Application of mulberry extract in preparation of anti-aging product

Abstract

The invention provides application of a mulberry extract in preparation of an anti-aging product. The mulberry extract has a good anti-aging effect. The mulberry extract comprises alkaloid with the weight content being 3% or above, and / or polysaccharide with the weight content not higher than 70%, and / or flavone with the weight content not higher than 10%, and / or amino acid with the weight content not higher than 50%, and / or other components.

Description

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 the "decline of cell function", the irreversible cessation of proliferation (growth), that is, the changes in the functions of molecules such as proteins and genes, and 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 cell aging include: mitochondrial dysfunction, which breaks down NAD in the cytoplasm. + / NADH ratio, production of reactive oxygen species, and other potential mechanisms to drive aging; decreased sirtuin expression, loss of PARP activity, and altered cellular redox status leading to NAD + Loss of energy can also lead to aging; autophagy dysfunction can cause aging in some cases, but 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, which cannot meet people's urgent needs. Summary of the invention

[0004] The purpose of the present invention is to provide a use of mulberry extract in preparing anti-aging products. Mulberry extract has good anti-aging effect.

[0005] Taking the weight percentage of the sum of the components of the mulberry extract as 100%, the mulberry extract contains alkaloids with a weight content of more than 3% (optionally containing alkaloids with a weight content of 3-99%, further optionally containing alkaloids with a weight content of 15-99%, further optionally containing alkaloids with a weight content of 30-99%, further optionally containing alkaloids with a weight content of 40-99%, further optionally containing alkaloids with a weight content of 50-99%, further optionally containing alkaloids with a weight content of 60-99%),

[0006] and / or comprising a polysaccharide content of not more than 70% by weight (optionally comprising a polysaccharide content of 0.2-70% by weight, further optionally comprising a polysaccharide content of 0.2-50% by weight, further optionally comprising a polysaccharide content of 0.2-35% by weight, further optionally comprising a polysaccharide content of 0.2-25% by weight, further optionally comprising a polysaccharide content of 0.2-23% by weight, further optionally comprising a polysaccharide content of 20-25% by weight),

[0007] and / or contains flavonoids having a weight content of not more than 10% (optionally containing flavonoids having a weight content of 0.05-5%, further optionally containing flavonoids having a weight content of 0-2%, further optionally containing flavonoids having a weight content of 0.05-2%, further optionally containing flavonoids having a weight content of 0.5-1.5%, further optionally containing flavonoids having a weight content of 0-1%, further optionally containing flavonoids having a weight content of 0.05-1%),

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

[0009] and / or other components (the weight content is 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 the aging of human and / or animal bodies, tissue and organ aging and / or cell 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 the 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 content of aging pigments in the body;

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

[0021] 4) Improving the body's metabolic level, preferably promoting the increase of body heat production;

[0022] 5) Improving the mitochondrial function of tissues and organs; preferably improving the structure and number of mitochondria in tissues and organs;

[0023] Preferably, the mitochondrial DNA copy number and the level of mitochondrial complex enzymes are increased, or mitochondrial fusion is promoted and mitochondrial fission is reduced; optionally, the increase in mitochondrial DNA copy number includes promoting ND1,

[0024] Optionally, the increasing the level of mitochondrial complex enzymes includes promoting the expression of any one or several of mitochondrial complex enzymes I-ndufs8, II-sdhb, III-uqcrc1, and V-atp5a1; Optionally, the promoting mitochondrial fusion and reducing mitochondrial fission include regulating DRP1, TFAM, TFB1, TFB2, MFN1, OPA1, NRF2, PGC1-α, HO-1,

[0025] Any one or several gene expression of NQO1; Optionally, the tissue organ is preferably any one or several of liver, myocardium and kidney;

[0026] 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), 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, preferably reducing the expression of any one or more of ATF4, ATF6, CHOP, Bax, Caspase12, and Caspase3;

[0027] The tissue organ is preferably liver tissue and / or kidney tissue; the cell is preferably liver cell and / or kidney cell;

[0028] 7) Increase the expression of longevity protein sirt in tissues and organs, preferably promote sirt1, sirt3, sirt5,

[0029] The expression of any one or more of sirt6, wherein the tissue organ is preferably liver and / or heart;

[0030] 8) Increase the coenzyme NAD in tissues and organs + Level and / or NAD + / NADH ratio, optionally, the tissue organ is preferably liver and / or heart;

[0031] 9) Relieving cell cycle arrest and / or increasing the level of cellular oxidative stress; the cells are preferably liver cells, endothelial cells and / or neuronal cells;

[0032] 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;

[0033] 11) Prolonging telomere length in cells; the cells are preferably neuronal cells;

[0034] 12) increasing ATP levels in tissues and organs, preferably kidneys;

[0035] 13) Improve bone density.

[0036] The present invention also provides the use of mulberry extract or its active ingredients in any of the following:

[0037] 1) Use of mulberry extract or its active ingredients in the preparation of a product for inhibiting ROS levels in HepG2 cells in vitro;

[0038] 2) Use of mulberry extract or its active ingredients in the preparation of products for delaying in vitro liver cell aging;

[0039] 3) Use of mulberry extract or its active ingredients in the preparation of products for delaying the aging of in vitro endothelial cells, neuronal cells, liver cells, kidney cells, cardiomyocytes or myoblasts;

[0040] 4) Use of mulberry extract or its active ingredients in the preparation of products for reducing the content of aging pigments in Caenorhabditis elegans;

[0041] 5) Use of mulberry extract or its active ingredients in the preparation of products for extending the life span of nematodes;

[0042] 6) Use of mulberry extract or its active ingredients in preparing a product for reducing the aging score of mice;

[0043] 7) Use of mulberry extract or its active ingredients in the preparation of products for increasing thermogenesis in mice;

[0044] 8) Use of mulberry extract or its active ingredients in preparing a product for increasing bone density in mice;

[0045] Taking the weight percentage of the sum of the components of the mulberry extract as 100%, the mulberry extract contains alkaloids with a weight content of more than 3% (optionally containing alkaloids with a weight content of 3-99%, further optionally containing alkaloids with a weight content of 15-99%, further optionally containing alkaloids with a weight content of 30-99%, further optionally containing alkaloids with a weight content of 40-99%, further optionally containing alkaloids with a weight content of 50-99%, further optionally containing alkaloids with a weight content of 60-99%),

[0046] and / or comprising a polysaccharide content of not more than 70% by weight (optionally comprising a polysaccharide content of 0.2-70% by weight, further optionally comprising a polysaccharide content of 0.2-50% by weight, further optionally comprising a polysaccharide content of 0.2-35% by weight, further optionally comprising a polysaccharide content of 0.2-25% by weight, further optionally comprising a polysaccharide content of 0.2-23% by weight, further optionally comprising a polysaccharide content of 20-25% by weight),

[0047] and / or contains flavonoids having a weight content of not more than 10% (optionally containing flavonoids having a weight content of 0.05-5%, further optionally containing flavonoids having a weight content of 0-2%, further optionally containing flavonoids having a weight content of 0.05-2%, further optionally containing flavonoids having a weight content of 0.5-1.5%, further optionally containing flavonoids having a weight content of 0-1%, further optionally containing flavonoids having a weight content of 0.05-1%),

[0048] and / or contains no more than 50% amino acids by weight (optionally contains 0-30% amino acids by weight, further optionally contains 0-25% amino acids by weight, further optionally contains 0-20% amino acids by weight, further optionally contains 0-5% amino acids by weight, further optionally contains 3-25% amino acids by weight or further optionally contains 5-20% amino acids by weight),

[0049] and / or other components (the weight content is optionally 0-25%, further optionally 0-20%, further optionally 0-15%, further optionally 0-11%, further optionally 2-20%, further optionally 4-8%).

[0050] In a preferred embodiment of the present invention, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0051] Alkaloids 3-99%,

[0052] Polysaccharide 0.2-70%,

[0053] Flavonoids 0-10%,

[0054] Amino acids 0-50%,

[0055] Other components 0-25%;

[0056] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0057] Alkaloids 30-99%,

[0058] Polysaccharide 0.2-35%,

[0059] Flavonoids 0-2%,

[0060] Amino acids 0-30%,

[0061] Other components 0-20%;

[0062] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0063] Alkaloids 30-99%,

[0064] Polysaccharide 0.2-35%,

[0065] Flavonoids 0.05-2%,

[0066] Amino acids 0-25%,

[0067] Other components 0-20%;

[0068] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0069] Alkaloids 40-99%,

[0070] Polysaccharide 0.2-25%,

[0071] Flavonoids 0.05-1%,

[0072] Amino acids 0-25%,

[0073] Other components 0-15%;

[0074] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0075] Alkaloids 50-99%,

[0076] Polysaccharide 0.2-23%,

[0077] Flavonoids 0-1%,

[0078] Amino acids 0-20%,

[0079] Other components 0-11%;

[0080] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0081] Alkaloids 50-99%,

[0082] Polysaccharide 0.2-35%,

[0083] Flavonoids 0-2%,

[0084] Amino acids 0-30%,

[0085] Other components 0-20%;

[0086] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0087] Alkaloids 60-99%,

[0088] Polysaccharide 0.2-23%,

[0089] Flavonoids 0-1%,

[0090] Amino acids 0-5%,

[0091] Other components 0-11%;

[0092] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0093] Alkaloids 63-99%,

[0094] Polysaccharide 0.2-23%,

[0095] Flavonoids 0-1%,

[0096] Amino acids 0-5%,

[0097] Other components 0-11%;

[0098] More preferably, based on the sum of the weight percentage of the components of the mulberry extract being 100%, the weight content of the components in the mulberry extract is:

[0099] Alkaloids 50-65%,

[0100] Polysaccharide 20-25%,

[0101] Flavonoids 0.5-1.5%,

[0102] Amino acids 3-25%,

[0103] Other components 2-20%.

