left kidney, and then sutured layer by layer. This model was used to evaluate the crude extract JM11002, the single component JM11201 with abundant content and the four components (JM11101, JM11102, JM11101, and JM11104) after macroporous resin separation at the same crude drug amount, and the positive drug was the marketed drug empagliflozin (20 mg / kg).

[0183] After the experiment, serum biochemical indexes such as blood urea nitrogen (BUN), creatinine (Cr), and cystatin C (Cys-C) were detected to evaluate renal function.

[0184] Detection of renal tissue fibrosis: The renal tissue was immersed in 4% paraformaldehyde solution for fixation, embedded in paraffin, and cut into thin sections with a thickness of about 4 μm. Sirius red staining method was used to observe renal fibrosis: After dewaxing and hydration of the sections, Sirius red staining was performed for 1 h, followed by gradient dehydration, xylene transparency, and sealing. Observation was carried out under a microscope. The morphological changes of renal tissue (HE staining) are in progress.

[0185] As Figure 7 shown, the crude extract JM11002 (2 g / kg) had the best efficacy, which was better than the positive drug empagliflozin, and significantly reduced the serum indicators related to renal function and fibrosis induced by UUO. The efficacy of the component JM11201 with high content was second. At the same crude drug concentration (10.88 mg / kg), creatinine and blood urea nitrogen were better than the positive drug; at the same concentration as the positive drug (20 mg / kg), creatinine was better than the positive drug, and cystatin C was comparable to the positive drug. The other 4 fractions JM11101, JM11102, JM11103, and JM11104 had efficacy at the same crude drug amount, but the serum data were not as good as those of the crude extract, indicating that the components in the extract of Stellaria yunnanensis Franch. of the present invention exerted a synergistic effect.

[0186] Example 10 Treatment of acute renal failure in mice induced by unilateral ischemia-reperfusion plus contralateral nephrectomy (UIRIx) with Stellaria yunnanensis Franch.

[0187] In this experiment, an acute renal failure model in mice was induced by establishing the method of unilateral renal ischemia-reperfusion plus contralateral nephrectomy (UIRIx) to investigate the effects of drugs on renal function, pathological indexes, fibrosis and other indexes, so as to judge the effects of drugs on chronic renal failure. UIRIx-induced acute renal failure model in mice: After the mice were adaptively raised, they were continuously anesthetized with isoflurane gas. After the corneal reflex of the mice disappeared, the mice were placed in the supine position and their limbs were fixed on the operating table. The hair in the abdominal surgical area was shaved off. An electric heating plate was used to prevent the body temperature of the mice from dropping, and the body temperature of the mice was maintained at about 37°C. After disinfection with iodophor twice, the muscle was cut open, and the intestinal tract was gently separated with a cotton swab. After removing the right kidney of the mice, the left renal pedicle was bluntly dissected, and the renal pedicle was clamped with a non-invasive arterial clamp for 30 minutes. The success of renal pedicle clamping was judged by the naked eye as the color of the kidney gradually deepened from pink to finally purple-black; a gauze soaked with warm normal saline was covered at the surgical incision to prevent dehydration of renal tissue. After 30 minutes, the arterial clamp was removed, and the surrounding organs were gently separated with a cotton swab to observe the condition of the kidney. If the kidney returned from purple-black to the red color before clamping within 30 seconds, it indicated successful renal reperfusion. Finally, the abdominal organs such as the kidney were repositioned, the abdominal cavity was closed, and the wound was sutured. This model was used to evaluate the crude extract JM11002, the abundant and single component JM11201, and the four fractions (JM11101, JM11102, JM11101, and JM11104) after macroporous resin fractionation at the same crude drug amount. The positive drug was the marketed drug empagliflozin (20 mg / kg). The corresponding concentration of the medicinal liquid was pre-administered 3 days before surgery, and the mice were sacrificed 24 h after modeling.

[0188] Results: After the experiment, the appearance of the kidney, kidney-body ratio, KIM-1 (kidney injury molecule-1), and NGAL (neutrophil gelatinase-associated lipocalin) were detected. As Figure 8 shown, the crude extract JM11002 (2 g / kg) and each fraction (except JM11104) had pharmacological effects and could significantly reduce the increase in kidney-body ratio induced by UIRIx modeling, which was comparable to the positive drug empagliflozin. At the same crude drug amount concentration, JM11201 (10.88 mg / kg) was comparable to the positive drug EMPA; at the same concentration as the positive drug (20 mg / kg), creatinine was better than the positive drug, and cystatin C was comparable to the positive drug. The other three fractions, JM11101, JM11102, and JM11103, had pharmacological effects at the same crude drug amount, but the serum data were not as good as those of the crude extract JM11002.

