Use of mulberry extract in preparation of Anti-aging product

Abstract

The use of a mulberry extract in the preparation of an anti-aging product. The mulberry extract comprises more than 3% by weight of an alkaloid, and / or no more than 70% by weight of a polysaccharide, and / or no more than 10% by weight of a flavone, and / or no more than 50% by weight of an amino acid, and / or other components.

Description

Use of mulberry extract in preparing anti-aging products Technical Field

[0001] The invention relates to use of mulberry extract in preparing anti-aging products. Background Art

[0002] The mechanism of human aging is "decline of cell function", the irreversible cessation of proliferation (growth), that is, changes in the functions of molecules such as proteins and genes, as well as organelles. Aging is a dynamic process, gradual, multi-step, and ultimately irreversible. The physiological mechanisms behind the aging process mainly focus on: genomic instability, telomere shortening, epigenetic changes, loss of protein homeostasis, decreased nutrient sensing ability, mitochondrial abnormalities, cell aging, stem cell exhaustion, and changes in cell communication. Studies have shown that the metabolic drivers of cellular aging include: mitochondrial dysfunction, which breaks down NAD in the cytoplasm. + / NADH ratio, production of reactive oxygen species, and other potential mechanisms driving aging; decreased sirtuin expression, loss of PARP activity, and altered cellular redox status lead to NAD + Loss of energy can also lead to aging; autophagy dysfunction can cause aging in some cases, but can also prevent aging in other cases; high concentrations of non-physiological oxygen promote aging, and so on.

[0003] Currently, there are few effective anti-aging products that cannot meet people's urgent needs. Summary of the Invention

[0004] The present invention aims to provide a use of mulberry extract in the preparation of anti-aging products. Mulberry extract has good anti-aging effects.

[0005] Based on the weight percentage of the sum of the components of the mulberry extract as 100%, the mulberry extract contains 3% or more of alkaloids by weight (optionally containing 3-99% of alkaloids by weight, further optionally containing 15-99% of alkaloids by weight, further optionally containing 30-99% of alkaloids by weight, further optionally containing 40-99% of alkaloids by weight, further optionally containing 50-99% of alkaloids by weight, further optionally containing 60-99% of alkaloids by weight),

[0006] and / or contains no more than 70% polysaccharide by weight (optionally containing 0.2-70% polysaccharide by weight, further optionally containing 0.2-50% polysaccharide by weight, further optionally containing 0.2-35% polysaccharide by weight, further optionally containing 0.2-25% polysaccharide by weight, further optionally containing 0.2-23% polysaccharide by weight, further optionally containing 20-25% polysaccharide by weight),

[0007] and / or contains flavonoids in an amount not higher than 10% by weight (optionally containing flavonoids in an amount of 0.05-5% by weight, further optionally containing flavonoids in an amount of 0-2% by weight, further optionally containing flavonoids in an amount of 0.05-2% by weight, further optionally containing flavonoids in an amount of 0.5-1.5% by weight, further optionally containing flavonoids in an amount of 0-1% by weight, further optionally containing flavonoids in an amount of 0.05-1% by weight),

[0008] and / or contains no more than 50% by weight of amino acids (optionally containing 0-30% by weight of amino acids, further optionally containing 0-25% by weight of amino acids, further optionally containing 0-20% by weight of amino acids, further optionally containing 0-5% by weight of amino acids, further optionally containing 3-25% by weight of amino acids or further optionally containing 5-20% by weight of amino acids),

[0009] and / or other components (the weight content can be optionally 0-25%, further optionally 0-20%, further optionally 0-15%, further optionally 0-11%, further optionally 2-20%, further optionally 4-8%).

[0010] In addition, the present invention also provides an anti-aging method, comprising administering a therapeutically and / or preventively effective amount of an anti-aging product comprising the mulberry extract to a patient in need thereof.

[0011] Another aspect of the present invention provides use of the mulberry extract for anti-aging.

[0012] Optionally, the anti-aging product is selected from at least one of food, medicine, beverage, or health care product. Optionally, the anti-aging product is a food additive; Optionally, the anti-aging product is a health care product.

[0013] Optionally, the anti-aging includes delaying aging of the human body and / or animal body, aging of tissues and organs, and / or cellular aging;

[0014] Preferably, the aging is natural aging; optionally, the aging is caused by any one or more of oxidative stress damage, mitochondrial dysfunction, DNA damage, and telomere dysfunction; optionally, the oxidative stress damage or mitochondrial dysfunction may be caused by any one or more of excessive fatty acids, D-galactose, and adenine; optionally, the DNA damage or telomere dysfunction may be caused by doxorubicin (DOX) and / or multiple cell replication and passage;

[0015] Preferably, the tissues and organs include any one or more of heart, liver, kidney, nerve tissue, bone tissue, vascular tissue, and muscle tissue;

[0016] Preferably, the cells are any one or more of liver cells, kidney cells, endothelial cells, neuronal cells, cardiomyocytes, and myoblasts;

[0017] Preferably, the anti-aging effect is specifically manifested as any one or more of the following:

[0018] 1) Prolong the life of the body;

[0019] 2) Reduce the body's aging pigment content;

[0020] 3) Improve the body's motor behavior ability;

[0021] 4) improving the body's metabolic rate, preferably promoting an increase in body heat production;

[0022] 5) Improving mitochondrial function in tissues and organs; preferably improving the structure and number of mitochondria in tissues and organs; preferably, increasing the mitochondrial DNA copy number and the level of mitochondrial complex enzymes, or promoting mitochondrial fusion and reducing mitochondrial fission; optionally, said increasing the mitochondrial DNA copy number comprises promoting the expression of any one or more of ND1, COXI, and COXII; optionally, said increasing the level of mitochondrial complex enzymes comprises promoting the expression of any one or more of mitochondrial complex enzymes I-ndufs8, II-sdhb, III-uqcrc1, and V-atp5a1; optionally, said promoting mitochondrial fusion and reducing mitochondrial fission comprises regulating the expression of any one or more of DRP1, TFAM, TFB1, TFB2, MFN1, OPA1, NRF2, PGC1-α, HO-1, and NQO1 genes; optionally, said tissues and organs are preferably any one or more of the liver, myocardium, and kidney;

[0023] 6) Improving the antioxidant capacity of tissues, organs and / or cells; preferably, reducing the level of any one or more of 4-HNE (4-hydroxynonenoic acid), ROS (reactive oxygen species), and MDA (malondialdehyde), and preferably increasing the expression of any one or more of SOD, CAT (catalase), and GSS (reduced glutathione); optionally, improving the antioxidant capacity of tissues and organs further includes alleviating endoplasmic reticulum stress in tissues and organs, preferably reducing the expression of any one or more of ATF4, ATF6, CHOP, Bax, Caspase12, and Caspase3;

[0024] The tissue organ is preferably liver tissue and / or kidney tissue; the cells are preferably liver cells and / or kidney cells;

[0025] 7) increasing the expression of the longevity protein SIRT in tissues and organs, preferably promoting the expression of any one or more of SIRT1, SIRT3, SIRT5, and SIRT6, wherein the tissues and organs are preferably the liver and / or heart;

[0026] 8) Increase the coenzyme NAD in tissues and organs + levels and / or NAD + / NADH ratio, optionally, the tissue organ is preferably liver and / or heart;

[0027] 9) Alleviating cell cycle arrest and / or increasing cellular oxidative stress levels; the cells are preferably liver cells, endothelial cells and / or neuronal cells;

[0028] 10) reducing the expression of p53 and / or p16 genes in cells, or increasing the expression of lamin B1 gene; the cells are preferably endothelial cells and / or neuronal cells;

[0029] 11) extending telomere length in cells; the cells are preferably neuronal cells;

[0030] 12) increasing ATP levels in tissues and organs, preferably the kidneys;

[0031] 13) Improve bone density.

[0032] The present invention also provides any of the following uses of the mulberry extract or its active ingredients:

[0033] 1) Use of mulberry extract or its active ingredients in the preparation of a product for inhibiting ROS levels in HepG2 cells in vitro;

[0034] 2) Use of mulberry extract or its active ingredients in the preparation of products for delaying in vitro liver cell aging;

[0035] 3) Use of mulberry extract or its active ingredients in the preparation of products for delaying aging of endothelial cells, neuronal cells, liver cells, kidney cells, cardiomyocytes or myoblasts in vitro;

[0036] 4) Use of mulberry extract or its active ingredients in the preparation of a product for reducing the content of aging pigments in Caenorhabditis elegans;

[0037] 5) Use of mulberry extract or its active ingredients in the preparation of products for extending nematode lifespan;

[0038] 6) Use of mulberry extract or its active ingredients in the preparation of a product for reducing aging scores in mice;

[0039] 7) Use of mulberry extract or its active ingredients in the preparation of products for increasing thermogenesis in mice;

[0040] 8) Use of mulberry extract or its active ingredients in the preparation of a product for increasing bone density in mice;

[0041] Based on the weight percentage of the sum of the components of the mulberry extract as 100%, the mulberry extract contains 3% or more of alkaloids by weight (optionally containing 3-99% of alkaloids by weight, further optionally containing 15-99% of alkaloids by weight, further optionally containing 30-99% of alkaloids by weight, further optionally containing 40-99% of alkaloids by weight, further optionally containing 50-99% of alkaloids by weight, further optionally containing 60-99% of alkaloids by weight),

[0042] and / or contains no more than 70% polysaccharide by weight (optionally containing 0.2-70% polysaccharide by weight, further optionally containing 0.2-50% polysaccharide by weight, further optionally containing 0.2-35% polysaccharide by weight, further optionally containing 0.2-25% polysaccharide by weight, further optionally containing 0.2-23% polysaccharide by weight, further optionally containing 20-25% polysaccharide by weight),

[0043] and / or contains flavonoids in an amount not higher than 10% by weight (optionally containing flavonoids in an amount of 0.05-5% by weight, further optionally containing flavonoids in an amount of 0-2% by weight, further optionally containing flavonoids in an amount of 0.05-2% by weight, further optionally containing flavonoids in an amount of 0.5-1.5% by weight, further optionally containing flavonoids in an amount of 0-1% by weight, further optionally containing flavonoids in an amount of 0.05-1% by weight),

[0044] and / or contains no more than 50% by weight of amino acids (optionally containing 0-30% by weight of amino acids, further optionally containing 0-25% by weight of amino acids, further optionally containing 0-20% by weight of amino acids, further optionally containing 0-5% by weight of amino acids, further optionally containing 3-25% by weight of amino acids or further optionally containing 5-20% by weight of amino acids),

[0045] and / or other components (the weight content can be optionally 0-25%, further optionally 0-20%, further optionally 0-15%, further optionally 0-11%, further optionally 2-20%, further optionally 4-8%).

[0046] In a preferred embodiment of the present invention, based on the sum of the weight percentages of the components of the mulberry extract being 100%, the weight contents of the components in the mulberry extract are as follows:

[0047] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0048] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0049] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0050] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0051] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0052] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0053] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0054] More preferably, based on the sum of the weight percentages of the components of the mulberry extract being 100%, the weight contents of the components in the mulberry extract are:

[0055] More preferably, based on the sum of the weight percentages of the components of the mulberry extract being 100%, the weight contents of the components in the mulberry extract are:

[0056] More preferably, based on the sum of the weight percentages of the components of the mulberry extract being 100%, the weight percentages of the components in the mulberry extract are:

[0057] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0058] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0059] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0060] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0061] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0062] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0063] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0064] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0065] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0066] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0067] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0068] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0069] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0070] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0071] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0072] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0073] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0074] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0075] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0076] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0077] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0078] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0079] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0080] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0081] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0082] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0083] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0084] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0085] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0086] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0087] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0088] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0089] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0090] Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0091] Preferably, the alkaloids include 1-deoxynojirimycin (DNJ), N-methyl-1-deoxynojirimycin (N-methly-1-deoxynojirimycin), fagomine (FAG), 3-epi-fagomine, 1,4-dideoxy-1,4-imino-D-arabinitol (DAB), calystegin B2, calystegin C1, 2-oxy-(α-D- One or more of 2-O-(α-D-galactopyranosyl)-1-deoxynojirimycin, 6-O-(β-D-glucopyranosyl)-1-deoxynojirimycin, and 1,4-dideoxy-1,4-imino-(2-O-β-D-glucopyranosyl)-D-arabinitol.