[0104] More preferably, based on the sum of the weight percentage of the components of the mulberry extract being 100%, the weight content of the components in the mulberry extract is:

[0105] Alkaloids 50-65%,

[0106] Polysaccharide 20-25%,

[0107] Flavonoids 0.5-1.5%,

[0108] Amino acids 5-20%,

[0109] Other components 4-8%.

[0110] More preferably, based on the sum of the weight percentage of the components of the mulberry extract being 100%, the weight content of the components in the mulberry extract is:

[0111] Alkaloids 30-90%,

[0112] Polysaccharide 0.2-35%,

[0113] Flavonoids 0-2%,

[0114] Amino acids 0-30%,

[0115] Other components 0-20%;

[0116] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0117] Alkaloids 30-90%,

[0118] Polysaccharide 0.2-35%,

[0119] Flavonoids 0.05-2%,

[0120] Amino acids 0-25%,

[0121] Other components 0-20%;

[0122] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0123] Alkaloids 30-80%,

[0124] Polysaccharide 0.2-35%,

[0125] Flavonoids 0-2%,

[0126] Amino acids 0-30%,

[0127] Other components 0-11%;

[0128] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0129] Alkaloids 30-80%,

[0130] Polysaccharide 5-35%,

[0131] Flavonoids 0.1-2%,

[0132] Amino acids 4-25%,

[0133] Other components 8-11%;

[0134] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0135] Alkaloids 30-70%,

[0136] Polysaccharide 0.2-35%,

[0137] Flavonoids 0-2%,

[0138] Amino acids 0-25%,

[0139] Other components 0-8%;

[0140] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0141] Alkaloids 30-70%,

[0142] Polysaccharide 20-35%,

[0143] Flavonoids 0.6-2%,

[0144] Amino acids 5-25%,

[0145] Other components 4-8%;

[0146] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0147] Alkaloids 30-65%,

[0148] Polysaccharide 0.2-35%,

[0149] Flavonoids 0-2%,

[0150] Amino acids 0-25%,

[0151] Other components 0-8%;

[0152] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0153] Alkaloids 30-65%,

[0154] Polysaccharide 20-35%,

[0155] Flavonoids 0-2%,

[0156] Amino acids 0-25%,

[0157] Other components 0-8%;

[0158] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0159] Alkaloids 30-60%,

[0160] Polysaccharide 0.2-35%,

[0161] Flavonoids 0-2%,

[0162] Amino acids 0-25%,

[0163] Other components 0-8%;

[0164] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0165] Alkaloids 30-60%,

[0166] Polysaccharide 20-35%,

[0167] Flavonoids 0-2%,

[0168] Amino acids 0-25%,

[0169] Other components 0-8%;

[0170] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0171] Alkaloids 35-70%,

[0172] Polysaccharide 0.2-35%,

[0173] Flavonoids 0-2%,

[0174] Amino acids 0-25%,

[0175] Other components 0-8%;

[0176] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0177] Alkaloids 35-70%,

[0178] Polysaccharide 20-35%,

[0179] Flavonoids 0.6-2%,

[0180] Amino acids 5-25%,

[0181] Other components 4-8%;

[0182] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0183] Alkaloids 40-70%,

[0184] Polysaccharide 0.2-25%,

[0185] Flavonoids 0.05-1%,

[0186] Amino acids 0-25%,

[0187] Other components 0-10%;

[0188] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0189] Alkaloids 40-70%,

[0190] Polysaccharide 20-25%,

[0191] Flavonoids 0.05-1%,

[0192] Amino acids 5-25%,

[0193] Other components 0-10%;

[0194] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0195] Alkaloids 30-65%,

[0196] Polysaccharide 0.2-35%,

[0197] Flavonoids 0-2%,

[0198] Amino acids 0-25%,

[0199] Other components 0-8%;

[0200] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0201] Alkaloids 30-65%,

[0202] Polysaccharide 20-35%,

[0203] Flavonoids 0.6-2%,

[0204] Amino acids 5-25%,

[0205] Other components 4-8%;

[0206] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0207] Alkaloids 15-99%

[0208] Polysaccharide 0.2-20%,

[0209] Flavonoids 0-10%,

[0210] Amino acids 0-45%,

[0211] Other components 0-15%;

[0212] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0213] Alkaloids 15-90%

[0214] Polysaccharide 3-20%,

[0215] Flavonoids 0-10%,

[0216] Amino acids 0-45%,

[0217] Other components 0-15%;

[0218] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0219] Alkaloids 15-80%

[0220] Polysaccharide 5-35%,

[0221] Flavonoids 0-10%,

[0222] Amino acids 0-45%,

[0223] Other components 0-15%;

[0224] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0225] Alkaloids 15-70%

[0226] Polysaccharide 0.2-20%,

[0227] Flavonoids 0-10%,

[0228] Amino acids 0-45%,

[0229] Other components 0-15%;

[0230] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0231] Alkaloids 15-70%

[0232] Polysaccharide 0.2-20%,

[0233] Flavonoids 0-10%,

[0234] Amino acids 5-45%,

[0235] Other components 0-15%;

[0236] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0237] Alkaloids 15-65%

[0238] Polysaccharide 0.2-20%,

[0239] Flavonoids 0-10%,

[0240] Amino acids 0-45%,

[0241] Other components 0-15%;

[0242] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0243] Alkaloids 15-65%

[0244] Polysaccharide 0.2-20%,

[0245] Flavonoids 0-10%,

[0246] Amino acids 5-45%,

[0247] Other components 0-15%;

[0248] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0249] Alkaloids 15-30%,

[0250] Polysaccharide 0.2-40%,

[0251] Flavonoids 0-10%,

[0252] Amino acids 20-50%,

[0253] Other components 0-15%;

[0254] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0255] Alkaloids 3-70%,

[0256] Polysaccharide 0.2-70%,

[0257] Flavonoids 0-10%,

[0258] Amino acids 0-10%,

[0259] Other components 0-10%;

[0260] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0261] Alkaloids 3-70%,

[0262] Polysaccharides 20-70%,

[0263] Flavonoids 0-10%,

[0264] Amino acids 5-10%,

[0265] Other components 0-10%;

[0266] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0267] Alkaloids 3-30%,

[0268] Polysaccharides 35-70%,

[0269] Flavonoids 0-10%,

[0270] Amino acids 0-25%,

[0271] Other components 0-10%;

[0272] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0273] Alkaloids 3-30%,

[0274] Polysaccharides 35-70%,

[0275] Flavonoids 0-10%,

[0276] Amino acids 0-25%,

[0277] Other components 0-10%;

[0278] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0279] Alkaloids 3-40%,

[0280] Polysaccharides 25-70%,

[0281] Flavonoids 0-10%,

[0282] Amino acids 0-25%,

[0283] Other components 0-10%;

[0284] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0285] Alkaloids 3-40%,

[0286] Polysaccharides 25-70%,

[0287] Flavonoids 0-10%,

[0288] Amino acids 10-25%,

[0289] Other components 0-10%;

[0290] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0291] Alkaloids 3-50%,

[0292] Polysaccharides 20-70%,

[0293] Flavonoids 0-10%,

[0294] Amino acids 0-20%,

[0295] Other components 0-10%;

[0296] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0297] Alkaloids 3-50%,

[0298] Polysaccharides 20-70%,

[0299] Flavonoids 0-10%,

[0300] Amino acids 10-20%,

[0301] Other components 0-10%;

[0302] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0303] Alkaloids 3-65%,

[0304] Polysaccharides 20-70%,

[0305] Flavonoids 0-10%,

[0306] Amino acids 0-10%,

[0307] Other components 0-10%;

[0308] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0309] Alkaloids 3-65%,

[0310] Polysaccharides 20-70%,

[0311] Flavonoids 0-10%,

[0312] Amino acids 5-10%,

[0313] Other components 0-10%;

[0314] Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

[0315] Alkaloids 80-99%,

[0316] Polysaccharide 0.2-10%,

[0317] Flavonoids 0-1%,

[0318] Amino acids 0-5%,

[0319] Other components 0-15%.

[0320] Preferably, the alkaloids include 1-deoxynojirimycin (DNJ), 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- 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.

[0321] Wherein, preferably, the weight percentage of DNJ is not less than 50% (preferably 60-99%) of the total alkaloids.

[0322] Preferably, the heavy metal content of the mulberry extract does not exceed 10 ppm.

[0323] 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.

[0324] In the present invention, the mulberry extract can be provided in the form of commercially available mulberry branch total alkaloids (national medicine standard Z20200001) or mulberry branch total alkaloids tablets (national medicine standard Z20200002).

[0325] Optionally, 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 through 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.

[0326] 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; 4) concentrating and / or drying the supernatant. Optionally, before subjecting the resin effluent of step 2) to alcohol precipitation, it is concentrated and / or dried.

[0327] 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.), Morus alba Var. Pendula Dippel, Morus alba Var. Pendula Dippel, Morus alba, and mulberry varieties bred from the above mulberry species, and a combination of one or more of the above mulberry species bred intraspecifically or interspecifically;

[0328] Optionally, the moraceae plant is one or more selected from 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 Sangte You No. 2. Leaves, roots, branches, bark, buds, stems, fruits and other parts of the moraceae plant can be used, and mulberry branches, mulberry leaves or white mulberry bark can be used.

[0329] 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.

[0330] In one embodiment, the mulberry extract is prepared according to the following steps: crushing the moraceae plant, heating and refluxing extraction 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 part, adding ethanol, precipitating to remove impurities, concentrating and / or drying to obtain an extract.