[0189] Example 11 Safety Evaluation of Silene tatarinowii Regel

[0190] In this experiment, continuous high-dose administration was carried out for 14 days. By examining the effects of the drug on the organ-body ratios of important organs, serum indicators, and pathological sections, the safety of the drug was judged. Randomly select 8 C57BL / 6J mice weighing about 22 g as a group, with half male and half female. Administer JM11002 at a dose of 10 g / kg by gavage for 14 days. Observe the toxicity reaction and death of the mice every day, and record the daily body weight, food intake, and water intake. Autopsy the dead animals in time. Sacrifice the mice in each group on the 15th day after administration, dissect them, and observe whether there are lesions in each important organ of the mice macroscopically. At the same time, weigh the heart, liver, spleen, lungs, and kidneys, calculate the organ-body ratio according to the formula of organ / body weight, and evaluate the serum indicators of liver and kidney functions.

[0191] Results: Continuous administration was carried out for two weeks at a dose 5 times the optimal efficacy dose (10 g / kg). During the experiment, there were no toxic reactions, and there were no abnormalities in the daily body weight, food intake, and water intake. Dissect the mice on the 15th day. As Figure 9 and Figure 10 shown, the colors, textures, and morphologies of the important organs (heart, liver, spleen, lungs, and kidneys) were no different from those of the normal control group. There were also no significant differences in the ratios of the organs to the body weight, and there were no significant differences in the serum data of liver and kidney functions. Therefore, we believe that the safety window of JM11002 is more than 5 times, and the safety is good.

[0192] All the documents mentioned in the present invention are cited in this application as references, as if each document was cited separately as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. Use of a Stellaria yunnanensis Franch extract in the preparation of a pharmaceutical composition for delaying aging or preventing or treating aging and / or frailty-related diseases.

2. The use according to claim 1, characterized in that The delaying of aging includes one or more selected from the following groups: Improve the health of the body; Improves health physiological parameters; Enhance or improve mobility capabilities; Improve the ability to resist stress; Delay or reverse the aging of normal somatic cells; Reducing the expression of cell senescence markers (such as SA-β-Gal); and / or Extend lifespan.

3. The use according to claim 1, characterized in that The aging and / or frailty-related diseases are selected from the group consisting of renal failure, heart failure, neurodegenerative diseases, and musculoskeletal diseases.

4. The use according to claim 1, characterized in that The extract is selected from the following group: a water extract of Herba Lycopodii, an organic solvent extract of Herba Lycopodii, a water-organic mixed solvent extract of Herba Lycopodii, or active components separated from the Herba Lycopodii extract (such as Herba Lycopodii polysaccharides, Herba Lycopodii alkaloids, Herba Lycopodii sterone compounds, Herba Lycopodii phenolic acid compounds, Herba Lycopodii flavonoid compounds, Herba Lycopodii polypeptide compounds, or a combination thereof) or a single component (phenols, cyclic peptides, sterones, indole alkaloids, flavonoid glycosides, phenylethanol glycosides, or a combination thereof), or a combination thereof.

5. The use as claimed in claim 1, characterized in that: The extract comprises beta-ecdysterone.

6. The use according to claim 1, wherein the extract is a 0-100 v / v% alcohol-water extract of Herba Lycopodii.

7. An extract of Herba Lycopodii, wherein the extract is a 0-100 v / v% alcohol-water extract of Herba Lycopodii.

8. The extract according to claim 7, characterized in that Includes one or more technical features selected from the following group: The Thousand Needle Thread Grass can be the whole plant, above-ground part, underground part, root, stem, leaf, flower, or a combination thereof, preferably the root; The extract is a 50-100 v / v% alcohol-water extract, a 65-85 v / v% alcohol-water extract, or a 70-80 v / v% alcohol-water extract of Herba Lycopodii, preferably a 75±2 v / v% alcohol-water extract; The alcohol is a C1-C4 alkanol, preferably, the alcohol is selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, n-butanol, or a combination thereof; and / or The mass volume ratio of the herb to alcohol-water is 1 g:1-20 mL, preferably 1 g:2-10 mL.

9. A composition comprising the Herba Lycopodii extract according to claim 7 or 8, and a pharmaceutically or cosmetically acceptable carrier.

10. Use of the extract according to claim 7 or 8 or the composition according to claim 9 in the preparation of a pharmaceutical composition for preventing or treating renal failure.

11. Use of the extract according to claim 7 or 8 or the composition according to claim 9 in preparing anti-aging cosmetics.

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