[0092] Wherein, preferably, the weight percentage of DNJ is not less than 50% (preferably 60-99%) of the total alkaloids.

[0093] Preferably, the heavy metal content of the mulberry extract does not exceed 10 ppm.

[0094] The anti-aging product is in the form of an oral dosage form; optionally, the anti-aging product is in the form of a tablet, capsule, lozenge, powder, tea bag, oral solution, oral emulsion, pill, granule, syrup or powder.

[0095] In the present invention, the mulberry extract can be provided in the form of commercially available total alkaloids of Morus alba (National Medicine Standard Z20200001) or total alkaloids of Morus alba tablets (National Medicine Standard Z20200002).

[0096] Alternatively, the mulberry extract can be prepared according to the method described in CN 110393738A. In one embodiment, the preparation of the mulberry extract comprises the following steps: preparing a crude extract of a moraceae plant; optionally, separating the extract with a cationic resin and / or an anionic resin; optionally, subjecting the resin effluent to alcohol precipitation and collecting the supernatant; and optionally, concentrating and / or drying the supernatant.

[0097] Optionally, in the present invention, the preparation of the mulberry extract comprises the following steps: 1) preparing a crude extract of a moraceae plant; 2) separating the crude extract through a cationic resin and / or an optional anionic resin to obtain a resin effluent; and optionally, step 3) subjecting the resin effluent of step 2) to alcohol precipitation and collecting the supernatant; and 4) concentrating and / or drying the supernatant. Optionally, the resin effluent of step 2) may be concentrated and / or dried before being subjected to alcohol precipitation.

[0098] Optionally, the Moraceae plant is Morus multicaulis Perrott., Morus alba L., Morus atropurpurea Roxb., Morus mizuho Hotta, Morus wittiorum Hand Mazz., Morus laevigata Wall, Morus nigra Linn., Morus cathayana Hemsi., Morus serrata Roxb., Morus mongolica Schneid., Morus bombycis Koidz., Morus notabilis Schneid., Morus nigriformis Koidz., Morus yunnanensis Koidz., Morus australis Poir., Morus mongolica (Bur.) Schneid. var.diabolica Koidz.), large-leaf mulberry, weeping mulberry (Morus alba Var. Pendula Dippel), white-veined mulberry, mulberry varieties bred from the above mulberry species, and a combination of one or more of the following:

[0099] Optionally, the moraceae plant is one or more selected from the group consisting of Guangdong mulberry, Shandong mulberry, white mulberry, fine-toothed mulberry, mountain mulberry, or hybrid mulberry, and the hybrid mulberry can be Yuesang No. 11, Guisang You No. 62, or Sang Te You No. 2. Any part of the moraceae plant, including leaves, roots, branches, bark, buds, stems, and fruits, can be used, and mulberry branches, mulberry leaves, or white mulberry bark can be used.

[0100] In the present invention, the mulberry extract can be selected from mulberry branch extract, white mulberry bark extract, mulberry leaf extract or a mixed extract thereof.

[0101] In one embodiment, the mulberry extract is prepared according to the following steps: crushing the moraceae plant, heating and refluxing the extract with water and / or alcohol solution or acid water, the amount of solvent is 3-20 times that of the original medicinal material, repeating the extraction 1-3 times, combining the extracts, concentrating, applying a cation exchange resin, eluting with 0.2-3N ammonia water, applying the eluate to an anion exchange resin, collecting the non-adsorbed portion, adding ethanol, precipitating to remove impurities, concentrating and / or drying to obtain the extract.

[0102] In one embodiment, the mulberry extract is prepared according to the following steps: crushing the moraceae plant, heating and refluxing the extract with water and / or alcohol solution or acid water, the amount of solvent is 3-20 times that of the original medicinal material, repeating the extraction 1-3 times, combining the extracts, concentrating, applying a cation exchange resin, eluting with 0.2-3N ammonia water, applying the eluate to an anion exchange resin, collecting the non-adsorbed portion, concentrating and / or drying to obtain the extract.

[0103] In one embodiment, the mulberry extract is prepared according to the following steps: crushing the moraceae plant, heating and refluxing the extract with water and / or alcohol solution or acid water, the amount of solvent is 3-20 times that of the original medicinal material, repeating the extraction 1-3 times, combining the extracts, concentrating, applying a cation exchange resin, eluting with 0.2-3N ammonia water, concentrating and / or drying the eluate to obtain the extract.

[0104] In one embodiment, the mulberry extract is prepared according to the following steps: crushing the moraceae plant, heating and refluxing the extract with water, the amount of solvent is 3-20 times (optionally 4-15 times, further optionally 4-12 times) of the original medicinal material, repeating the extraction 1-3 times (the extraction time can be 0.5-3 hours each time, further optionally 1-2 hours each time), combining the extracts, concentrating, applying a cation exchange resin, eluting with 0.2-3N ammonia water, applying the eluate to an anion exchange resin, collecting the non-adsorbed portion (i.e., the anion resin effluent), adding ethanol, precipitating to remove impurities, concentrating and / or drying to obtain an extract.

[0105] Optionally, the crude extract after the concentration treatment can also be subjected to alcohol precipitation treatment before the resin separation treatment in step 2). During the alcohol precipitation treatment, ethanol is added to the crude extract, stirred and mixed, and the stirring is stopped and allowed to stand for a certain time to precipitate the insoluble matter therein. Optionally, the volume mass ratio of the added ethanol to the plant raw material is 0.2-20 times, optionally 0.4-10 times, in terms of L / kg. Further optionally, an alcohol precipitation tank is used for alcohol precipitation treatment. Optionally, the stirring speed in the alcohol precipitation treatment is 10-600rpm, optionally 40-500rpm, further optionally 80-400rpm or 300rpm.

[0106] Optionally, after the cationic resin is loaded into the column, it is activated in the order of washing with an acidic solution, washing with an alkaline solution, and washing with an acidic solution. Optionally, the alkaline solution is washed until the pH of the eluate is 8.0-9.5, optionally 8.5-9.5; optionally, the alkaline solution is selected from aqueous ammonia solution, sodium hydroxide solution, potassium hydroxide solution, or sodium carbonate solution; optionally, the concentration of the alkaline solution is 0.5-4 mol / L, optionally 1-2 mol / L. Optionally, the acidic solution is washed until the pH of the eluate is 3.0-7.0, optionally 4.5-6.5. Optionally, the acidic solution is selected from hydrochloric acid solution, phosphoric acid solution, or sodium hydrogen phosphate-citrate buffer; optionally, the concentration of the acidic solution is 0.5-4 mol / L, optionally 1.5-2 mol / L. Optionally, after the final acidic solution wash, the cationic resin can be rinsed with 3-5 column volumes of deionized water.

[0107] Optionally, the cationic resin is a combination of one or more of 732 strong acid styrene cation exchange resin, 002SC strong acid styrene cation exchange resin, 734 strong acid styrene cation exchange resin, D001 macroporous strong acid styrene cation exchange resin or D113 macroporous weak acid cation exchange resin and D254 macroporous strong basic quaternary ammonium cation exchange resin.

[0108] Optionally, the weight ratio of the cationic resin to the plant raw material is 1:1-30 (optionally, 1:1-25, 1:2-20, 1:2-15, 1:2-10, 1:2-7, 1:2-3).

[0109] After the crude plant extract is loaded onto the cationic resin, the loaded cationic resin is eluted with an eluent. Optionally, the eluent is a salt solution or alkaline solution containing cations, and can be selected from one or more of sodium chloride, ammonium chloride, ammonium sulfate, ammonium nitrate, ammonia water, potassium chloride, and sodium hydroxide.

[0110] Optionally, the concentration of the eluent is 0.04-5 mol / L (optionally 0.5-2.5 mol / L, 0.2-3 mol / L, further optionally 0.5-2.5 mol / L).

[0111] Optionally, the eluent flow rate is 1-15 BV / h (optionally 5-10 BV / h, further optionally 5-6 BV / h).

[0112] Optionally, the weight of the eluent used for cationic resin separation is 0.1-30 times the weight of the plant raw material, and optionally, the eluent is 0.5-10 times the weight of the plant raw material.

[0113] The collection starting point can be determined according to the pH of the cationic resin effluent. For example, when an alkaline solution such as aqueous ammonia is used for elution, the eluate is collected when the pH of the cationic column effluent is detected to be greater than 7, or the collection starting point of the effluent is determined based on a color development or precipitation reaction. Optionally, when the volume of the collected liquid reaches 0.1-10 times (further optionally, 0.2-5 times) the weight of the plant raw material fed, collection is stopped, and the collected liquid is optionally purified by an anion column.

[0114] When purifying by anion column, optionally, after the anion resin is loaded into the column, activation is performed in the order of washing with alkaline solution, washing with acidic solution, and washing with alkaline solution.

[0115] Optionally, washing with an alkaline solution until the pH of the eluate is 8.0-9.5, optionally 8.5-9.5;

[0116] Optionally, the alkaline solution is selected from ammonia solution, sodium hydroxide solution, potassium hydroxide solution or sodium carbonate solution; optionally, the concentration of the alkaline solution is 0.5-4 mol / L, optionally 1-2 mol / L.

[0117] Optionally, the acidic solution is washed until the pH of the eluate is 3.0-7.0, optionally 4.5-6.5. Optionally, the acidic solution is selected from hydrochloric acid solution, phosphoric acid solution, disodium hydrogen phosphate-citric acid buffer, and optionally, the concentration of the acidic solution is 0.5-4 mol / L, optionally 1-2 mol / L.

[0118] Optionally, the anion resin is a combination of one or more of 711 type strong basic styrene anion resin, 717 type strong basic styrene anion exchange resin, D201 type macroporous strong basic styrene anion exchange resin or D218 type macroporous strong basic acrylic anion exchange resin, D301-G type macroporous weak acid styrene anion exchange resin and D301 type macroporous weak basic styrene anion exchange resin.

[0119] Optionally, the weight ratio of the anion resin to the plant raw material is 1:1-80 (optionally, 1:1-64, 1:1-32, 1:1-24, 1:5-16, 1:3).

[0120] When the liquid flows out of the anion resin, collection begins. Optionally, collection is stopped when the volume of the collected liquid reaches 0.05-10 times (optionally, 0.1-5 times) the weight of the plant raw material input.

[0121] Optionally, the weight ratio of ethanol used in the alcohol precipitation treatment to the plant raw material is 1:4-600 (optionally 1:20-300, further optionally 1:20-50, 1:40, 1:80, 1:22). During the alcohol precipitation treatment, the stirring speed is 10-600 rpm (optionally 40-500 rpm, 80-400 rpm). The alcohol precipitation treatment time is 12-24 hours.