[0331] In one embodiment, the mulberry extract is prepared according to the following steps: crushing the moraceae plant, heating and refluxing extraction 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 part, concentrating and / or drying to obtain an extract.

[0332] In one embodiment, the mulberry extract is prepared according to the following steps: crushing the moraceae plant, heating and refluxing extraction 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 an extract.

[0333] In one embodiment, the mulberry extract is prepared according to the following steps: crushing the moraceae plant, heating and refluxing extraction 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 0.5-3h each time, further optionally 1-2h 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 part (i.e., the anion resin effluent), adding ethanol, precipitating to remove impurities, concentrating and / or drying to obtain an extract.

[0334] 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 period of 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, and 0.4-10 times can be selected 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, and further optionally 80-400rpm or 300rpm.

[0335] 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, and can be 8.5-9.5; Optionally, the alkaline solution is selected from an aqueous ammonia solution, a sodium hydroxide solution, a potassium hydroxide solution, or a sodium carbonate solution; Optionally, the concentration of the alkaline solution is 0.5-4 mol / L, and can be 1-2 mol / L. Optionally, the acidic solution is washed until the pH of the eluate is 3.0-7.0, and can be 4.5-6.5. Optionally, the acidic solution is selected from a hydrochloric acid solution, a phosphoric acid solution, and a sodium hydrogen phosphate-citric acid buffer solution, and Optionally, the concentration of the acidic solution is 0.5-4 mol / L, and can be 1.5-2 mol / L. Optionally, the cationic resin can also be rinsed with 3-5 column volumes of deionized water after the last acidic solution wash.

[0336] 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.

[0337] 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).

[0338] After the plant crude 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.

[0339] 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).

[0340] Optionally, the eluent flow rate is 1-15 BV / h (optionally 5-10 BV / h, further optionally 5-6 BV / h).

[0341] Optionally, the weight of the eluent used for cationic resin separation is 0.1-30 times the weight of the plant raw material feed. Optionally, the eluent is 0.5-10 times the weight of the plant raw material feed for elution.

[0342] The collection starting point can be determined according to the pH of the effluent from the cationic resin. For example, when an alkaline solution such as aqueous ammonia is used for elution, the eluate is collected when the pH of the effluent from the cationic column is detected to be greater than 7, or the collection starting point of the effluent is determined according to 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 input, the collection is stopped, and the collected liquid is optionally purified by an anion column.

[0343] When purifying by anion column, optionally, after the anion resin is loaded into the column, it is activated in the order of washing with alkaline solution, washing with acidic solution, and washing with alkaline solution.

[0344] Optionally, washing with an alkaline solution until the pH of the washing solution is 8.0-9.5, optionally 8.5-9.5;

[0345] 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.

[0346] 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 the concentration of the acidic solution is 0.5-4 mol / L, optionally 1-2 mol / L.

[0347] 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.

[0348] 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).

[0349] When the liquid flows out of the anion resin, the collection begins. Optionally, 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, the collection is stopped.

[0350] 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 10-600rpm (optionally 40-500rpm, 80-400rpm). The alcohol precipitation treatment time is 12-24h.

[0351] Furthermore, before the alcohol precipitation treatment, the anion resin effluent is centrifuged or filtered through a microfiltration membrane to remove impurities, and then concentrated through a reverse ion osmosis membrane. The specific gravity of the concentrated liquid can be 1.0-1.3, and can be optionally 1.1-1.25.

[0352] 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 conforms to the route of administration or the mode of administration. The carrier can be a solid or liquid excipient. Solid excipients, for example, include 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, polyvinylpyrrolidone, starch, magnesium stearate, sodium carboxymethyl starch and talc; liquid excipients, for example, include water, ethanol, syrup and glycerol.

[0353] In the present invention, the mulberry extract is referred to as SZ-A.

[0354] Beneficial effects:

[0355] 1. The results of the nematode experiment showed that mulberry extract has the effect of reducing the content of aging pigments in the body of Caenorhabditis elegans and prolonging the life span of nematodes. The results of the mouse experiment showed that mulberry extract effectively improved the survival rate of aging mice, significantly improved their aging scores, increased the basal metabolic capacity and bone density of mice, and thus enhanced the motor behavior of mice. Overall, it improved the mental state, activity, loose hair color, muscle grip, vision, hearing, bite, dermatitis, hunchback, gait and other aging manifestations of mice.

[0356] 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 4-HNE, a lipid peroxidation product in liver tissue; relieve endoplasmic reticulum stress: reduce tissue and cell ROS and relieve stress damage.

[0357] 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

[0358] Figure 1 This is an electron microscopic image of the ultrastructure of mitochondria in the liver of the experimental mouse in Experimental Example 1;

[0359] Figure 2 In Experimental Example 1, the expression of mitochondrial DNA copy number ND-1, COXI, COXII, the expression of mitochondrial complex enzymes I-ndufs8, II-sdhb, III-uqcrc1, V-atp5a1, and the expression of mitochondrial fission, fusion and mitochondrial generation related genes DRP1, TFAM, TFB1, TFB2, MFN1, OPA1, NRF2, PGC1-α, HO-1 and NQO1 in the experimental mice; ## P<0.01 vs. NC group; *P<0.05, **P<0.01, ***P<0.001 vs. HFD group.

[0360] Figure 3 In Experimental Example 1, SZ-A reduces the level of 4-HNE, a lipid peroxidation product in liver tissue; # P<0.05, vs. NC group (chow group); **P<0.01 vs. HFD group.

[0361] Figure 4 In Experimental Example 1, mulberry extract relieves liver endoplasmic reticulum stress. # P<0.05 vs. NC group (CHOW group), *P<0.05, **P<0.01 vs. HFD group.

[0362] Figure 5 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.

[0363] Figure 6 In Experimental Example 1, SZ-A increased the NAD of high-fat-fed mice + 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.

[0364] Figure 7This is for 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 the number, arrangement and structure of mitochondria 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 tissues of CKD rats, mean±SEM, n=6, ***p<0.001, **p<0.01, *p<0.05vs CKD.

[0365] Figure 8 It is that SZ-A in Experimental Example 3 alleviates the increase of ROS in liver cells caused by PA / OA; # P<0.05 vs. BSA group, **P<0.01 vs. PA / OA group.

[0366] Fig. 9 In Experimental Example 3, SZ-A decreased the expression of p16 and p53 genes; # P<0.05, ## P<0.01 vs. D-gal-500 (or dox-1000) group, ***P<0.001 vs. NC group.

[0367] Fig.10 The effect of SZ-A on the gene expression of P53 (A) and LaminB1 (B) in the D-gal-induced HT22 neuronal cell aging model in Experimental Example 3;

[0368] Fig.11 It 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);

[0369] Fig.12 This is the staining result of the effect of SZ-A on D-gal-induced H9C2 cell senescence in Experimental Example 3;

[0370] Fig.13The results of the effect of SZ-A on D-gal-induced C2C12 cell senescence in Experimental Example 3 (A in the figure is a cell staining diagram, and B is a quantitative statistical diagram. Compared with the model group, ***P<0.001, ****P<0.0001);

[0371] Fig.14 The staining results of flow cytometry detection of the effect of SZ-A on D-gal-induced C2C12 cell senescence in Experimental Example 3 are statistical graphs of mean fluorescence intensity. Compared with the model group (D20), **P<0.01, ***P<0.001);

[0372] Fig.15 This is the result of SZ-A reducing the aging pigment content in Caenorhabditis elegans in Experimental Example 4; *P<0.05, **P<0.01, vs. Control.

[0373] Fig.16 This is the result of the apparent aging score after administration of SZ-A for 1 month in Experimental Example 5;

[0374] Fig.17 This is the result of the apparent aging score after administration of SZ-A for 5 months in Experimental Example 5;

[0375] Fig.18 This is the heat production result of mice administered SZ-A for 4 months in Experimental Example 5 (compared with the model group G2, *P<0.05, **P<0.01);

[0376] Fig.19 This is the bone density test result of mice administered SZ-A for 5 months in Experimental Example 5. DETAILED DESCRIPTION

[0377] The present invention is further described in detail below through the accompanying drawings and examples. Through these exemplary descriptions, the characteristics and advantages of the present invention will become more clear and distinct.

[0378] 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.

[0379] 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.

[0380] 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).

[0381] Preparation Example 1

[0382] Take 1000kg of fresh mulberry branches (fine-toothed mulberry branches, Yuesang 11), crush them, add 4000L of water, extract them by heating reflux for 2h, combine the extracts, filter and remove insoluble matter to obtain a crude extract. The crude extract is heat concentrated to a solid mass percentage of 4%, and kept at 50℃ as the loading liquid for the cationic resin column.

[0383] Use 150kg of D113 macroporous weakly acidic phenyl propylene cationic resin to load the column, wash with 2mol / L hydrochloric acid solution until the pH of the eluate is 4.5; wash with 1mol / L sodium hydroxide solution until the pH of the eluate is 8.5; wash with 2mol / L hydrochloric acid solution until the pH of the eluate is 4.5; then rinse with 5 times the column volume of deionized water to complete the activation. Load the concentrated extract, then elute with 1000L 2.5mol / L ammonia water at a rate of 6BV / h. Collect the eluate when the pH of the cation column effluent is >7. When the collected liquid reaches 900L, stop collecting and directly purify the collected liquid through the anion column.

[0384] Use 62.5 kg of D218 macroporous strongly alkaline acrylic anion resin to load the column, wash with 1.5 mol / L sodium hydroxide solution until the pH of the eluate is 9.0; wash with 1.5 mol / L hydrochloric acid solution until the pH of the eluate is 3.5; wash with 1.5 mol / L sodium hydroxide solution until the pH of the eluate is 9.0; complete activation. Load the collected cationic resin eluate onto the anion resin, and collect the effluent until the effluent reaches 870 L.