[0122] Furthermore, before the alcohol precipitation treatment, the anion resin effluent is subjected to a step of centrifugal impurity removal or microfiltration membrane filtration, followed by concentration by a reverse ion osmosis membrane. The specific gravity of the concentrated liquid can be 1.0-1.3, optionally 1.1-1.25.

[0123] Optionally, the anti-aging product further comprises a carrier acceptable for food, medicine, beverage or health product. The carrier is an inactive ingredient that is non-toxic to the human body and is consistent with the route of administration or mode of administration. The carrier can be a solid or liquid excipient. Solid excipients include, for example, microcrystalline cellulose, mannitol, lactose, pregelatinized starch, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, sodium carboxymethyl starch, aspartame, calcium hydrogen phosphate, sodium lactate, poloxamer, sodium lauryl sulfate, sodium carboxymethyl cellulose, gelatin, xanthan gum, povidone, starch, magnesium stearate, sodium carboxymethyl starch and talc; liquid excipients include, for example, water, ethanol, syrup and glycerin.

[0124] In the present invention, the mulberry extract is referred to as SZ-A. Beneficial effects:

[0125] 1. Nematode experiments showed that mulberry extract reduced the amount of aging pigment in the nematode Caenorhabditis elegans, extending its lifespan. Mouse experiments showed that mulberry extract effectively increased the survival rate of aging mice, significantly improved their aging scores, increased their basal metabolic capacity and bone density, and enhanced their motor behavior. Overall, it improved the mice's mental state, activity, hair color and looseness, muscle grip, vision, hearing, bite, dermatitis, hunchback, gait, and other aging symptoms.

[0126] 2. The effect of mulberry extract on mitochondrial function and aging-related indicators in mice. The study found that mulberry extract can regulate mitochondrial function: significantly improve the mitochondrial function of myocardium, liver, kidney and other tissues, maintain homeostasis, increase the number; promote the production of longevity proteins and NAD + Expression: Increase the expression of longevity protein SIRT and its coenzyme NAD + expression level; improve antioxidant capacity: SZ-A can reduce the level of lipid peroxidation product 4-HNE in liver tissue; alleviate endoplasmic reticulum stress: reduce tissue and cell ROS and alleviate stress damage.

[0127] 3. Cell experiments show that mulberry extract can alleviate the aging of cardiomyocytes, myoblasts, liver cells, kidney cells, endothelial cells and neuronal cells; and reduce the ROS level of HepG2 cells and renal tubular epithelial cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0128] FIG1 is an electron microscopic image of the mitochondrial ultrastructure of the liver of experimental mice in Experimental Example 1;

[0129] Figure 2 shows the expression of mitochondrial DNA copy number ND-1, COXI, COXII, mitochondrial complex enzymes I-ndufs8, II-sdhb, III-uqcrc1, V-atp5a1, and mitochondrial fission, fusion and mitochondrial generation related genes DRP1, TFAM, TFB1, TFB2, MFN1, OPA1, NRF2, PGC1-α, HO-1 and NQO1 in experimental mice in Experimental Example 1; ## P<0.01 vs. NC group; *P<0.05, **P<0.01, ***P<0.001 vs. HFD group.

[0130] FIG3 is a graph showing that SZ-A reduces the level of 4-HNE, a lipid peroxidation product, in liver tissue in Experimental Example 1; # P<0.05, vs. NC group (chow group); **P<0.01 vs. HFD group.

[0131] Figure 4 shows that mulberry extract alleviates liver endoplasmic reticulum stress in Experimental Example 1. # P<0.05 vs. NC group (CHOW group), *P<0.05, **P<0.01 vs. HFD group.

[0132] Figure 5 shows that the mulberry extract in Experimental Example 1 increases the expression of the longevity protein SIRT in the liver and heart; # P<0.05, ## P<0.01 vs. NC group (CHOW group), *P<0.05 vs. HFD group.

[0133] Figure 6: SZ-A increases NAD in high-fat fed mice in Experiment 1 + Levels and NAD + / NADH ratio; # P<0.05, ## P<0.01 vs. NC group (CHOW group), *P<0.05, **P<0.01, vs. HFD group.

[0134] Figure 7 shows the improvement of renal oxidative stress and mitochondrial function of CKD rats by long-term administration of SZ-A in Experimental Example 2, (A) SOD level in renal tissue and (B) MDA level in renal tissue, mean ± SEM, n = 5, ***p < 0.001, **p < 0.01, *p < 0.05 vs CKD, (C) RTqPCR detection of mRNA expression levels of GSS and CAT in rat renal tissue, mean ± SEM, n = 6, ***p < 0.001, **p < 0.01, *p < 0.05 vs CKD, (D) SZ-A improves H2O2-induced ROS production in HK2 and NRK-52E cells, (E) SZ-A improves mitochondrial number, arrangement and structure in the kidneys of CKD rats, 7000x, scale bar: 2μm, 15000x, scale bar: 1μm, (F) SZ-A increases ATP content and (G) mitochondrial copy number in kidney tissue of CKD rats, mean ± SEM, n = 6, ***p < 0.001, **p < 0.01, *p < 0.05 vs CKD.

[0135] FIG8 is a graph showing that SZ-A alleviates the increase of ROS in liver cells caused by PA / OA in Experimental Example 3; # P<0.05 vs. BSA group, **P<0.01 vs. PA / OA group.

[0136] Figure 9: SZ-A reduces p16 and p53 gene expression in Experimental Example 3; # P<0.05, ## P<0.01 vs.D-gal-500 (or dox-1000) group, ***P<0.001 vs.NC group.

[0137] Figure 10 is the effect of SZ-A on P53 (A) and LaminB1 (B) gene expression in the D-gal-induced HT22 neuronal cell aging model in Experimental Example 3;

[0138] Figure 11 is the result of telomere length detection in the HT22 cell replicative aging model in Experimental Example 3 (compared with NC (blank control group), *P<0.05, **P<0.01);

[0139] Figure 12 is the staining results of the effect of SZ-A on D-gal-induced H9C2 cell senescence in Experimental Example 3;

[0140] Figure 13 shows the effect of SZ-A on D-gal-induced C2C12 cell senescence in Experimental Example 3 (A is a cell staining image, B is a quantitative statistical graph, compared with the model group, ***P<0.001, ****P<0.0001);

[0141] FIG14 is the staining results of flow cytometry detection of the effect of SZ-A on D-gal-induced C2C12 cell senescence in Experimental Example 3, which is a statistical graph of mean fluorescence intensity. Compared with the model group (D20), **P<0.01, ***P<0.001);

[0142] FIG15 is a graph showing the results of SZ-A reducing the aging pigment content in C. elegans in Experimental Example 4; *P<0.05, **P<0.01, vs. Control.

[0143] FIG16 is the result of aging appearance score after administration of SZ-A for 1 month in Experimental Example 5;

[0144] FIG17 is the result of aging appearance score after administration of SZ-A for 5 months in Experimental Example 5;

[0145] Figure 18 shows the thermogenic results of mice administered SZ-A for 4 months in Experimental Example 5 (compared with the model group G2, *P<0.05, **P<0.01);

[0146] FIG19 shows the bone density test results of mice administered SZ-A for 5 months in Experimental Example 5. DETAILED DESCRIPTION

[0147] The present invention will be further described in detail below with reference to the accompanying drawings and examples, through which the features and advantages of the present invention will become more clearly apparent.

[0148] The word “exemplary” is used exclusively herein to mean “serving as an example, example, or illustration.” Any embodiment described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments.

[0149] In addition, the technical features involved in different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0150] The content of the components involved in the present invention is detected according to the published method (reference is made to the method described in patents with publication numbers CN111077247A and CN110393738A).

[0151] Preparation Example 1

[0152] 1000 kg of fresh mulberry branches (fine-toothed mulberry, Yuesang No. 11) were crushed and added to 4000 L of water. The extracts were then heated and refluxed for 2 hours. The combined extracts were filtered to remove insoluble matter to obtain a crude extract. The crude extract was heat-concentrated to a solids content of 4% by weight and then heated to 50°C to serve as the loading solution for a cationic resin column.

[0153] A 150 kg column of D113 macroporous weakly acidic phenyl propylene-based cationic resin was loaded and washed with 2 mol / L hydrochloric acid until the eluate pH reached 4.5; then with 1 mol / L sodium hydroxide until the eluate pH reached 8.5; and finally with 2 mol / L hydrochloric acid until the eluate pH reached 4.5. The column was then rinsed with 5 column volumes of deionized water to complete activation. The concentrated extract was loaded and then eluted with 1000 L of 2.5 mol / L ammonia at a rate of 6 BV / h. The eluate from the cation column was collected when the pH was >7. When the collected solution reached 900 L, the collection was stopped and the collected solution was directly passed through an anion column for purification.

[0154] Activate the column using 62.5 kg of D218 macroporous, strongly basic acrylic anion resin. Elute with 1.5 mol / L sodium hydroxide solution until the eluate has a pH of 9.0. Elute with 1.5 mol / L hydrochloric acid solution until the eluate has a pH of 3.5. Finally, elute with 1.5 mol / L sodium hydroxide solution until the eluate has a pH of 9.0. The collected eluate from the cationic resin is loaded onto the anionic resin, and the effluent is collected until 870 L of effluent are reached.

[0155] The collected liquid was centrifuged to remove impurities and then concentrated using a reverse ion osmosis membrane. The concentrated liquid had a specific gravity of 1.25 and was transferred to an alcohol precipitation tank. 25 L of anhydrous ethanol was added with a stirring paddle at 500 rpm. After the ethanol addition was complete, stirring was stopped and the mixture was allowed to settle for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain a mulberry twig extract concentrate (i.e., SZ-A extract).

[0156] The mulberry branch extract contains 52% alkaloids, 22% polysaccharides, 0.8% flavonoids, and 20% amino acids. Among the alkaloids, 1-DNJ, FAG, and DAB account for 60%, 17%, and 15%.

[0157] Preparation Example 2

[0158] Take 10kg of fresh mulberry branches (Sang Teyou No. 2), crush them, add 150L of water, add them twice, extract them by decoction for 3h each time, combine the extracts, and filter to remove insoluble matter. The extract is hot concentrated until the solid content reaches 8%, then transferred to an alcohol precipitation tank, and 2367.9g of absolute ethanol (3L) is added under a stirring paddle at 300rpm. After the ethanol addition is complete, stop stirring, precipitate with alcohol for 24h, and take the supernatant as the loading liquid for a cationic resin column. Use 5kg of 002SC type strong acid styrene cationic resin to fill the column, and activate the cationic resin according to the method of Preparation Example 1. Load the extract after concentrated alcohol precipitation, then use 100L of 5mol / L potassium chloride to elute, with an elution rate of 5BV / h, and detect the effluent with 20% silicotungstic acid. Start collecting when a white precipitate is generated. Stop collecting when the collected liquid reaches 25L, and the collected liquid is directly purified by anion column.

[0159] A 10 kg column of 711 strong base styrene anion resin was loaded and activated according to the method of Preparation Example 1. The collected cationic resin eluate was loaded onto the anion resin, and the effluent was collected until the effluent reached 15 L. The collected eluate was reloaded onto the cationic resin and separated twice more using the cationic resin and anion resin, respectively, according to the above method.

[0160] The collected liquid obtained after three column separations was centrifuged for impurity removal and then concentrated using a reverse ion osmosis membrane. The concentrated liquid had a specific gravity of 1.25 and was transferred to an alcohol precipitation tank. 125 g of anhydrous ethanol was added under a stirring paddle at 1000 rpm. After the ethanol addition was complete, stirring was stopped, and the mixture was allowed to precipitate for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain a mulberry branch extract. Separately, fresh Morus alba bark and mulberry leaf (Sang Teyou No. 2) were extracted using the same extraction method and parameters as described above.