[0385] The collected liquid was centrifuged to remove impurities and then concentrated by reverse ion osmosis membrane. The liquid after concentration had a specific gravity of 1.25, and was transferred to an alcohol precipitation tank. 25 L of anhydrous ethanol was added at 500 rpm. After the addition of ethanol, stirring was stopped, and the alcohol precipitation was carried out for 24 hours. The supernatant was taken and concentrated under reduced pressure to obtain a mulberry branch extract (i.e., SZ-A extract).

[0386] The mulberry branch extract has 52% alkaloids, 22% polysaccharides, 0.8% flavonoids and 20% amino acids in the extract. Among the alkaloids, 1-DNJ, FAG and DAB are 60%, 17% and 15%.

[0387] Preparation Example 2

[0388] Take 10kg of fresh mulberry branches (Sangteyou No. 2), crush them, add 150L of water, add them in 2 times, extract them by decoction for 3h each time, combine the extracts, and filter to remove insoluble matter. The extract is hot concentrated to 8% solid mass percentage, transferred to an alcohol precipitation tank, and 2367.9g of anhydrous ethanol (3L) is added under a stirring paddle of 300rpm. After the addition of ethanol, stop stirring, precipitate with alcohol for 24h, and take the supernatant as the sample liquid for the cationic resin column. Use 002SC type strong acid styrene cationic resin 5kg to load the column, and activate the cationic resin according to the method of Preparation Example 1. Load the extract treated with concentrated alcohol precipitation, then use 100L 5mol / L potassium chloride for elution, the elution rate is 5BV / h, and detect the effluent with 20% silicotungstic acid. Start collecting when a white precipitate is generated, and stop collecting when the collected liquid reaches 25L, and directly purify the collected liquid through an anion column.

[0389] Use 10 kg of 711 type strong basic styrene anion resin to load the column, and activate the anion resin according to the method of Preparation Example 1. Load the collected cationic resin eluate onto the anion resin, and collect the effluent until the effluent reaches 15 L. Load the collected liquid onto the cationic resin again, and separate twice more using the cationic resin and the anionic resin in turn according to the above method.

[0390] The collected liquid obtained after three column separations was centrifuged to remove impurities and then concentrated by 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 of 1000 rpm. After the addition of ethanol, stirring was stopped, and the alcohol precipitation was performed for 24 hours. The supernatant was taken and concentrated under reduced pressure to obtain a mulberry branch extract. In addition, fresh mulberry bark and mulberry leaf (Sangteyou No. 2) were extracted, and the extraction method and parameters were the same as the above method.

[0391] 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%, the content of FAG is 0.5%, and the content of DAB is 0.4%.

[0392] The obtained Morus alba extract has 95% alkaloids, 2% polysaccharides, 0.1% flavonoids and 1% amino acids. Among the alkaloids, 1-DNJ, FAG and DAB are 96%, 1.5% and 1.4%.

[0393] The obtained mulberry leaf extract has an alkaloid content of 90%, a polysaccharide content of 4%, a flavonoid content of 0.1%, and an amino acid content of 3%. Among the alkaloids, the content of 1-DNJ is 91%, FAG is 3.1%, and DAB is 2.8%.

[0394] Preparation Example 3

[0395] Take 1000kg of fresh mulberry branches (Guangdong mulberry), crush them, add 11500L of water, heat and reflux for 2h, combine the extracts, filter and remove insoluble matter to obtain a crude extract. The crude extract is first centrifuged to remove impurities, and then concentrated with a counter ion permeation membrane to a solid mass percentage of 1%, which is used as the loading solution for the cationic resin column.

[0396] 300 kg of D001 macroporous strong acid styrene cationic resin was loaded into the column, and the cationic resin 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 detected with 20% silicotungstic acid. When a white precipitate was generated, collection began, and when the collected liquid reached 1000 L, collection was stopped.

[0397] The collected liquid obtained after the cationic column separation is concentrated by nanofiltration membrane, and the extract is concentrated under reduced pressure to obtain an extract concentrate.

[0398] The obtained mulberry branch extract has 15% alkaloid content, 20% polysaccharide content, 7% flavonoid content, and 45% amino acid content. Among the alkaloids, the content of 1-DNJ is 55%, FAG is 23%, and DAB is 10%.

[0399] Preparation Example 4

[0400] 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.

[0401] Use 150kg of D113 macroporous weakly acidic phenyl propylene cationic resin to load the column, wash with 2mol / L hydrochloric acid solution until the pH of the eluate is 4.5; wash with 1mol / L sodium hydroxide solution until the pH of the eluate is 8.5; wash with 2mol / L hydrochloric acid solution until the pH of the eluate is 4.5; then rinse with 5 times the column volume of deionized water to complete the activation. Load the concentrated extract, then elute with 1000L 2.5mol / L ammonia water at a rate of 6BV / h. Collect the eluate when the pH of the cation column effluent is >7. When the collected liquid reaches 900L, stop collecting and directly purify the collected liquid through the anion column.

[0402] Use 125kg of D218 macroporous strongly alkaline acrylic anion resin to load the column, wash with 1.5mol / L sodium hydroxide solution until the pH of the eluate is 9.0; wash with 1.5mol / L hydrochloric acid solution until the pH of the eluate is 3.5; wash with 1.5mol / L sodium hydroxide solution until the pH of the eluate is 9.0; complete activation. Load the collected cationic resin eluate onto the anion resin, and collect the effluent with a pH greater than 8 until the effluent reaches 870L.

[0403] The collected liquid obtained after the anion column separation is filtered through a microfiltration membrane to remove impurities and then concentrated with a counter ion permeation membrane. The concentrated liquid has a specific gravity of 1.1 and is transferred to an alcohol precipitation tank. 15 kg of anhydrous ethanol is added at a stirring paddle of 400 rpm. After the addition of ethanol, stirring is stopped, and the alcohol precipitation is carried out for 24 hours. The supernatant is taken and concentrated under reduced pressure to obtain a mulberry branch extract. Sample content: alkaloid content is 80%, polysaccharide content is 5%, flavonoid content is 0.1%, and amino acid content is 4%. Among the alkaloids, the content of 1-DNJ is 75%, FAG is 12%, and DAB is 10%.

[0404] Preparation Example 5

[0405] 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.

[0406] Use 62.5 kg of D218 macroporous strongly alkaline acrylic anion resin to load the column, wash with 1.5 mol / L sodium hydroxide solution until the pH of the eluate is 9.0; wash with 1.5 mol / L hydrochloric acid solution until the pH of the eluate is 3.5; wash with 1.5 mol / L sodium hydroxide solution until the pH of the eluate is 9.0; complete activation. Load the collected extract concentrate onto the anion resin and collect the effluent.

[0407] The collected solution obtained after the anion column separation was filtered through a microfiltration membrane to remove impurities and then concentrated by a counter ion permeation membrane, and further concentrated and dried under reduced pressure to obtain a mulberry branch extract. Sample content: 3% alkaloids, 70% polysaccharides, 10% flavonoids, and 10% amino acids. Among the alkaloids, the content of 1-DNJ was 68%, FAG was 17%, and DAB was 8%.

[0408] Preparation Example 6

[0409] Take 1500kg of fresh mulberry branches (fine-toothed mulberry branches, Yuesang 11), crush them, add 6000L of water, extract them by heating reflux for 2h, combine the extracts, filter and remove insoluble matter to obtain a crude extract. The crude extract is heat concentrated to a solid mass percentage of 4%, and kept at 50℃ as the loading liquid for the cationic resin column.

[0410] Use 100kg of D113 macroporous weakly acidic phenyl propylene cationic resin to load the column, wash with 2mol / L hydrochloric acid solution until the pH of the eluate is 4.5; wash with 1mol / L sodium hydroxide solution until the pH of the eluate is 8.5; wash with 2mol / L hydrochloric acid solution until the pH of the eluate is 4.5; then rinse with 5 times the column volume of deionized water to complete the activation. Load the concentrated extract, then elute with 1000L 2.5mol / L ammonia water at a rate of 6BV / h. Collect the eluate when the pH of the cation column effluent is >7. Stop collecting when the collected liquid reaches 900L, and directly purify the collected liquid through the anion column.

[0411] Use 62.5kg of D218 macroporous strong alkaline acrylic anion resin to load the column, use 1.5mol / L sodium hydroxide solution to wash until the pH of the eluate is 9.0; use 1.5mol / L hydrochloric acid solution to wash until the pH of the eluate is 3.5; wash with 1.5mol / L sodium hydroxide solution until the pH of the eluate is 9.0; complete activation. Load the collected cationic resin eluate onto the anion resin, and collect the effluent until the effluent reaches 870L. Concentrate the effluent under reduced pressure to obtain a mulberry branch extract, in which the content of alkaloids is 30%, the content of polysaccharides is 35%, the content of flavonoids is 2%, and the content of amino acids is 25%. Among the alkaloids, the content of 1-DNJ is 62%, FAG is 20%, and DAB is 13%.

[0412] Preparation Example 7

[0413] Take 1000kg of fresh mulberry branches (fine-toothed mulberry branches, Yuesang 11), crush them, add 4000L of water, extract them by heating reflux for 2h, combine the extracts, filter and remove insoluble matter to obtain a crude extract. The crude extract is heat concentrated to a solid mass percentage of 4%, and kept at 50℃ as the loading liquid for the cationic resin column.

[0414] Use 100kg of D113 macroporous weakly acidic phenyl propylene cationic resin to load the column, wash with 2mol / L hydrochloric acid solution until the pH of the eluate is 4.5; wash with 1mol / L sodium hydroxide solution until the pH of the eluate is 8.5; wash with 2mol / L hydrochloric acid solution until the pH of the eluate is 4.5; then rinse with 5 times the column volume of deionized water to complete the activation. Load the concentrated extract, then elute with 1000L 2.5mol / L ammonia water at a rate of 6BV / h. Collect the eluate when the pH of the cation column effluent is >7. Stop collecting when the collected liquid reaches 900L, and directly purify the collected liquid through the anion column.

[0415] Use 62.5kg of D218 macroporous strong alkaline acrylic anion resin to load the column, wash with 1.5mol / L sodium hydroxide solution until the pH of the eluate is 9.0; wash with 1.5mol / L hydrochloric acid solution until the pH of the eluate is 3.5; wash with 1.5mol / L sodium hydroxide solution until the pH of the eluate is 9.0; complete activation. Load the collected cationic resin eluate onto the anion resin, and collect the effluent until the effluent reaches 870L. Concentrate the effluent under reduced pressure to obtain a mulberry branch extract, in which the content of alkaloids is 40%, the content of polysaccharides is 25%, the content of flavonoids is 0.5%, and the content of amino acids is 25%. Among the alkaloids, the content of 1-DNJ is 57%, FAG is 24%, and DAB is 16%.

[0416] Preparation Example 8

[0417] 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.

[0418] Use 150kg of D113 macroporous weakly acidic phenyl propylene cationic resin to load the column, wash with 2mol / L hydrochloric acid solution until the pH of the eluate is 4.5; wash with 1mol / L sodium hydroxide solution until the pH of the eluate is 8.5; wash with 2mol / L hydrochloric acid solution until the pH of the eluate is 4.5; then rinse with 5 times the column volume of deionized water to complete the activation. Load the concentrated extract, then elute with 1000L 2.5mol / L ammonia water at a rate of 6BV / h. Collect the eluate when the pH of the cation column effluent is >7. When the collected liquid reaches 900L, stop collecting and directly purify the collected liquid through the anion column.

[0419] Use 62.5 kg of D218 macroporous strongly alkaline acrylic anion resin to load the column, wash with 1.5 mol / L sodium hydroxide solution until the pH of the eluate is 9.0; wash with 1.5 mol / L hydrochloric acid solution until the pH of the eluate is 3.5; wash with 1.5 mol / L sodium hydroxide solution until the pH of the eluate is 9.0; complete activation. Load the collected cationic resin eluate onto the anion resin, and collect the effluent with a pH greater than 8 until the effluent reaches 870 L.

[0420] The collected liquid obtained after the anion column separation was filtered through a microfiltration membrane to remove impurities and then concentrated with 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 at 400 rpm. After the ethanol was added, stirring was stopped, and the alcohol was precipitated for 24 hours. The supernatant was taken and concentrated under reduced pressure to obtain a mulberry branch extract. Sample content: 63% alkaloids, 23% polysaccharides, 1% flavonoids, and 5% amino acids. Among the alkaloids, the content of 1-DNJ was 61.9%, FAG was 16.6%, and DAB was 11.1%.

[0421] Preparation Example 9

[0422] Take 1000kg of fresh mulberry branches (Yue Sang No. 11), crush them, add 4000L of water, extract them by heating reflux method for 2h, combine the extracts, filter and remove insoluble matter to obtain crude extract. The crude extract is heat concentrated to a solid content of 4%, and kept at 50℃ as the loading liquid for the cationic resin column.

[0423] Use 120kg of D113 macroporous weakly acidic phenyl propylene series cationic resin to load the column, wash with 2mol / L hydrochloric acid solution until the pH of the eluate is 4.5; wash with 1mol / L sodium hydroxide solution until the pH of the eluate is 8.5; wash with 2mol / L hydrochloric acid solution until the pH of the eluate is 4.5; and then rinse with 5 times the column volume of deionized water to complete the activation. Load the concentrated extract, then elute with 1000L 2.5mol / L ammonia water at an elution rate of 6BV / h. Collect the eluate when the pH of the cation column effluent is >7. When the collected liquid reaches 900L, stop collecting and directly purify the collected liquid through the anion column.

[0424] Use 45kg of D218 macroporous strongly alkaline acrylic anion resin to load the column, wash with 1.5mol / L sodium hydroxide solution until the pH of the eluate is 9.0; wash with 1.5mol / L hydrochloric acid solution until the pH of the eluate is 3.5; wash with 1.5mol / L sodium hydroxide solution until the pH of the eluate is 9.0; complete activation. Load the collected cationic resin eluate onto the anion resin and collect the effluent until the effluent reaches 870L.

[0425] The collected liquid obtained after separation by anion column is filtered through a microfiltration membrane to remove impurities and then concentrated with a counter ion permeation membrane. The concentrated liquid has a specific gravity of 1.1 and is transferred to an alcohol precipitation tank. 15 kg of anhydrous ethanol is added at 300 rpm. After the addition of ethanol, stirring is stopped, and the alcohol precipitation is carried out for 24 hours. The supernatant is taken and concentrated under reduced pressure to obtain an extract. Sample content: the mass percentage of alkaloids is 70%, the mass percentage of polysaccharides is 20%, the mass percentage of flavonoids is 0.6%, and the mass percentage of amino acids is 5%. Among the alkaloids, the content of 1-DNJ is 70%, FAG is 13%, and DAB is 10%.

[0426] Preparation Example 10

[0427] Take 80kg of fresh mulberry branches (Guisangyou No. 62), crush them, add 1000L of water, extract them by heating reflux method for 2h, combine the extracts, filter and remove insoluble matter to obtain crude extract. The crude extract is heat concentrated to a solid content of 4%, and kept at 50℃ as the loading liquid for the cationic resin column.

[0428] Use 50kg of D113 macroporous weakly acidic phenyl propylene series cationic resin to load the column, wash with 2mol / L hydrochloric acid solution until the pH of the eluate is 4.5; wash with 1mol / L sodium hydroxide solution until the pH of the eluate is 8.5; wash with 2mol / L hydrochloric acid solution until the pH of the eluate is 4.5; and then rinse with 5 times the column volume of deionized water to complete the activation. Load the concentrated extract, then elute with 600L 2.5mol / L ammonia water at a rate of 6BV / h. Collect the eluate when the pH of the cation column effluent is >7. When the collected liquid reaches 9800L, stop collecting and directly purify the collected liquid through the anion column.

[0429] Use 65kg of D218 macroporous strongly alkaline acrylic anion resin to load the column, wash with 1.5mol / L sodium hydroxide solution until the pH of the eluate is 9.0; wash with 1.5mol / L hydrochloric acid solution until the pH of the eluate is 3.5; wash with 1.5mol / L sodium hydroxide solution until the pH of the eluate is 9.0; complete activation. Load the collected cationic resin eluate onto the anion resin and collect the effluent until the effluent reaches 750L. Reload the collected liquid onto the cationic resin, and separate it again using the cationic resin and anionic resin in turn according to the above method.

[0430] The collected liquid obtained after the two separations 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. 400 g of anhydrous ethanol was added under a stirring paddle of 350 rpm. After the addition of ethanol, stirring was stopped, and the alcohol precipitation was continued for 24 hours. The supernatant was taken and concentrated under reduced pressure to obtain an extract. Sample content: the mass percentage of alkaloids was 90%, the mass percentage of polysaccharides was 3%, the mass percentage of flavonoids was 0.5%, and the mass percentage of amino acids was 2%.

[0431] Among the alkaloids, the content of 1-DNJ was 94%, FAG was 2%, and DAB was 1%.

[0432] Test Example 1

[0433] 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 basic feed, and the HFD group and the SZ-A group were fed with a high-fat diet (Research Diet, D12492, 60 kcal% Fat). After 14 weeks of feeding, the corresponding drugs were gavaged to each group of mice 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.

[0434] (1) SZ-A improves the structure and quantity of mitochondria in liver and myocardium

[0435] Electron microscopy method: To observe the ultrastructure of mitochondria, mouse liver and myocardial tissues were fixed in glutaraldehyde solution (2.5%) 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 taken under an 80 kV transmission electron microscope (TEM, Hitachi H7650, Tokyo, Japan). Figure 1 shown.

[0436] 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 × 3 min, 4 ° C, and discard the precipitate. Take the supernatant, 12000 g × 10 min, 4 ° C,

[0437] Centrifuge, and the precipitate is the crude mitochondria. Dissolve the precipitate in 100mL (50mmol / L glucose, 10mmol / L Na2EDTA, 25mmol / L Tris-HCl pH 8.0) solution and mix thoroughly. Add 200mL of freshly prepared (1% SDS, 0.2N NaOH) solution, shake gently, and ice bath for 5min. Add 150mL (3M NaAc pH 4.8) solution, shake gently, and ice bath for 5min. Centrifuge at 12000g×10min, 4℃. Take the supernatant and add an equal volume of (phenol, chloroform, isoamyl alcohol volume ratio is 25:24:1) solution, extract 2 to 4 times, there is no white substance on the interface, centrifuge at 10000g×5min. Take the supernatant and add 2 times the volume of 95% ethanol, let it stand at room temperature for 15min, and then centrifuge at 12000g×10min. Discard the supernatant, wash the precipitate once with 75% ethanol, and centrifuge at 12000g×5min. Discard the supernatant, take the precipitate, and vacuum dry it to obtain mitochondrial DNA. Dissolve it in an appropriate amount of TE buffer and store it at -20℃. Fluorescence quantitative PCR was used to detect the expression of mitochondrial copy number ND1, COXI, COXII, the expression of mitochondrial complex enzymes I-ndufs8, II-sdhb, III-uqcrc1, V-atp5a1, and the expression of mitochondrial fission, fusion and mitochondrial generation related genes DRP1, TFAM, TFB1, TFB2, MFN1, OPA1, NRF2, PGC1-α, HO-1 and NQO1, such as Figure 2 shown.