[0161] The obtained mulberry branch extract has an alkaloid content of 98%, a polysaccharide content of 0.2%, a flavonoid content of 0.05%, and an amino acid content of 0. Among the alkaloids, the content of 1-DNJ is 99%, FAG is 0.5%, and DAB is 0.4%.

[0162] The obtained Morus alba bark extract has an alkaloid content of 95%, a polysaccharide content of 2%, a flavonoid content of 0.1%, and an amino acid content of 1%. Among the alkaloids, the content of 1-DNJ is 96%, FAG is 1.5%, and DAB is 1.4%.

[0163] The mulberry leaf extract contains 90% alkaloids, 4% polysaccharides, 0.1% flavonoids, and 3% amino acids. Among the alkaloids, 1-DNJ, FAG, and DAB account for 91%, 3.1%, and 2.8%, respectively.

[0164] Preparation Example 3

[0165] 1000 kg of fresh mulberry branches (Mulberry twigs) were crushed, added to 11,500 L of water, and heated under reflux for 2 hours. The extracts were combined and filtered to remove insoluble matter to obtain a crude extract. This crude extract was first centrifuged to remove impurities and then concentrated using a counter-ion permeation membrane to a solids content of 1% by weight. This was then used as the loading solution for the cationic resin column.

[0166] A column was loaded with 300 kg of D001 macroporous, strongly acidic styrene-based cationic resin, which was activated according to the method of Preparation Example 1. The concentrated crude extract was loaded and eluted with 5000 L of 0.04 mol / L ammonium nitrate at a rate of 5 BV / h. The effluent was tested with 20% silicotungstic acid. Collection began when a white precipitate formed and stopped when the collected solution reached 1000 L.

[0167] The collected liquid obtained after the cationic column separation is concentrated by nanofiltration membrane and concentrated under reduced pressure to obtain an extract concentrate.

[0168] The mulberry branch extract contains 15% alkaloids, 20% polysaccharides, 7% flavonoids, and 45% amino acids. Among the alkaloids, 1-DNJ, FAG, and DAB account for 55%, 23%, and 10%, respectively.

[0169] Preparation Example 4

[0170] Take 333 kg of dry mulberry branches (Yue Sang No. 11), crush them, add 4000 L of water, and extract them twice by heating reflux method, each time reflux for 1 hour, combine the extracts, filter, and concentrate the extracts to 1 kg of crude drug / L.

[0171] A 150 kg column of D113 macroporous weakly acidic phenyl propylene-based cationic resin was loaded and washed with 2 mol / L hydrochloric acid until the eluate pH reached 4.5; then with 1 mol / L sodium hydroxide until the eluate pH reached 8.5; and finally with 2 mol / L hydrochloric acid until the eluate pH reached 4.5. The column was then rinsed with 5 column volumes of deionized water to complete activation. The concentrated extract was loaded and then eluted with 1000 L of 2.5 mol / L ammonia at a rate of 6 BV / h. The eluate from the cation column was collected when the pH was >7. When the collected solution reached 900 L, the collection was stopped and the collected solution was directly passed through an anion column for purification.

[0172] Activate the column using 125 kg of D218 macroporous, strongly basic acrylic anion resin. Elute with 1.5 mol / L sodium hydroxide solution until the eluate has a pH of 9.0. Elute with 1.5 mol / L hydrochloric acid solution until the eluate has a pH of 3.5. Finally, elute with 1.5 mol / L sodium hydroxide solution until the eluate has a pH of 9.0. The collected cationic resin eluate is loaded onto the anion resin, and the effluent with a pH greater than 8 is collected until the effluent reaches 870 L.

[0173] The collected liquid after anion column separation was filtered through a microfiltration membrane to remove impurities and then concentrated using a counter-ion osmosis membrane. The concentrated liquid had a specific gravity of 1.1 and was transferred to an alcohol precipitation tank. 15 kg of anhydrous ethanol was added with a stirring paddle at 400 rpm. After the ethanol addition was complete, stirring was stopped. The mixture was allowed to settle for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain a mulberry twig extract. The sample contained 80% alkaloids, 5% polysaccharides, 0.1% flavonoids, and 4% amino acids. Among the alkaloids, 1-DNJ accounted for 75%, FAG 12%, and DAB 10%.

[0174] Preparation Example 5

[0175] Take 400 kg of dry mulberry branches (Yue Sang No. 11), crush them, add 4000 L of water, and extract them twice by heating reflux method, each time reflux for 1 hour, combine the extracts, filter, and concentrate the extracts to 1 kg of crude drug / L.

[0176] Activate the column using 62.5 kg of D218 macroporous, strongly basic acrylic anion resin. Elute with 1.5 mol / L sodium hydroxide solution until the eluate has a pH of 9.0; then with 1.5 mol / L hydrochloric acid solution until the eluate has a pH of 3.5; and finally with 1.5 mol / L sodium hydroxide solution until the eluate has a pH of 9.0. The collected extract concentrate was loaded onto the anion resin, and the effluent was collected.

[0177] The collected solution after anion column separation was filtered through a microfiltration membrane to remove impurities, then concentrated using a counter-ion osmosis membrane. Further vacuum concentration and drying yielded a mulberry twig extract concentrate. The sample contained 3% alkaloids, 70% polysaccharides, 10% flavonoids, and 10% amino acids. Among the alkaloids, 1-DNJ accounted for 68%, FAG 17%, and DAB 8%.

[0178] Preparation Example 6

[0179] 1500 kg of fresh mulberry branches (fine-toothed mulberry, Yuesang No. 11) were crushed and added to 6000 L of water. The extracts were heated and refluxed for 2 hours. The combined extracts were filtered to remove insoluble matter to obtain a crude extract. The crude extract was heat-concentrated to a solids content of 4% by weight and then heated to 50°C as the loading solution for a cationic resin column.

[0180] A 100 kg column of D113 macroporous weakly acidic phenyl propylene-based cationic resin was loaded and washed with 2 mol / L hydrochloric acid until the eluate pH reached 4.5; then with 1 mol / L sodium hydroxide until the eluate pH reached 8.5; and finally with 2 mol / L hydrochloric acid until the eluate pH reached 4.5. The column was then rinsed with 5 column volumes of deionized water to complete activation. The concentrated extract was loaded and then eluted with 1000 L of 2.5 mol / L ammonia at a rate of 6 BV / h. The eluate from the cation column was collected when the pH was >7. When the collected solution reached 900 L, the collection was stopped and the collected solution was directly passed through an anion column for purification.

[0181] A column was loaded with 62.5 kg of D218 macroporous, strongly alkaline acrylic anion resin. The column was washed with a 1.5 mol / L sodium hydroxide solution until the eluate had a pH of 9.0; then washed with a 1.5 mol / L hydrochloric acid solution until the eluate had a pH of 3.5; and finally washed with a 1.5 mol / L sodium hydroxide solution until the eluate had a pH of 9.0. Activation was completed. The collected cationic resin eluate was loaded onto an anionic resin, and the effluent was collected until the effluent reached 870 L. The effluent was concentrated under reduced pressure to obtain a mulberry branch extract concentrate, which contained 30% alkaloids, 35% polysaccharides, 2% flavonoids, and 25% amino acids. Among the alkaloids, the content of 1-DNJ was 62%, FAG was 20%, and DAB was 13%.

[0182] Preparation Example 7

[0183] 1000 kg of fresh mulberry branches (fine-toothed mulberry, Yuesang No. 11) were crushed and added to 4000 L of water. The extracts were then heated and refluxed for 2 hours. The combined extracts were filtered to remove insoluble matter to obtain a crude extract. The crude extract was heat-concentrated to a solids content of 4% by weight and then heated to 50°C to serve as the loading solution for a cationic resin column.

[0184] A 100 kg column of D113 macroporous weakly acidic phenyl propylene-based cationic resin was loaded and washed with 2 mol / L hydrochloric acid until the eluate pH reached 4.5; then with 1 mol / L sodium hydroxide until the eluate pH reached 8.5; and finally with 2 mol / L hydrochloric acid until the eluate pH reached 4.5. The column was then rinsed with 5 column volumes of deionized water to complete activation. The concentrated extract was loaded and then eluted with 1000 L of 2.5 mol / L ammonia at a rate of 6 BV / h. The eluate from the cation column was collected when the pH was >7. When the collected solution reached 900 L, the collection was stopped and the collected solution was directly passed through an anion column for purification.

[0185] A column was loaded with 62.5 kg of D218 macroporous, strongly alkaline acrylic anion resin and washed with 1.5 mol / L sodium hydroxide solution until the eluate pH reached 9.0; then washed with 1.5 mol / L hydrochloric acid solution until the eluate pH reached 3.5; and finally washed with 1.5 mol / L sodium hydroxide solution until the eluate pH reached 9.0 to complete activation. The collected cationic resin eluate was loaded onto an anion resin, and the effluent was collected until the effluent reached 870 L. The effluent was concentrated under reduced pressure to obtain a mulberry branch extract concentrate, which contained 40% alkaloids, 25% polysaccharides, 0.5% flavonoids, and 25% amino acids. Among the alkaloids, the content of 1-DNJ was 57%, FAG was 24%, and DAB was 16%.

[0186] Preparation Example 8

[0187] Take 333 kg of dry mulberry branches (Yue Sang No. 11), crush them, add 4000 L of water, and extract them twice by heating reflux method, each time reflux for 1 hour, combine the extracts, filter, and concentrate the extracts to 1 kg of crude drug / L.

[0188] A 150 kg column of D113 macroporous weakly acidic phenyl propylene-based cationic resin was loaded and washed with 2 mol / L hydrochloric acid until the eluate pH reached 4.5; then with 1 mol / L sodium hydroxide until the eluate pH reached 8.5; and finally with 2 mol / L hydrochloric acid until the eluate pH reached 4.5. The column was then rinsed with 5 column volumes of deionized water to complete activation. The concentrated extract was loaded and then eluted with 1000 L of 2.5 mol / L ammonia at a rate of 6 BV / h. The eluate from the cation column was collected when the pH was >7. When the collected solution reached 900 L, the collection was stopped and the collected solution was directly passed through an anion column for purification.

[0189] Activate the column using 62.5 kg of D218 macroporous, strongly basic acrylic anion resin. Elute with 1.5 mol / L sodium hydroxide solution until the eluate has a pH of 9.0. Elute with 1.5 mol / L hydrochloric acid solution until the eluate has a pH of 3.5. Finally, elute with 1.5 mol / L sodium hydroxide solution until the eluate has a pH of 9.0. The collected cationic resin eluate is loaded onto the anion resin, and the effluent with a pH greater than 8 is collected until the effluent reaches 870 L.

[0190] The collected liquid after anion column separation was filtered through a microfiltration membrane to remove impurities and then concentrated using a counter-ion osmosis membrane. The concentrated liquid had a specific gravity of 1.1 and was transferred to an alcohol precipitation tank. 15 kg of anhydrous ethanol was added with a stirring paddle at 400 rpm. After the ethanol addition was complete, stirring was stopped. The mixture was allowed to settle for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain a mulberry branch extract. The sample contained 63% alkaloids, 23% polysaccharides, 1% flavonoids, and 5% amino acids. Among the alkaloids, 1-DNJ accounted for 61.9%, FAG was 16.6%, and DAB was 11.1%.