[0438] Figure 1 and Figure 2 The results showed that SZ-A could 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.

[0439] (2) Improve the antioxidant capacity of liver tissue

[0440] 4-HNE immunohistochemistry: The liver tissue of the 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 sliced ​​at a thickness of 5 μm for 4-HNE staining. The primary antibody 4-HNE (ab46545, Abcam, 1:200) was incubated at 4°C overnight. The peroxidase substrate kit was used for staining, and the results were photographed under a microscope. Figure 3 shown.

[0441] Figure 3 The results showed that SZ-A could reduce the level of 4-HNE, a lipid peroxidation product in liver tissue, and restore the antioxidant capacity.

[0442] (3) Alleviate liver endoplasmic reticulum stress

[0443] GRP78 immunohistochemistry: The liver tissues of the experimental mice were fixed in 10% neutral buffered formalin overnight, washed once with PBS, and stored in 70% ethanol at 4°C. The tissues were dehydrated and embedded in paraffin. The embedded tissues were sliced ​​at a thickness of 5 μm for immunohistochemical staining. The primary antibody was incubated at 4°C overnight. The peroxidase substrate kit was used for staining and the microscope was used for photography. Figure 4 shown.

[0444] mRNA expression detection: use Total RNA was isolated from liver tissue of experimental mice by using a 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 included 30 s at 95°C, 10 s at 95°C, and 30 s at 60°C for a total of 40 cycles. The purity of the PCR product was determined by melting curve analysis. 2 -ΔΔCT Calculate the relative amount of each gene, such as Figure 4 shown.

[0445] Figure 4 The results showed that the endoplasmic reticulum stress marker GRP78 and pro-apoptotic protein CHOP increased in the model group, the function of the downstream anti-apoptotic protein Bcl-2 was inhibited, and the pro-apoptotic protein Bax was activated, and endoplasmic reticulum stress induced hepatocyte apoptosis. SZ-A reduced the expression of endoplasmic reticulum stress-related pro-apoptotic genes (ATF4, ATF6, CHOP, Bax, Caspase12, Caspase3) in liver tissue and alleviated HFD-induced hepatocyte stress injury.

[0446] (4) Increase the expression of the longevity protein sirt in the liver and heart

[0447] 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 included 30 s at 95°C, 10 s at 95°C, and 30 s at 60°C for a total of 40 cycles. The purity of the PCR product was determined by melting curve analysis. 2 -ΔΔCT Calculate the relative amount of each gene, such as Figure 5 shown.

[0448] The results showed that mulberry extract SZ-A could significantly increase the expression levels of longevity genes sirt1, sirt3, sirt5 and sirt6 in myocardial and liver tissues.

[0449] (6) Increase NAD + Level

[0450] NAD + Detection method: After washing the tissue with pre-cooled PBS on ice, weigh about 10-30 mg of tissue sample, cut it into pieces with scissors, put 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 taken as the sample to be tested. + , NADH content or NAD + Determination of the ratio of NADH / NADH: 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 at room temperature or 4°C for 5 minutes, 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 appropriately, then add 10 μL of the colorimetric solution to each well, mix well, and incubate at 37°C in the dark for 30 minutes. At this time, orange-yellow formazan will be formed. Measure the absorbance at 450 nm. Figure 6 shown.

[0451] The results showed that SZ-A could increase NAD + Levels and NAD + / NADH ratio. Studies have shown that as we age, the NAD + The concentration of NAD decreases by about 10% to 80%. Many diseases are related to NAD + Reduced levels of NAD are associated with metabolic disorders, cancer, and neurodegenerative diseases. + Levels can improve physiological function and increase healthy life span. + The ratio of NAD / NADH 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.

[0452] 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)

[0453] 1. Experimental Design

[0454] 1. Experimental Materials

[0455] 1.1 Reagents

[0456] CCK8 kit (Beiren Chemical Technology Co., Ltd.), adenine (sigma), DMEM / F-12 medium (ThermoFisher Scientific), fetal bovine serum (Thermo Fisher Scientific), ATP detection kit (Biyuntian), DNA extraction kit (QIAamp), ROS detection reagent DCFH-DA (Solebo), SOD detection kit, MDA detection kit (Biyuntian), CD45, CD68, COL1A1, Fibronectine, α-SMA, COL4A1 primary antibodies, secondary fluorescent antibodies (Abcam).

[0457] 1.2 Experimental drugs

[0458] The mulberry branch extract prepared in Preparation Example 1 was prepared in different concentrations and dissolved in water.

[0459] 1.3 Experimental animals

[0460] Wistar rats, male, 200 g, were purchased from Beijing Sibeifu Experimental Animal Technology Co., Ltd. and kept 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.

[0461] 2. Experimental methods

[0462] Animal Experimentation:

[0463] Wistar male rats were divided into 6 groups, 8 in each group, and administered by gavage. The specific experimental groups, dosage and frequency of administration are shown in Table 1. After one week of adaptive feeding of the rats, the model was established and the administration was continued for 4 weeks. After the experiment, whole blood and kidney tissue were separated, and the levels of uric acid, urea nitrogen and creatinine in serum were determined using kits.

[0464] Table 1

[0465]

[0466] 2. Experimental Results

[0467] The effects of long-term administration of SZ-A on renal oxidative stress and mitochondrial function in CKD rats are as follows:

[0468] Figure 7As shown in Figures 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 with antioxidant effect in the kidney of the model group rats was significantly reduced, the gene expression of H2O2 oxidase CAT and glutathione synthetase GSS was significantly reduced, and 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.

[0469] Figure 7 The results of the D cell experiment showed that SZ-A could reduce the production of ROS (marked with green fluorescence in the figure) induced by H2O2 for both renal tubular epithelial cells, verifying its direct anti-oxidative stress effect.

[0470] In addition, transmission electron microscopy results showed that ( Figure 7 As shown in Figure E, the number of mitochondria in the kidneys of rats in the model group decreased, their arrangement was disordered, their morphology was deformed and twisted, they were slightly swollen, their matrix became partially pale, their cristae expanded, and some of their mitochondria were broken and disappeared. SZ-A can improve the above mitochondrial lesions.

[0471] The ATP and mitochondrial DNA copy number of rat kidney tissue were detected, and the results showed that ( Figure 7 (F and G) SZ-A can significantly improve ATP deficiency and reduced mitochondrial copy number in the kidneys of CKD rats and alleviate mitochondrial damage.

[0472] Test Example 3 Cell Test

[0473] (1) SZ-A reduces ROS levels in HepG2 cells

[0474] Cell culture and ROS detection: HepG2 cells were cultured in DMEM containing 10% FBS and 1% penicillin / streptomycin, maintained at 37°C and 5% CO2. PA / OA stock solution was prepared and then coupled with 1% fatty acid-free bovine serum albumin (BSA). Cells were treated with SZ-A (the mulberry branch extract in Preparation Example 8, with a drug concentration of 50 μg / mL in terms of alkaloids), and an appropriate amount of PA / OA (palmitic acid / oleic acid) was added to the cell DMEM culture medium. DCFH-DA was diluted with serum-free culture medium at a ratio of 1:1000 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 should be sufficient to cover the cells. Usually, no less than 1 ml of diluted DCFH-DA was added to one well of a six-well plate. Incubate in a 37°C cell culture incubator for 20 minutes. Wash the cells three times with serum-free cell culture medium to fully remove the DCFH-DA that did not enter the cells. Flow cytometry was used to detect Figure 8shown.

[0475] The results showed that SZ-A can alleviate the increase of ROS in liver cells caused by PA / OA. Mitochondria are the energy basis for maintaining the physiological activities of cells and the entire body, and are also the main site of ROS generation. Excessive ROS generation can cause lipid peroxidation, leading to mitochondrial degeneration, leakage and rupture, which are important causes of cell aging. The results of this experiment showed that SZ-A can effectively remove mitochondrial ROS.

[0476] (2) SZ-A alleviates aging of endothelial cells and neurons

[0477] 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. HT22 cell replicative senescence model was established by continuous subculture.

[0478] 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 culture medium was changed every other day, and the cells were subcultured when they were 80% confluent. DOX was dissolved in DMEM culture medium to prepare 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. Different concentrations of mulberry branch extract of Preparation Example 8 were added while DOX treatment.

[0479] 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. In order to simulate the aging phenotype, HT22 cells were inoculated into 6-well plates and incubated with D-gal for 48 hours. The mulberry branch extract of Preparation Example 8 was added at the same time as D-gal treatment (dosing concentration 200 μg / mL, calculated as alkaloid). In the same way, the mulberry branch extracts of Preparation Example 6, Preparation Example 8 and Preparation Example 10 (dosing concentration 45 μg / mL, 83 μg / mL, 166 μg / mL, 333 μg / mL, calculated as extract) were treated.

[0480] Fluorescence quantitative PCR was used to detect the mRNA of p16, lamin B1 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 included 95°C for 30 s, 95°C for 10 s, and 60°C for 30 s for a total of 40 cycles. The purity of the PCR product was determined by melting curve analysis. 2 -ΔΔCT The relative amount of each gene was calculated.