[0191] Preparation Example 9

[0192] 1000 kg of fresh mulberry branches (Yue Sang No. 11) were crushed and added to 4000 L of water. The extracts were then heated and refluxed for 2 hours. The combined extracts were filtered to remove insoluble matter to obtain a crude extract. The crude extract was then heat-concentrated to a solids content of 4% and maintained at 50°C as the loading solution for a cationic resin column.

[0193] A 120 kg column of D113 macroporous weakly acidic phenyl propylene-based cationic resin was loaded and washed with 2 mol / L hydrochloric acid until the eluate pH reached 4.5; then with 1 mol / L sodium hydroxide solution until the eluate pH reached 8.5; then with 2 mol / L hydrochloric acid until the eluate pH reached 4.5; and finally with 5 column volumes of deionized water to complete the activation. The concentrated extract was loaded and then eluted with 1000 L of 2.5 mol / L ammonia at a rate of 6 BV / h. The eluate from the cation column was collected when the pH was > 7. Collection was stopped when 900 L of the collected solution reached and the collected solution was directly passed through an anion column for purification.

[0194] Activate the column using a 45 kg column of D218 macroporous, strongly basic acrylic anion resin. Elute with 1.5 mol / L sodium hydroxide solution until the eluate has a pH of 9.0. Elute with 1.5 mol / L hydrochloric acid solution until the eluate has a pH of 3.5. Elute with 1.5 mol / L sodium hydroxide solution until the eluate has a pH of 9.0. The collected cation resin eluate is loaded onto the anion resin, and the effluent is collected until 870 L of effluent are reached.

[0195] After anion column separation, the collected liquid was filtered through a microfiltration membrane to remove impurities and then concentrated using a counter-ion permeation membrane. The concentrated liquid had a specific gravity of 1.1 and was transferred to an alcohol precipitation tank. 15 kg of anhydrous ethanol was added with a stirring paddle at 300 rpm. After the ethanol addition was complete, stirring was stopped. The alcohol precipitation was allowed to proceed for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain an extract. The sample contained 70% alkaloids by weight, 20% polysaccharides by weight, 0.6% flavonoids by weight, and 5% amino acids by weight. Among the alkaloids, the content of 1-DNJ was 70%, FAG was 13%, and DAB was 10%.

[0196] Preparation Example 10

[0197] 80 kg of fresh mulberry branches (Guisangyou No. 62) were crushed and added to 1000 L of water. The mixture was extracted by heating and refluxing for 2 h. The extracts were combined and filtered to remove insoluble matter to obtain a crude extract. The crude extract was heat-concentrated to a solids content of 4% and then heated at 50°C to serve as the loading solution for a cationic resin column.

[0198] A 50 kg column of D113 macroporous weakly acidic phenyl propylene-based cationic resin was loaded and washed with 2 mol / L hydrochloric acid until the eluate pH reached 4.5; then with 1 mol / L sodium hydroxide until the eluate pH reached 8.5; then with 2 mol / L hydrochloric acid until the eluate pH reached 4.5; and finally with 5 column volumes of deionized water to complete the activation. The concentrated extract was loaded and then eluted with 600 L of 2.5 mol / L ammonia at a rate of 6 BV / h. The eluate was collected when the pH of the cation column effluent was >7. Collection was stopped when 9800 L of the collected solution reached and the collected solution was directly passed through an anion column for purification.

[0199] Activate the column using a 65 kg column of D218 macroporous, strongly basic acrylic anion resin. Elute with 1.5 mol / L sodium hydroxide solution until the eluate has a pH of 9.0; then with 1.5 mol / L hydrochloric acid solution until the eluate has a pH of 3.5; and finally with 1.5 mol / L sodium hydroxide solution until the eluate has a pH of 9.0. The collected eluate from the cationic resin is loaded onto the anionic resin, and the effluent is collected until 750 L of effluent has been collected. Reload the collected eluate onto the cationic resin and repeat the separation using the cationic and anionic resins, respectively, as described above.

[0200] The collected liquid from these two separations was filtered through a microfiltration membrane to remove impurities and then concentrated using a counter-ion osmosis membrane. The concentrated liquid had a specific gravity of 1.1 and was transferred to an alcohol precipitation tank. 400 g of anhydrous ethanol was added with a stirring paddle at 350 rpm. Stirring was stopped after the ethanol addition, and the mixture was allowed to settle for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain an extract. Sample content: 90% by weight of alkaloids, 3% by weight of polysaccharides, 0.5% by weight of flavonoids, and 2% by weight of amino acids.

[0201] Among the alkaloids, the content of 1-DNJ is 94%, FAG is 2%, and DAB is 1%.

[0202] Test Example 1

[0203] Six-week-old healthy male C57 mice were randomly divided into a normal group (NC), a model group (HFD), and a mulberry extract (SZ-A) group, with 10 mice in each group. The mulberry extract was the mulberry extract of Preparation Example 8. The mice in the normal group were fed with a basal feed, and the HFD group and the SZ-A group were fed with a high-fat diet (Research Diet, D12492, 60kcal% Fat). After 14 weeks of feeding, the corresponding drugs were given to each group of mice by gavage every day for 6 consecutive weeks. The SZ-A group was gavaged with 400 mg / kg / d of total alkaloids, and the normal group and the model group were gavaged with the corresponding dose of solvent saline. During the drug treatment, the general condition of the mice was observed. After the end of the administration, all mice were fasted for 12 hours, weighed, blood was collected from the eyeballs, serum was separated by centrifuge, and the liver and heart tissues of each mouse were removed for the following related tests.

[0204] (1) SZ-A improves the structure and quantity of mitochondria in the liver and myocardium

[0205] Electron Microscopy: To observe mitochondrial ultrastructure, mouse liver and myocardial tissues were fixed in 2.5% glutaraldehyde solution at 4°C for 4 h and postfixed in 1% osmium hydroxide in 0.1 M phosphate buffer at 4°C for 1 h. After dehydration, infiltration, and embedding, liver tissue samples were cut and stained with uranyl acetate. Images were obtained using an 80 kV transmission electron microscope (TEM H7650, Hitachi, Tokyo, Japan), as shown in Figure 1.

[0206] Mitochondrial DNA extraction and detection: Take 50 mg of test mouse tissue, add 1 mL of cold homogenate, homogenize with a glass homogenizer, then centrifuge at 1000 g for 3 min at 4 °C, discard the precipitate. Take the supernatant, centrifuge at 12000 g for 10 min at 4 °C,

[0207] Centrifuge to obtain the crude mitochondria. Dissolve the pellet in 100 mL of a solution containing 50 mmol / L glucose, 10 mmol / L Na2EDTA, and 25 mmol / L Tris-HCl (pH 8.0) and mix thoroughly. Add 200 mL of freshly prepared 1% SDS and 0.2 N NaOH, shake gently, and place on ice for 5 minutes. Add 150 mL of 3 M NaAc (pH 4.8), shake gently, and place on ice for 5 minutes. Centrifuge at 12,000 g for 10 minutes at 4°C. Remove the supernatant and add an equal volume of a solution containing phenol, chloroform, and isoamyl alcohol in a 25:24:1 volume ratio. Extract the supernatant 2 to 4 times. Once the white material is removed from the interface, centrifuge at 10,000 g for 5 minutes. Remove the supernatant and add two volumes of 95% ethanol. Let stand at room temperature for 15 minutes, then centrifuge at 12,000 g for 10 minutes. Discard the supernatant, wash the pellet once with 75% ethanol, and centrifuge at 12,000 g for 5 min. Discard the supernatant, collect the pellet, and vacuum-dry it to obtain mitochondrial DNA. Dissolve it in an appropriate amount of TE buffer and store at -20°C. Fluorescence quantitative PCR was used to detect mitochondrial copy number, including expression of ND1, COXI, and COXII; expression of mitochondrial complex enzymes I-ndufs8, II-sdhb, III-uqcrc1, and V-atp5a1; and expression of genes involved in mitochondrial fission, fusion, and mitochondrial biogenesis, including DRP1, TFAM, TFB1, TFB2, MFN1, OPA1, NRF2, PGC1-α, HO-1, and NQO1, as shown in Figure 2.

[0208] The results in Figures 1 and 2 indicate that SZ-A can protect the mitochondrial structure of liver and myocardium, increase the mitochondrial DNA copy number and the level of complex enzymes, promote mitochondrial fusion, and reduce fission.

[0209] (2) Improve the antioxidant capacity of liver tissue

[0210] 4-HNE Immunohistochemistry: Liver tissue from experimental mice was fixed in 10% neutral buffered formalin overnight, washed once with PBS, and stored in 70% ethanol at 4°C. The tissue was dehydrated and embedded in paraffin. The embedded tissue was sectioned at 5 μm thickness for 4-HNE staining. The primary antibody, 4-HNE (ab46545, Abcam, 1:200), was incubated overnight at 4°C. Staining was performed using a peroxidase substrate kit, and microscopic images were taken, as shown in Figure 3.

[0211] The results in Figure 3 show that SZ-A can reduce the level of 4-HNE, a lipid peroxidation product in liver tissue, and restore antioxidant capacity.

[0212] (3) Alleviate liver endoplasmic reticulum stress

[0213] GRP78 immunohistochemistry: Liver tissue from test mice was fixed in 10% neutral buffered formalin overnight, washed once with PBS, and stored in 70% ethanol at 4°C. The tissue was dehydrated and embedded in paraffin. The embedded tissue was sectioned at 5 μm thickness for immunohistochemical staining. The primary antibody was incubated overnight at 4°C. Staining was performed using a peroxidase substrate kit, and microscopic images were taken, as shown in Figure 4.

[0214] mRNA expression detection: use Total RNA was isolated from the liver tissue of experimental mice (Invitrogen, Carlsbad, CA, USA). RNA was quantified by Nano-300 microspectrophotometer (AllSheng, Hangzhou, China), and 1 μg of RNA was reverse transcribed into cDNA using a reverse transcription system (Promega, Madison, WI, USA). The PCR amplification protocol consisted of 40 cycles of 95°C for 30 s, 95°C for 10 s, and 60°C for 30 s. The purity of the PCR products was determined by melting curve analysis. 2 -ΔΔCT The relative amount of each gene was calculated as shown in Figure 4.

[0215] The results in Figure 4 show that in the model group, the ER stress marker GRP78 and the pro-apoptotic protein CHOP were elevated, the downstream anti-apoptotic protein Bcl-2 function was inhibited, and the pro-apoptotic protein Bax was activated, leading to ER stress-induced hepatocyte apoptosis. SZ-A reduced the expression of ER stress-related pro-apoptotic genes (ATF4, ATF6, CHOP, Bax, Caspase12, and Caspase3) in liver tissue, alleviating HFD-induced hepatocyte stress damage.

[0216] (4) Increase the expression of the longevity protein SIRT in the liver and heart

[0217] mRNA expression detection: use Total RNA was isolated from myocardial and liver tissues of experimental mice (Invitrogen, Carlsbad, CA, USA). RNA was quantified by Nano-300 microspectrophotometer (AllSheng, Hangzhou, China), and 1 μg of RNA was reverse transcribed into cDNA using a reverse transcription system (Promega, Madison, WI, USA). The PCR amplification protocol consisted of 40 cycles of 95°C for 30 s, 95°C for 10 s, and 60°C for 30 s. The purity of the PCR products was determined by melting curve analysis. 2 -ΔΔCT The relative amount of each gene was calculated as shown in Figure 5.

[0218] The results showed that mulberry extract SZ-A can significantly increase the expression levels of longevity genes sirt1, sirt3, sirt5, and sirt6 in myocardial and liver tissues.