[0481] The primer sequences are as follows:

[0482] p16, Forward sequence as shown in SEQ 1: 5′-TCAAGACATCGTGCGATATTTG-3′;

[0483] The reverse sequence is shown in SEQ 2: 5'-TTAGCTCTGCTCTTGGGATTG-3';

[0484] P53, forward sequence as shown in SEQ 3: 5′-GCCATCTACAAGAAGTCACAGCAA-3′;

[0485] The reverse sequence is shown in SEQ 4: 5'-AGGCACAAACACGAACCTCAA-3';

[0486] LaminB1, forward sequence as shown in SEQ 5: 5′-TCGCAAAAGCATGTATGAAGAG-3′;

[0487] The reverse sequence is shown in SEQ 6: 5'-CTCAAGTTTGGCATGGTAAGTC-3';

[0488] GADPH, forward sequence as shown in SEQ 7: 5'-TCCCACTCTTCCACCTTC-3';

[0489] The reverse sequence is shown in SEQ 8: 5'-CTGTAGCCGTATTCATTGTC-3'.

[0490] Telomere length was detected in the HT22 cell replicative aging model: HT22 cells were cultured for 15 generations, and Preparation Example 8 SZ-A (dosing concentration was 83 μg / mL and 166 μg / mL, calculated as extract) was added to detect telomere length in HT22 cells after drug treatment. Telomere length was determined according to the ratio of telomere product / single copy gene product (T / S) obtained by quantitative PCR.

[0491] like Fig. 9 As shown, the results showed that mulberry branch extract SZ-A can significantly reduce the expression of p53 gene. At the same time, mulberry branch extract SZ-A has a tendency to reduce the expression of p16 gene and alleviate the cell cycle arrest of senescent cells.

[0492] like Fig.10 As shown in Figure 2, mulberry extract SZ-A can reduce p53 expression to varying degrees and increase lamin B1 gene expression ( Fig.10 ), relieves the cell cycle arrest of senescent cells and has a significant anti-cell aging effect.

[0493] Fig.11 The results showed that after 15 generations of continuous subculture of HT22 cells, SZ-A of Preparation Example 8 (dosing concentrations of 40 μg / mL, 83 μg / mL, and 166 μg / mL, calculated as extract) could significantly prolong telomere length and delay replicative cell senescence.

[0494] (3) Effects of mulberry extract SZ-A on aging mouse cardiomyocytes H9C2 cells

[0495] Cell culture: H9C2 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 (30 mg / ml, referred to as D30) was dissolved in DMEM culture medium. In order to simulate the aging phenotype, H9C2 cells were inoculated into 6-well plates and incubated with D-gal for 48 hours. While D-gal treatment, different concentrations (160 μg / mL, 320 μg / mL, calculated by extract) of the mulberry branch extract SZ-A of Preparation Examples 2, 6, 8, and 10 were added.

[0496] SA-β-galactosidase staining: For cells cultured in 6-well plates, remove the cell culture medium, wash once with PB, add 1 ml of β-galactosidase staining fixative, and fix at room temperature for 15 minutes. Remove the cell fixative and wash the cells 3 times with PBS or HBSS for 3 minutes each. Remove the PBS and add 1 ml of staining working solution to each well. Incubate at 37°C overnight. You can seal the 6-well plate with parafilm or plastic wrap to prevent evaporation.

[0497] Fig.12 The results showed that after 48h of D-gal (30mg / ml) induction, the senescence of H9C2 cells increased significantly, and the mulberry extract SZ-A (dosing concentration 160μg / mL, 320μg / mL, calculated on the basis of the extract) prepared in different examples of the present invention could significantly reduce the degree of cell senescence ( Fig.12 Green staining represents senescent cells).

[0498] (4) Effects of mulberry extract SZ-A on aging mouse myoblasts C2C12 cells

[0499] Growth medium (high glucose DMEM medium + 10% fetal bovine serum) was added to C2C12 cells and incubated in a constant temperature incubator at 37°C and 5% CO2. The cells were cultured to about 85%, and the cells were collected and grouped into a control group (NC), an aging model group (D-gal), and an SZ-A group (D-gal + SZ-A). Subsequently, the culture medium of each group was replaced with a differentiation medium (high glucose DM EM medium + 2% horse serum). According to the purpose of the experiment, D-gal and SZ-A (Mulberry branch extract obtained in Preparation Examples 2, 6, and 8) were added to the differentiation medium before the end of the experiment.

[0500] The groups are as follows:

[0501] NC: control group;

[0502] Model: Aging model group;

[0503] Preparation Example 2: SZ-A group, SZ-A is the mulberry branch extract in Preparation Example 2;

[0504] Preparation Example 6: SZ-A group, SZ-A is the mulberry branch extract in Preparation Example 6;

[0505] Preparation Example 8: SZ-A group, SZ-A is the mulberry branch extract in Preparation Example 8;

[0506] Cell senescence detection method:

[0507] a. SA-β-galactosidase staining

[0508] The aging model group added 100mM D-gal to the culture medium, and the SZ-A group added 166μg / mL (based on the extract) SZ-A and 100mM D-gal to the culture medium for 48h. SA-β-galactosidase staining was used to detect the senescence degree of cells in different groups.

[0509] b. Flow cytometry

[0510] The aging model group added D-gal at a concentration of 100 mM to the culture medium, and the SZ-A group added SZ-A at a concentration of 83 μg / mL (based on the extract) and 100 mM D-gal to the culture medium for 48 h. The cells were trypsinized after 48 h of senescence induction to produce a single cell suspension. The cells were washed and then resuspended in 1X PBS at a concentration of 0.5×10 6 -1.0×10 6 Cells were washed in PBS containing 1% BSA, centrifuged at 400 × g for 5 minutes, and the supernatant was removed. Cells were resuspended in 100 μL of fixative solution, incubated at room temperature in the dark for 10 minutes, and washed in PBS containing 1% BSA to remove the fixative. Cells were resuspended in 100 μL of working solution and incubated in a 37°C CO2-free incubator with CellEvent senescence buffer diluted 1 / 1000. TM Senescence green probe staining for 90 minutes. After incubation, centrifuge at 400×g for 5 minutes, remove the working solution, wash the cells with PBS containing 1% BSA, and after the last wash, resuspend the cells with 500μL PBS and prepare for detection. Use the flow cytometer to set the FITC channel (excitation wavelength 488nm, emission wavelength around 530nm).

[0511] Fig.13 The green staining represents senescent cells. Fig.13 The results showed that the mulberry branch extracts of Preparation Examples 2, 6 and 8 could significantly inhibit the senescence of C2C12 myoblasts.

[0512] Fig.14 The results showed 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 could significantly reduce the senescence of cells.

[0513] Test Example 4 Nematode Experiment

[0514] (1) Nematode brown lipid detection

[0515] Senescence pigment is a kind of aging-related spontaneous fluorescent substance in organisms and is generally considered to be a biomarker for evaluating the degree of aging. The more senescence pigment accumulates in the body, the shorter the life span of the organism. Therefore, we can evaluate whether the test substance has anti-aging effect by detecting and comparing the senescence pigment content in Caenorhabditis elegans at the early stage of aging (the tenth day of adulthood).

[0516] Blank control group (Control): Contains L4 (D0) stage Caenorhabditis elegans and 100 μL SM buffer

[0517] Positive control group (Metformin group): containing L4 (D0) stage Caenorhabditis elegans, 100 μL SM buffer and 50 mM Metformin (metformin)

[0518] The test substance test group contained L4 (D0) stage Caenorhabditis elegans, 100 μL SM buffer and SZ-A prepared in Preparation Example 8.

[0519] Different treatments were added to the wells, mixed thoroughly, and then covered with a culture dish lid, sealed with a sealing film, and wrapped with tin foil. The culture dish was cultured in a shaker at 20°C and 150 rpm in the dark until the end point (Day 10). After the culture was completed, each group of Caenorhabditis elegans was taken out and collected in an EP tube. After washing with SM buffer, the supernatant was removed and about 750 μL of worm liquid was left. 200 μL of 1 mm zirconium oxide grinding beads were added and placed in a tissue grinder. After grinding at a frequency of 30 Hz for 2 min, 100 μL of supernatant was taken and placed in a black 96-well plate. The fluorescence intensity at 355 nm and 460 nm was detected with an ELISA instrument, and the protein content in the supernatant was measured according to the BCA kit.

[0520] Anti-aging test results Fig.15 As shown in the figure, the aging pigment content of the positive control group was significantly reduced compared with the blank control group, indicating that the test was effective. The aging pigment content of the SZ-A group was also significantly reduced compared with the blank control group, indicating that SZ-A has the effect of reducing the aging pigment content in Caenorhabditis elegans.

[0521] (2) Nematode life span detection

[0522] C. elegans growth medium (NGM): Dissolve 3g sodium chloride, 17g agar powder, and 2.5g polypeptone in 975mL distilled water, and sterilize by high pressure steam. After cooling to about 55°C, add 1mL 1mol / L calcium chloride, 1mL 1mol / L magnesium sulfate, 1mL 5mg / mL cholesterol, and 25mL potassium phosphate buffer. Mix well and pour into a sterile culture dish.

[0523] 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 1.5mL centrifuge tube, add 1mL Caenorhabditis elegans lysis solution (10% hypochlorous acid: 1mol / L sodium hydroxide = 1:1), centrifuge at 4000r / min for 1min; remove the lysis solution, wash three times with M9 buffer (4000r / min, centrifuge for 1min), suck out the eggs on the bottom layer with a pipette, apply them on a culture medium containing Escherichia coli OP50 with a diameter of 1cm, and culture at 20℃ for 48-60h to obtain young adults.

[0524] Exposure method of Caenorhabditis elegans: The synchronized Caenorhabditis elegans were transferred to a fresh culture medium containing Escherichia coli OP50, M9 buffer was added to the control group, and different concentrations of SZ-A, i.e., the mulberry branch extract in Preparation Example 1, were added to the exposure groups, treated at 20°C for 24h, and subsequent experiments were carried out.