[0219] (6) Increase NAD + Level

[0220] NAD + Detection method: After washing the tissue with ice-cold PBS, weigh about 10-30 mg of tissue sample, chop it with scissors, place it in a homogenizer, and add 400 μL of NAD + / NADH extract was homogenized at room temperature or on ice. Then centrifuged at 12,000g, 4℃ for 5-10 minutes and the supernatant was used as the sample to be tested. + , NADH content or NAD + Determination of the ratio of NADH to NAD+: Pipette 50-100 μL of the sample to be tested into a centrifuge tube and heat it in a 60°C water bath or PCR instrument for 30 minutes to decompose NAD+. If insoluble matter is produced after heating, centrifuge at 10,000g for 5 minutes at room temperature or 4°C, and pipette 20 μL of the supernatant as the sample to be tested into a 96-well plate. Incubate at 37°C in the dark for 10 minutes. The purpose of this incubation step is to decompose the NAD+ in the sample. + Converted to NADH. Mix the colorimetric solution thoroughly, then add 10 μL of colorimetric solution to each well. Mix thoroughly, and incubate at 37°C in the dark for 30 minutes. Orange-yellow formazan will form. Measure the absorbance at 450 nm, as shown in Figure 6.

[0221] The results showed that SZ-A could increase NAD + Levels and NAD + / NADH ratio. Studies have shown that as people age, NAD + The concentration of NAD decreases by about 10% to 80%. Many diseases are related to + Reduced levels of NAD are associated with metabolic disorders, cancer, and neurodegenerative diseases. + NAD levels can improve physiological functions and increase healthy lifespan. + The NAD / NADH ratio is the key to regulating the production of ATP in cells, regulating the redox state and combating oxidative stress. + The anti-aging effect is through PARPs and sirtuins.

[0222] Experimental Example 2 Effects of long-term administration of SZ-A on renal oxidative stress and mitochondrial function in rats with chronic kidney disease (CKD)

[0223] 1. Experimental Design

[0224] 1. Experimental Materials

[0225] 1.1 Reagents

[0226] CCK8 kit (Beiren Chemical Technology Co., Ltd.), adenine (Sigma), DMEM / F-12 medium (Thermo Fisher Scientific), fetal bovine serum (Thermo Fisher Scientific), ATP detection kit (Beyotime), DNA extraction kit (QIAamp), ROS detection reagent DCFH-DA (Solarbio), SOD detection kit, MDA detection kit (Beyotime), primary antibodies for CD45, CD68, COL1A1, Fibronectine, α-SMA, and COL4A1, and secondary fluorescent antibodies (Abcam).

[0227] 1.2 Experimental drugs

[0228] The mulberry branch extract prepared in Preparation Example 1 was prepared at different concentrations and dissolved in water.

[0229] 1.3 Experimental animals

[0230] Wistar rats, male, 200 g, were purchased from Beijing Sibeifu Experimental Animal Technology Co., Ltd. and housed in the SPF animal room of Zhongsheng Beidong (Beijing) Technology Co., Ltd. with free access to ordinary feed and drinking water. The experimental period was 5 weeks.

[0231] 2. Experimental methods

[0232] Animal experiments:

[0233] Male Wistar rats were divided into six groups of eight rats each and administered intragastrically. Specific experimental groups, dosage, and dosing frequency are shown in Table 1. After one week of adaptive feeding, the rats were modeled and administered simultaneously for four consecutive weeks. Whole blood and kidney tissue were isolated, and serum uric acid, urea nitrogen, and creatinine levels were measured using kits.

[0234] Table 1

[0235] 2. Experimental Results

[0236] The effects of long-term administration of SZ-A on renal oxidative stress and mitochondrial function in CKD rats are as follows:

[0237] As shown in Figure 7 AC, the enzymes and products related to oxidative stress in the kidney tissue of rats were detected. The results showed that compared with the NC group, the activity of superoxide dismutase SOD, which has an antioxidant effect, in the kidneys of rats in the model group was significantly reduced, and the gene expressions of H2O2 oxidase CAT and glutathione synthetase GSS were significantly reduced. Correspondingly, the peroxidation product MDA was significantly increased; SZ-A treatment can increase the expression of SOD, CAT and GSS, reduce the level of MDA, and play a role in improving the body's oxidative stress.

[0238] The results of the cell experiment in Figure 7D show that for both renal tubular epithelial cells, SZ-A can reduce the production of ROS (labeled with green fluorescence in the figure) induced by H2O2, verifying its direct anti-oxidative stress effect.

[0239] Furthermore, transmission electron microscopy results (Figure 7E) showed that the mitochondria in the kidneys of the model group were reduced in number, disordered in arrangement, deformed and distorted in morphology, slightly swollen, with partially pale matrix, dilated cristae, and some ruptures and disappearances. SZ-A can improve these mitochondrial changes.

[0240] ATP and mitochondrial DNA copy number in rat kidney tissue were detected, and the results showed (as shown in Figure 7F and G) that SZ-A could significantly improve ATP deficiency and reduced mitochondrial copy number in the kidneys of CKD rats, and alleviate mitochondrial damage.

[0241] Test Example 3 Cell Test

[0242] (1) SZ-A reduces ROS levels in HepG2 cells

[0243] Cell Culture and ROS Detection: HepG2 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin / streptomycin at 37°C and 5% CO2. A PA / OA stock solution was prepared and then conjugated to 1% fatty acid-free bovine serum albumin (BSA). The cells were treated with SZ-A (the mulberry branch extract from Preparation Example 8, at a drug concentration of 50 μg / mL, calculated as alkaloids). An appropriate amount of PA / OA (palmitic acid / oleic acid) was added to the DMEM culture medium. DCFH-DA was diluted 1:1000 in serum-free culture medium to a final concentration of 10 μmol / L. The cell culture medium was removed and an appropriate volume of diluted DCFH-DA was added. The volume added was sufficient to cover the cells; typically, at least 1 mL of diluted DCFH-DA was added to one well of a six-well plate. The cells were incubated at 37°C in a cell culture incubator for 20 minutes. The cells were washed three times with serum-free culture medium to fully remove any DCFH-DA that had not entered the cells. Detection was performed using flow cytometry, as shown in FIG8 .

[0244] The results showed that SZ-A can alleviate the increase in ROS in liver cells caused by PA / OA. Mitochondria serve as the energy source for maintaining cellular and overall physiological activities and are also the primary site of ROS generation. Excessive ROS production can cause lipid peroxidation, leading to mitochondrial degeneration, leakage, and rupture, all of which are key factors in cellular aging. These results demonstrate that SZ-A can effectively scavenge mitochondrial ROS.

[0245] (2) SZ-A alleviates aging of endothelial cells and neuronal cells

[0246] Senescent cells will undergo cell cycle arrest. Doxorubicin (DOX) was used to induce human endothelial cells (EA.hy926) and D-galactose (D-gal) was used to induce mouse hippocampal neuronal cell line (HT22) to simulate cell senescence. Continuous subculture was used to establish a replicative senescence model of HT22 cells.

[0247] EA.hy926 cells were cultured in DMEM supplemented with fetal bovine serum (10% v / v), penicillin (100 U / mL), and streptomycin (100 μg / mL). The medium was changed every other day, and cells were subcultured when they reached 80% confluence. DOX was dissolved in DMEM to prepare a DOX stock solution. To simulate the aging phenotype, EA.hy926 cells were seeded into 6-well plates and incubated with 1000 mM DOX (DOX-1000) for 24 hours. Simultaneously with DOX treatment, different concentrations of the mulberry branch extract from Preparation Example 8 were added.

[0248] HT22 cells were cultured in DMEM supplemented with fetal bovine serum (10% v / v), penicillin (100 U / mL) and streptomycin (100 μg / mL). The culture medium was changed every other day, and the cells were subcultured when they were 80% confluent. D-gal was dissolved in DMEM culture medium. To simulate the aging phenotype, HT22 cells were seeded into 6-well plates and incubated with D-gal for 48 hours. Simultaneously with the D-gal treatment, the mulberry branch extract of Preparation Example 8 was added (dosage concentration 200 μg / mL, calculated as alkaloid). In the same manner, the mulberry branch extracts of Preparation Example 6, Preparation Example 8 and Preparation Example 10 were treated (dosage concentrations 45 μg / mL, 83 μg / mL, 166 μg / mL, 333 μg / mL, calculated as extract).

[0249] Fluorescence quantitative PCR was used to detect the mRNA of p16, LaminB1 and p53: Total RNA was isolated from liver tissue (Invitrogen, Carlsbad, CA, USA). RNA was quantified by Nano-300 microspectrophotometer (AllSheng, Hangzhou, China), and 1 μg of RNA was reverse transcribed into cDNA using a reverse transcription system (Promega, Madison, WI, USA). The PCR amplification protocol consisted of 40 cycles of 95°C for 30 s, 95°C for 10 s, and 60°C for 30 s. The purity of the PCR products was determined by melting curve analysis. 2 -ΔΔCT The relative amount of each gene was calculated.

[0250] The primer sequences are as follows:

[0251] p16, forward sequence as shown in SEQ 1: 5′-TCAAGACATCGTGCGATATTTG-3′;

[0252] The reverse sequence is shown in SEQ 2: 5'-TTAGCTCTGCTCTTGGGATTG-3';

[0253] P53, forward sequence is shown in SEQ 3: 5′-GCCATCTACAAGAAGTCACAGCAA-3′;

[0254] The reverse sequence is shown in SEQ 4: 5'-AGGCACAAACACGAACCTCAA-3';

[0255] LaminB1, forward sequence is shown in SEQ 5: 5′-TCGCAAAAGCATGTATGAAGAG-3′;

[0256] The reverse sequence is shown in SEQ 6: 5'-CTCAAGTTTGGCATGGTAAGTC-3';

[0257] GADPH, forward sequence is shown in SEQ 7: 5'-TCCCACTCTTCCACCTTC-3';

[0258] The reverse sequence is shown in SEQ 8: 5'-CTGTAGCCGTATTCATTGTC-3'.

[0259] Telomere length was tested in the HT22 cell replicative aging model: HT22 cells were serially subcultured for 15 generations and SZ-A (preparation example 8) was added (dosage concentrations of 83 μg / mL and 166 μg / mL, calculated as extract). Telomere length was measured in HT22 cells after drug treatment. Telomere length was determined based on the ratio of telomere product to single-copy gene product (T / S) obtained by quantitative PCR.

[0260] As shown in FIG9 , the results showed that the mulberry branch extract SZ-A could significantly reduce the expression of the p53 gene. At the same time, the mulberry branch extract SZ-A had a tendency to reduce the expression of the p16 gene, thereby alleviating the cell cycle arrest of senescent cells.

[0261] As shown in FIG10 , mulberry extract SZ-A can reduce p53 expression to varying degrees, increase lamin B1 gene expression ( FIG10 ), and alleviate cell cycle arrest in senescent cells, showing a significant anti-cellular aging effect.

[0262] The results in Figure 11 show that after 15 generations of continuous subculture of HT22 cells, SZ-A of Preparation Example 8 (dosage concentrations of 40 μg / mL, 83 μg / mL, and 166 μg / mL, calculated as extract) can significantly prolong telomere length and delay replicative cell senescence.

[0263] (3) Effects of mulberry extract SZ-A on aging mouse cardiomyocytes H9C2 cells

[0264] Cell culture: H9C2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with fetal bovine serum (10% v / v), penicillin (100 U / mL), and streptomycin (100 μg / mL). The culture medium was changed every other day, and cells were subcultured when they reached 80% confluence. D-gal (30 mg / ml, abbreviated as D30) was dissolved in DMEM. To simulate the aging phenotype, H9C2 cells were seeded into 6-well plates and incubated with D-gal for 48 hours. Simultaneously with D-gal treatment, different concentrations (160 μg / mL, 320 μg / mL, calculated on the basis of the extract) of the mulberry twig extract SZ-A of Preparation Examples 2, 6, 8, and 10 were added.

[0265] SA-β-galactosidase staining: For cells cultured in a 6-well plate, aspirate the cell culture medium, wash once with PBS, add 1 ml of β-galactosidase staining fixative, and fix at room temperature for 15 minutes. Aspirate the fixative and wash the cells three times with PBS or HBSS for 3 minutes each. Aspirate the PBS and add 1 ml of staining solution to each well. Incubate at 37°C overnight. Cover the 6-well plate with parafilm or plastic wrap to prevent evaporation.

[0266] The results in Figure 12 show that the senescence of H9C2 cells increased significantly after 48 hours of D-gal (30 mg / ml) induction. The mulberry extract SZ-A prepared in different examples of the present invention (dosage concentrations of 160 μg / mL and 320 μg / mL, calculated as extract) can significantly reduce the degree of cell senescence (in Figure 12, green staining represents senescent cells).

[0267] (4) Effects of mulberry extract SZ-A on aging mouse myoblasts C2C12 cells

[0268] C2C12 cells were incubated in a growth medium (high-glucose DMEM medium + 10% fetal bovine serum) at 37°C in a constant-temperature incubator with 5% CO2. After the cells reached approximately 85% confluence, they were harvested and grouped into a control group (NC), an aging model group (D-gal), and an SZ-A group (D-gal + SZ-A). The culture medium in each group was then replaced with a differentiation medium (high-glucose DMEM medium + 2% horse serum). Depending on the experimental objectives, D-gal and SZ-A (the mulberry twig extract obtained in Preparation Examples 2, 6, and 8) were added to the differentiation medium before the end of the experiment.

[0269] The groups are as follows:

[0270] NC: control group;

[0271] Model: aging model group;

[0272] Preparation Example 2: SZ-A group, SZ-A is the mulberry branch extract in Preparation Example 2;

[0273] Preparation Example 6: SZ-A group, SZ-A is the mulberry branch extract in Preparation Example 6;

[0274] Preparation Example 8: SZ-A group, SZ-A is the mulberry branch extract in Preparation Example 8;

[0275] Cell senescence detection method:

[0276] a. SA-β-galactosidase staining

[0277] The aging model group received 100 mM D-gal in the culture medium, while the SZ-A group received 166 μg / mL (based on the extract) of SZ-A and 100 mM D-gal in the culture medium for 48 hours. SA-β-galactosidase staining was used to assess the degree of senescence in the different cell groups.

[0278] b. Flow cytometry

[0279] The senescence model group received 100 mM D-gal in the culture medium, while the SZ-A group received 83 μg / mL (based on the extract) of SZ-A and 100 mM D-gal in the culture medium for 48 hours. After 48 hours of senescence induction, cells were trypsinized to generate single-cell suspensions. Cells were washed and resuspended in 1X PBS at a concentration of 0.5 × 10 6 -1.0×10 6 cells / 100 μL. Wash the cells in PBS containing 1% BSA, centrifuge at 400 × g for 5 minutes, and remove the supernatant. Resuspend the cells in 100 μL of fixative, incubate in the dark at room temperature for 10 minutes, and wash the cells with PBS containing 1% BSA to remove the fixative. Resuspend the cells in 100 μL of working solution. After resuspension, stain the cells with CellEventTM Senescence Green Probe diluted 1 / 1000 in CellEvent Senescence Buffer in a 37°C CO2-free incubator for 90 minutes. After incubation, centrifuge at 400 × g for 5 minutes, remove the working solution, and wash the cells with PBS containing 1% BSA. After the final wash, resuspend the cells in 500 μL of PBS and prepare for detection on the machine. Use the flow cytometer to set the FITC channel (excitation wavelength 488 nm, emission wavelength around 530 nm).

[0280] The green staining in FIG13 represents senescent cells. The results in FIG13 show that the mulberry branch extracts of Preparation Examples 2, 6 and 8 can significantly inhibit the senescence of C2C12 myoblasts.

[0281] The results in FIG14 show that the senescence of C2C12 cells increased significantly after 48 hours of D-gal induction, and the mulberry extract SZ-A of the present invention can significantly reduce cell senescence.

[0282] Test Example 4: Nematode Experiment

[0283] (1) Nematode brown fat detection

[0284] Senescence pigment is an aging-related autofluorescent substance in organisms and is generally considered a biomarker for evaluating the degree of aging. The greater the accumulation of senescence pigment in the body, the shorter the lifespan of the organism. Therefore, by detecting and comparing the senescence pigment content in Caenorhabditis elegans at the early stage of aging (the tenth day of adulthood), we can evaluate whether the test substance has anti-aging effects.

[0285] Blank control group (Control): Contains L4 (D0) stage Caenorhabditis elegans and 100 μL SM buffer

[0286] Positive control group (Metformin group): Contains L4 (D0) stage Caenorhabditis elegans, 100 μL SM buffer and 50 mM Metformin (metformin)

[0287] The test substance test group contained L4 (D0) stage Caenorhabditis elegans, 100 μL SM buffer and SZ-A prepared in Preparation Example 8.

[0288] Different treatments were added to the wells, mixed thoroughly, and then covered with a culture dish lid, sealed with parafilm, and wrapped with tin foil. The culture dish was incubated in a shaker at 20°C, 150 rpm, and protected from light until the end point (Day 10). After the incubation period, each group of C. elegans was removed and collected in an EP tube. After washing with SM buffer, the supernatant was removed, leaving approximately 750 μL of worm fluid. 200 μL of 1 mm zirconium oxide grinding beads were added to the tissue grinder and ground at 30 Hz for 2 minutes. After centrifugation at 12,000 rpm in a refrigerated centrifuge for 2 minutes, 100 μL of the supernatant was collected and placed in a black 96-well plate. The fluorescence intensity at 355 nm and 460 nm was measured using a microplate reader. The protein content in the supernatant was also measured using a BCA kit.

[0289] The results of the anti-aging experiment are shown in Figure 15. Compared with the blank control group, the positive control group showed a significant decrease in senescence pigment content, demonstrating the effectiveness of the experiment. The SZ-A group also showed a significant decrease in senescence pigment content compared to the blank control group, indicating that SZ-A is effective in reducing senescence pigment content in C. elegans.

[0290] (2) Nematode lifespan detection

[0291] C. elegans Growth Medium (NGM): Dissolve 3g sodium chloride, 17g agar powder, and 2.5g polypeptone in 975mL of distilled water. Autoclave. After cooling to approximately 55°C, add 1mL 1mol / L calcium chloride, 1mL 1mol / L magnesium sulfate, 1mL 5mg / mL cholesterol, and 25mL potassium phosphate buffer. Mix thoroughly and pour into a sterile Petri dish.

[0292] Synchronization of Caenorhabditis elegans: When a large number of eggs appear in the body of Caenorhabditis elegans, add an appropriate amount of M9 buffer to the culture medium, elute the Caenorhabditis elegans and collect them in a sterilized 1.5 mL centrifuge tube, add 1 mL of Caenorhabditis elegans lysis buffer (10% hypochlorous acid: 1 mol / L sodium hydroxide = 1:1), centrifuge at 4000 r / min for 1 min; remove the lysis buffer, wash three times with M9 buffer (4000 r / min, centrifuge for 1 min), aspirate the eggs on the bottom layer with a pipette, smear them on a culture medium containing Escherichia coli OP50 with a diameter of 1 cm, and culture at 20°C for 48 to 60 h to obtain young adults.

[0293] Exposure method for Caenorhabditis elegans: The synchronized Caenorhabditis elegans were transferred to fresh culture medium containing Escherichia coli OP50. M9 buffer was added to the control group, and different concentrations of SZ-A (the mulberry branch extract in Preparation Example 1) were added to the exposure groups. The cells were treated at 20°C for 24 hours before subsequent experiments.

[0294] Effects of SZ-A on the lifespan of Caenorhabditis elegans

[0295] After exposure, C. elegans were randomly assigned to a culture medium containing E. coli OP50. Each group contained 33 nematodes, with three replicates per group. The cells were incubated at 20°C, along with a control group. Survival was calculated starting from the day of transfer (day 0 of the lifespan experiment). The number of nematodes that survived and died (a C. elegans that remained motionless upon contact with a platinum wire was considered dead) was recorded daily. The experiment continued until the last nematode died. Curves were plotted based on the number of nematodes that survived and died. The experiment was repeated three times.

[0296] The experimental results are shown in Table 2 below, which show that mulberry extract SZ-A can prolong the lifespan of nematodes.

[0297] Table 2 Experimental groups and results of the effects of SZ-A on the lifespan of Caenorhabditis elegans Note: Compared with the Contor1 group, **P<0.01, ***P<0.001, ns, no significant difference.

[0298] The results in Table 2 show that after treatment with the mulberry extract SZ-A of the present invention, the average lifespan of nematodes increased significantly, indicating that the mulberry extract SZ-A has the effect of extending the lifespan of Caenorhabditis elegans.

[0299] Experimental Example 5: Mouse Experiment

[0300] 1. Animal Grouping and Treatment

[0301] Fifteen-month-old C57 mice were randomly divided into seven groups (see Table 3 for specific groups). Each group consisted of 15 mice, and the drug was administered orally once daily. The mice were weighed weekly at the same time during the feeding period. After administration, the mice were fasted for 16 hours. Their eyes were enucleated, and blood samples were collected. Serum was separated by centrifugation at 3500 rpm and 4°C for 15 minutes and stored at -80°C. Tissues, including the brain and liver, were rapidly dissected and washed with saline. After blotting with filter paper, the tissues were weighed. Some samples were fixed in 10% formalin and stored at 4°C for morphological observation; others were frozen at -80°C for subsequent analysis. Fifteen three-month-old mice served as a young control group.

[0302] 2. Behavioral sign indicator detection

[0303] Appearance Observation: The mice were observed for mental state, activity, hair color and looseness, and aging scores were calculated. The specific scoring criteria included a comprehensive evaluation of the mice's mental state, activity, hair color and looseness, muscle grip strength, vision, hearing, bite, dermatitis, hunchback, gait, and other visible tissue abnormalities. Each indicator was scored on a scale of 0 to 10. Objective scoring was performed based on the severity of the condition, with higher scores indicating a higher degree of aging.

[0304] 3. Metabolic Cage Experiment

[0305] Four months after administration, the mice were placed in metabolic cages, and the oxygen consumption rate and carbon dioxide production rate were measured to calculate the heat production capacity of the mice.

[0306] Table 3 Experimental groups and drug administration of naturally aging mice

[0307] The results in Figure 16 show that after one month of administration, the aging scores of mice in the G4 group were significantly lower than those in the model group (G2 natural aging group). The results in Figure 17 show that after five months of administration, the aging scores of the G5, G6, and G7 groups were significantly lower than those in the model group. Therefore, the mulberry extract SZ-A of the present invention can significantly delay the phenotype of naturally aging mice.

[0308] The results in Figure 18 show that the heat production capacity of the model group (G2 natural aging group) was significantly lower than that of the young control group (G1); compared with the model group, the nighttime heat production of the G4 and G6 drug-administered groups increased significantly, indicating that the drug-administered groups can improve the body's metabolic level.