[0525] Effects of SZ-A on the lifespan of Caenorhabditis elegans

[0526] The exposed C. elegans were randomly selected and placed on a culture medium containing E. coli OP50, with 33 nematodes per group and 3 parallels per group, cultured at 20°C, and a control group was set up. The number of days the nematodes survived was calculated from the day of transfer (day 0 of the lifespan experiment), and the number of surviving and dead C. elegans was recorded every day (the C. elegans was considered dead if it did not move when touched by the platinum wire). The experiment lasted until the last C. elegans died. A curve was drawn based on the number of surviving and dead nematodes, and the experiment was repeated three times.

[0527] The experimental results are shown in Table 2 below, and the results show that mulberry extract SZ-A can prolong the lifespan of nematodes.

[0528] Table 2 Experimental groups and results of the effects of SZ-A on the life span of Caenorhabditis elegans

[0529]

[0530]

[0531] Note: Compared with the Contorl group, **P<0.01, ***P<0.001, ns, no significant difference.

[0532] The results in Table 2 show that after being treated 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.

[0533] Experimental Example 5: Mouse Experiment

[0534] 1. Animal Grouping and Treatment

[0535] 15-month-old C57 mice were randomly divided into 7 groups (see Table 3 for specific groups). Each group had 15 mice, and the drug was administered once a day by gavage. The body weight of the mice was weighed at the same time every week during the feeding period. After the administration, the mice were fasted for 16 hours, the eyeballs were removed, and blood samples were collected. The serum was separated after centrifugation at 3500r / min and 4℃ for 15 minutes and stored in a -80℃ refrigerator. The brain, liver and other tissues were quickly dissected and washed with physiological saline. After drying with filter paper, the mass was weighed. Some samples were fixed in a 10% formalin solution by volume and stored in a 4℃ refrigerator for morphological observation; some samples were frozen in a -80℃ refrigerator for subsequent index analysis. At the same time, 15 3-month-old mice were taken as the young control group.

[0536] 2. Behavioral sign indicator detection

[0537] Apparent observation: observe the mental state, activity, loose hair color, etc. of the mice, and score the aging of the mice. The specific scoring criteria are: comprehensive evaluation of the mental state, activity, loose hair color, muscle grip, vision, hearing, bite, dermatitis, hunchback, gait, and other tissue abnormalities visible to the naked eye. Each indicator is scored from 0 to 10 points. According to the severity, objective scoring is performed, and the higher the score, the higher the degree of aging.

[0538] 3. Metabolic Cage Experiment

[0539] Four months after administration, the mice were placed in metabolic cages, and the oxygen consumption rate and carbon dioxide generation rate were measured to calculate the heat production capacity of the mice.

[0540] Table 3 Experimental groups and drug administration of naturally aged mice

[0541]

[0542]

[0543] Fig.16 The results showed 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). Fig.17 The results showed that after 5 months of administration, the aging scores of the G5, G6 and G7 groups were significantly lower than those of the model group. Therefore, the mulberry extract SZ-A of the present invention can significantly delay the phenotype of naturally aging mice.

[0544] Fig.18 The results showed 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 night 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.

[0545] Bone density test:

[0546] Use the instrument iNSiGHT VET DXA / OT20-2F7113-01. Turn on the gas anesthesia machine and put the mouse into the induction box for anesthesia. After the mouse is fully anesthetized, confirm the mouse number, put it on the photographic board, and close the cabinet door. Avoid overlapping of the mouse's limbs and trunk, and fully stretch the mouse's body. Keep the head, body, and tail in a straight line as much as possible. The mouse's head should be close to the anesthesia mask to maintain anesthesia. Open the software, create the mouse information, and click "Measure" in the animal list interface. After confirming the mouse information and measurement information, click "Start Measurement". Do not open the cabinet door during the test. Analyze the data after the measurement.

[0547] Fig.19 The results showed that after 5 months of 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.

[0548] result:

[0549] During the aging process, mice will gradually show weakness in the limbs, decreased responsiveness, yellow 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 motor behavior ability of mice. It also generally improves the mental state, activity, loose hair color, muscle grip, vision, hearing, bite, dermatitis, hunchback, gait and other aging manifestations of mice.

[0550] The present invention has been described above in conjunction with preferred embodiments, but these embodiments are only exemplary and serve only as an illustration. On this basis, the present invention may be subjected to a variety of substitutions and improvements, 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: Taking the weight percentage of the sum of the components of the mulberry extract as 100%, the mulberry extract contains alkaloids with a weight content of more than 3% (optionally containing alkaloids with a weight content of 3-99%, further optionally containing alkaloids with a weight content of 15-99%, further optionally containing alkaloids with a weight content of 30-99%, further optionally containing alkaloids with a weight content of 40-99%, further optionally containing alkaloids with a weight content of 50-99%, further optionally containing alkaloids with a weight content of 60-99%), and / or comprising a polysaccharide content of not more than 70% by weight (optionally comprising a polysaccharide content of 0.2-70% by weight, further optionally comprising a polysaccharide content of 0.2-50% by weight, further optionally comprising a polysaccharide content of 0.2-35% by weight, further optionally comprising a polysaccharide content of 0.2-25% by weight, further optionally comprising a polysaccharide content of 0.2-23% by weight, further optionally comprising a polysaccharide content of 20-25% by weight), and / or contains flavonoids having a weight content of not more than 10% (optionally containing flavonoids having a weight content of 0.05-5%, further optionally containing flavonoids having a weight content of 0-2%, further optionally containing flavonoids having a weight content of 0.05-2%, further optionally containing flavonoids having a weight content of 0.5-1.5%, further optionally containing flavonoids having a weight content of 0-1%, further optionally containing flavonoids having a weight content of 0.05-1%), and / or contains no more than 50% amino acids by weight (optionally contains 0-30% amino acids by weight, further optionally contains 0-25% amino acids by weight, further optionally contains 0-20% amino acids by weight, further optionally contains 0-5% amino acids by weight, further optionally contains 3-25% amino acids by weight or further optionally contains 5-20% amino acids by weight), and / or other components (the weight content is 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 the 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 content of aging pigments in the body; 3) Improve the body's motor behavior ability; 4) Improving the body's metabolic level, preferably promoting the increase of body heat production; 5) Improving mitochondrial function of 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, the increasing the mitochondrial DNA copy number includes promoting the expression of any one or several of ND1, COXI, and COXII; optionally, the increasing the level of mitochondrial complex enzymes includes promoting the expression of any one or several of mitochondrial complex enzymes I-ndufs8, II-sdhb, III-uqcrc1, and V-atp5a1; optionally, the promoting mitochondrial fusion and reducing mitochondrial fission includes regulating the expression of any one or several of DRP1, TFAM, TFB 1, 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 tissues and / or kidney tissues; 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, the 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 + Level and / or NAD + / NADH ratio, the tissue organ is preferably liver and / or heart; 9) Relieving cell cycle arrest and / or increasing the level of cellular oxidative stress; 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) Prolonging telomere length in cells; the cells are preferably neuronal cells; 12) increasing ATP levels in tissues and organs, preferably kidneys; 13) Improve bone density.

4. Use of mulberry extract or its active ingredient as described 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 the aging of in vitro endothelial cells, neuronal cells, liver cells, kidney cells, cardiomyocytes or myoblasts; 4) Use of mulberry extract or its active ingredients in the preparation of products 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 the life span of nematodes; 6) Use of mulberry extract or its active ingredients in preparing a product for reducing the aging score of 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 preparing a product for increasing bone density in mice; Taking the weight percentage of the sum of the components of the mulberry extract as 100%, the mulberry extract contains alkaloids with a weight content of more than 3% (optionally containing alkaloids with a weight content of 3-99%, further optionally containing alkaloids with a weight content of 15-99%, further optionally containing alkaloids with a weight content of 30-99%, further optionally containing alkaloids with a weight content of 40-99%, further optionally containing alkaloids with a weight content of 50-99%, further optionally containing alkaloids with a weight content of 60-99%), and / or comprising a polysaccharide content of not more than 70% by weight (optionally comprising a polysaccharide content of 0.2-70% by weight, further optionally comprising a polysaccharide content of 0.2-50% by weight, further optionally comprising a polysaccharide content of 0.2-35% by weight, further optionally comprising a polysaccharide content of 0.2-25% by weight, further optionally comprising a polysaccharide content of 0.2-23% by weight, further optionally comprising a polysaccharide content of 20-25% by weight), and / or contains flavonoids having a weight content of not more than 10% (optionally containing flavonoids having a weight content of 0.05-5%, further optionally containing flavonoids having a weight content of 0-2%, further optionally containing flavonoids having a weight content of 0.05-2%, further optionally containing flavonoids having a weight content of 0.5-1.5%, further optionally containing flavonoids having a weight content of 0-1%, further optionally containing flavonoids having a weight content of 0.05-1%), and / or contains no more than 50% amino acids by weight (optionally contains 0-30% amino acids by weight, further optionally contains 0-25% amino acids by weight, further optionally contains 0-20% amino acids by weight, further optionally contains 0-5% amino acids by weight, further optionally contains 3-25% amino acids by weight or further optionally contains 5-20% amino acids by weight), and / or other components (the weight content is 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 percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is: Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is: Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is: Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is: Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is: Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is: Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is: Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is: Preferably, taking the sum of the weight percentage of the components of the mulberry extract as 100%, the weight content of the components in the mulberry extract is:

6. The use according to any one of claims 1 to 5, characterized in that: The alkaloids include 1-deoxynojirimycin (DNJ), 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-galactoside) and 1,4-dideoxy-1,4-imino-D-arabinitol (DAB). 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 administration 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 of 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).

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