[0309] Bone density test:

[0310] Use the iNSiGHT VET DXA / OT20-2F7113-01 instrument. Turn on the gas anesthesia machine and place the mouse in the induction box for anesthesia. Once the mouse is fully anesthetized, confirm the mouse number, place it on the imaging board, and close the cabinet door. Avoid overlapping of the mouse's limbs and torso, and keep the mouse's body fully extended. Keep the head, body, and tail in a straight line as much as possible. Keep the mouse's head close to the anesthesia mask to maintain anesthesia. Open the software, create mouse information, and click "Measure" in the animal list. After confirming the mouse and measurement information, click "Start Measurement." Do not open the cabinet door during the test. Analyze the data after the measurement is completed.

[0311] The results in Figure 19 show that 5 months after administration, the bone density of mice in the aging model group (G2 group) was significantly lower than that of mice in the young group (G1 group), and the bone density of the G3, G5 and G6 administration groups was significantly higher than that of the aging model group.

[0312] result:

[0313] During the aging process, mice will gradually show weakness in the limbs, decreased responsiveness, yellowing and dull fur, and even hair loss. SZ-A can effectively improve the survival rate of aging mice, significantly improve their aging scores, increase the basal metabolic capacity and bone density of mice, and thus enhance the mice's motor behavior ability. It also generally improves the mice's mental state, activity, hair color and looseness, muscle grip, vision, hearing, bite, dermatitis, hunchback, gait and other aging manifestations.

[0314] The present invention has been described above with reference to preferred embodiments, but these embodiments are merely exemplary and serve only as illustrations. On this basis, various replacements and improvements can be made to the present invention, all of which fall within the scope of protection of the present invention.

Claims

1. Use of mulberry extract or its active ingredient in preparing anti-aging products, characterized in that: Based on the weight percentage of the sum of the components of the mulberry extract as 100%, the mulberry extract contains 3% or more of alkaloids by weight (optionally containing 3-99% of alkaloids by weight, further optionally containing 15-99% of alkaloids by weight, further optionally containing 30-99% of alkaloids by weight, further optionally containing 40-99% of alkaloids by weight, further optionally containing 50-99% of alkaloids by weight, further optionally containing 60-99% of alkaloids by weight), and / or contains no more than 70% polysaccharide by weight (optionally containing 0.2-70% polysaccharide by weight, further optionally containing 0.2-50% polysaccharide by weight, further optionally containing 0.2-35% polysaccharide by weight, further optionally containing 0.2-25% polysaccharide by weight, further optionally containing 0.2-23% polysaccharide by weight, further optionally containing 20-25% polysaccharide by weight), and / or contains flavonoids in an amount not higher than 10% by weight (optionally containing flavonoids in an amount of 0.05-5% by weight, further optionally containing flavonoids in an amount of 0-2% by weight, further optionally containing flavonoids in an amount of 0.05-2% by weight, further optionally containing flavonoids in an amount of 0.5-1.5% by weight, further optionally containing flavonoids in an amount of 0-1% by weight, further optionally containing flavonoids in an amount of 0.05-1% by weight), and / or contains no more than 50% by weight of amino acids (optionally containing 0-30% by weight of amino acids, further optionally containing 0-25% by weight of amino acids, further optionally containing 0-20% by weight of amino acids, further optionally containing 0-5% by weight of amino acids, further optionally containing 3-25% by weight of amino acids or further optionally containing 5-20% by weight of amino acids), and / or other components (the weight content can be optionally 0-25%, further optionally 0-20%, further optionally 0-15%, further optionally 0-11%, further optionally 2-20%, further optionally 4-8%).

2. The use according to claim 1, characterized in that The anti-aging product is selected from at least one of food, medicine, beverage or health care product.

3. The use according to claim 1 or 2, characterized in that The anti-aging includes delaying the aging of human and / or animal bodies, tissue and organ aging and / or cell aging; Preferably, the aging is natural aging; Optionally, the aging is caused by any one or more of oxidative stress damage, mitochondrial dysfunction, DNA damage, and telomere dysfunction; Optionally, the oxidative stress damage or mitochondrial dysfunction may be caused by any one or more of excessive fatty acids, D-galactose, and adenine; Alternatively, the DNA damage or telomere dysfunction may be caused by doxorubicin (DOX) and / or multiple cell replication passages; Preferably, the tissues and organs include any one or more of heart, liver, kidney, nerve tissue, bone tissue, vascular tissue, and muscle tissue; Preferably, the cells are any one or more of liver cells, kidney cells, endothelial cells, neuronal cells, cardiomyocytes, and myoblasts; Preferably, the anti-aging effect is specifically manifested as any one or more of the following: 1) Prolong the life of the body; 2) Reduce the body's aging pigment content; 3) Improve the body's motor behavior ability; 4) improving the body's metabolic rate, preferably promoting an increase in body heat production; 5) Improving mitochondrial function in tissues and organs; preferably improving the structure and number of mitochondria in tissues and organs; preferably, increasing the mitochondrial DNA copy number and the level of mitochondrial complex enzymes, or promoting mitochondrial fusion and reducing mitochondrial fission; optionally, increasing the mitochondrial DNA copy number includes promoting the expression of any one or more of ND1, COXI, and COXII; optionally, increasing the level of mitochondrial complex enzymes includes promoting the expression of any one or more of mitochondrial complex enzymes I-ndufs8, II-sdhb, III-uqcrc1, and V-atp5a1; optionally, promoting mitochondrial fusion and reducing mitochondrial fission includes regulating the expression of any one or more of DRP1, TFAM, TFB1, TFB2, MFN1, OPA1, NRF2, PGC1-α, HO-1, and NQO1 genes; The tissue organ is preferably any one or more of the liver, myocardium and kidney; 6) Improving the antioxidant capacity of tissues, organs and / or cells, wherein the tissues and organs are preferably liver tissue and / or kidney tissue; the cells are preferably liver cells and / or kidney cells; the improving the antioxidant capacity of tissues and organs is preferably reducing the level of any one or more of 4-HNE (4-hydroxynonenoic acid), ROS (reactive oxygen species), and MDA (malondialdehyde), and preferably increasing the expression of any one or more of SOD, CAT (catalase), and GSS (reduced glutathione); optionally, improving the antioxidant capacity of tissues and organs also includes alleviating endoplasmic reticulum stress in tissues and organs, and preferably reducing the expression of any one or more of ATF4, ATF6, CHOP, Bax, Caspase12, and Caspase3; 7) increasing the expression of the longevity protein SIRT in tissues and organs, preferably promoting the expression of any one or more of SIRT1, SIRT3, SIRT5, and SIRT6, wherein the tissues and organs are preferably the liver and / or heart; 8) Increase the coenzyme NAD in tissues and organs + levels and / or NAD + / NADH ratio, the tissue organ is preferably the liver and / or heart; 9) Alleviating cell cycle arrest and / or increasing cellular oxidative stress levels; the cells are preferably liver cells, endothelial cells and / or neuronal cells; 10) reducing the expression of p53 and / or p16 genes in cells, or increasing the expression of lamin B1 gene; the cells are preferably endothelial cells and / or neuronal cells; 11) extending telomere length in cells; the cells are preferably neuronal cells; 12) increasing ATP levels in tissues and organs, preferably the kidneys; 13) Improve bone density.

4. Use of mulberry extract or its active ingredient in any of the following: 1) Use of mulberry extract or its active ingredients in the preparation of a product for inhibiting ROS levels in HepG2 cells in vitro; 2) Use of mulberry extract or its active ingredients in the preparation of products for delaying in vitro liver cell aging; 3) Use of mulberry extract or its active ingredients in the preparation of products for delaying aging of endothelial cells, neuronal cells, liver cells, kidney cells, cardiomyocytes or myoblasts in vitro; 4) Use of mulberry extract or its active ingredients in the preparation of a product for reducing the content of aging pigments in Caenorhabditis elegans; 5) Use of mulberry extract or its active ingredients in the preparation of products for extending nematode lifespan; 6) Use of mulberry extract or its active ingredients in the preparation of a product for reducing aging scores in mice; 7) Use of mulberry extract or its active ingredients in the preparation of products for increasing thermogenesis in mice; 8) Use of mulberry extract or its active ingredients in the preparation of a product for increasing bone density in mice; Based on the weight percentage of the sum of the components of the mulberry extract as 100%, the mulberry extract contains 3% or more of alkaloids by weight (optionally containing 3-99% of alkaloids by weight, further optionally containing 15-99% of alkaloids by weight, further optionally containing 30-99% of alkaloids by weight, further optionally containing 40-99% of alkaloids by weight, further optionally containing 50-99% of alkaloids by weight, further optionally containing 60-99% of alkaloids by weight), and / or contains no more than 70% polysaccharide by weight (optionally containing 0.2-70% polysaccharide by weight, further optionally containing 0.2-50% polysaccharide by weight, further optionally containing 0.2-35% polysaccharide by weight, further optionally containing 0.2-25% polysaccharide by weight, further optionally containing 0.2-23% polysaccharide by weight, further optionally containing 20-25% polysaccharide by weight), and / or contains flavonoids in an amount not higher than 10% by weight (optionally containing flavonoids in an amount of 0.05-5% by weight, further optionally containing flavonoids in an amount of 0-2% by weight, further optionally containing flavonoids in an amount of 0.05-2% by weight, further optionally containing flavonoids in an amount of 0.5-1.5% by weight, further optionally containing flavonoids in an amount of 0-1% by weight, further optionally containing flavonoids in an amount of 0.05-1% by weight), and / or contains no more than 50% by weight of amino acids (optionally containing 0-30% by weight of amino acids, further optionally containing 0-25% by weight of amino acids, further optionally containing 0-20% by weight of amino acids, further optionally containing 0-5% by weight of amino acids, further optionally containing 3-25% by weight of amino acids or further optionally containing 5-20% by weight of amino acids), and / or other components (the weight content can be optionally 0-25%, further optionally 0-20%, further optionally 0-15%, further optionally 0-11%, further optionally 2-20%, further optionally 4-8%).

5. The use according to any one of claims 1 to 4, characterized in that Taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight percentages of the components in the mulberry extract are as follows: Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are: Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are: Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are: Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are: Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are: Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are: Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are: Preferably, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

6. The use according to any one of claims 1 to 5, characterized in that The alkaloids include 1-deoxynojirimycin (DNJ), N-methyl-1-deoxynojirimycin (N-methly-1-deoxynojirimycin), fagomine (FAG), 3-epi-fagomine, 1,4-dideoxy-1,4-imino-D-arabinitol (DAB), calystegin B2, calystegin C1, 2-oxo-(α-D-galacto- One or more of 2-O-(α-D-galactopyranosyl)-1-deoxynojirimycin, 6-O-(β-D-glucopyranosyl)-1-deoxynojirimycin, and 1,4-dideoxy-1,4-imino-(2-O-β-D-glucopyranosyl)-D-arabinitol. Preferably, the weight percentage of DNJ is not less than 50% (preferably 60-99%) of the total alkaloids.

7. The use according to any one of claims 1 to 6, characterized in that The anti-aging product is in the form of an oral dosage form; preferably, the anti-aging product is in the form of a tablet, capsule, lozenge, powder, tea bag, oral solution, oral emulsion, pill, granule, syrup or powder.

8. The use according to any one of claims 1 to 7, characterized in that: The preparation method of the mulberry extract comprises the following steps: 1) preparing a crude extract of a Moraceae plant; 2) separating the crude extract through a cationic resin and / or an optional anionic resin to obtain the mulberry extract.

9. The use according to claim 8, characterized in that: The method further comprises the steps of: 3) performing alcohol precipitation on the resin effluent from step 2) and collecting the supernatant; 4) The supernatant is concentrated and dried. Optionally, the method further comprises the step of concentrating and drying the resin effluent from step 2).

Images