Use of mulberry extract in preparing drug for treating and / or preventing abnormal bone metabolism-related diseases

Abstract

Provided is use of a mulberry extract in preparing a drug for treating and / or preventing abnormal bone metabolism-related diseases. The mulberry extract comprises, based on the sum of the weight percentages of components in the mulberry extract being 100%: 3-99% of alkaloid, 0.2-70% of polysaccharide, 0-10% of flavone, 0-50% of amino acid, and 0-25% of other components.

Description

Use of mulberry extract in preparing medicine for treating and / or preventing diseases related to abnormal bone metabolism Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to use of a mulberry extract in preparing a medicine for treating and / or preventing diseases related to abnormal bone metabolism. Background Art

[0002] Bone is a tissue that continuously performs remodeling metabolic activities, involving two opposing processes: bone formation (osteoblasts) and bone resorption (osteoclasts). Under normal circumstances, bone resorption and bone formation are closely linked by various hormones (such as PTH, vitamin D, steroids, and calcitonin) and intrinsic regulatory factors (such as cytokines and growth factors). Bone resorption is regulated by RANKL and M-CSF and inhibited by OPG. Bone formation is induced by many biological factors and is also regulated by M-CSF, ALP, osteocalcin, osteopontin, and osteonectin. An imbalance between bone resorption and bone formation leads to bone metabolic diseases such as osteoporosis, osteoarthritis, rheumatoid arthritis, and bone displacement.

[0003] Osteoporosis (OP) is a common metabolic bone disease characterized by decreased bone mass and microarchitectural disruption, leading to increased bone brittleness, decreased bone strength, and increased susceptibility to fractures. The stability of bone metabolism is regulated by the balance between bone formation by osteoblasts and bone resorption by osteoclasts. Primary osteoporosis is the most common clinical type, accounting for approximately 80% of cases. It can be divided into type I high-turnover osteoporosis (common in postmenopausal women), characterized by increased bone resorption, and type II low-turnover osteoporosis (common in the elderly), characterized by decreased bone formation.

[0004] Sarcopenia is a clinical syndrome characterized by a progressive and widespread loss of muscle mass and strength, leading to physical disability, decreased quality of life, and an increased risk of mortality. Sarcopenia and osteoporosis are closely related degenerative diseases, primarily manifesting through the interaction between muscle and bone. Loss of muscle mass can lead to and accelerate the development of osteoporosis, while decreased bone strength can contribute to muscle atrophy and functional degeneration.

[0005] There are currently no reports on the use of mulberry extracts in the treatment of diseases related to abnormal bone metabolism. Summary of the Invention

[0006] The purpose of the present invention is to provide a use of a mulberry extract in preparing a medicament for treating and / or preventing diseases related to abnormal bone metabolism. The mulberry extract of the present invention has a significant effect on improving diseases related to abnormal bone metabolism.

[0007] In the present invention, based on the weight percentage of the sum of the components of the mulberry extract as 100%, the mulberry extract contains 3% or more of alkaloids by weight (optionally containing 3-99% of alkaloids by weight, further optionally containing 15-99% of alkaloids by weight, further optionally containing 30-99% of alkaloids by weight, further optionally containing 40-99% of alkaloids by weight, further optionally containing 50-99% of alkaloids by weight, further optionally containing 60-99% of alkaloids by weight, further optionally containing 35-70% of alkaloids by weight, further optionally containing 60-75% of alkaloids by weight);

[0008] and / or contains no more than 70% polysaccharide by weight (optionally containing 0.2-70% polysaccharide by weight, further optionally containing 0.2-50% polysaccharide by weight, further optionally containing 0.2-35% polysaccharide by weight, further optionally containing 0.2-25% polysaccharide by weight, further optionally containing 0.2-23% polysaccharide by weight, further optionally containing 20-25% polysaccharide by weight),

[0009] and / or contains flavonoids in an amount not higher than 10% by weight (optionally containing flavonoids in an amount of 0.05-5% by weight, further optionally containing flavonoids in an amount of 0-2% by weight, further optionally containing flavonoids in an amount of 0.05-2% by weight, further optionally containing flavonoids in an amount of 0.5-1.5% by weight, further optionally containing flavonoids in an amount of 0-1% by weight, further optionally containing flavonoids in an amount of 0.05-1% by weight),

[0010] and / or contains no more than 50% by weight of amino acids (optionally containing 0-30% by weight of amino acids, further optionally containing 0-25% by weight of amino acids, further optionally containing 0-20% by weight of amino acids, further optionally containing 0-5% by weight of amino acids, further optionally containing 3-25% by weight of amino acids or further optionally containing 5-20% by weight of amino acids),

[0011] 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% by weight.

[0012] Optionally, the disease related to abnormal bone metabolism is any one of osteoporosis, sarcopenia, sarcopenia-osteoporosis, muscular dystrophy, myasthenia, osteomalacia, rickets, primary hyperparathyroidism bone disease, fluorosis, osteoarthritis and bone displacement; it can be osteoporosis, sarcopenia or sarcopenia-osteoporosis.

[0013] In addition, diseases related to abnormal bone metabolism may also include kidney disease, liver disease, diabetes, rheumatoid arthritis and other diseases that cause decreased bone density.

[0014] Optionally, the use of the mulberry extract in the preparation of a medicament for treating and / or preventing diseases related to abnormal bone metabolism includes: the use of the mulberry extract in any one of the following (b1) to (b3):

[0015] (b1) use in the preparation of a medicament for increasing bone density;

[0016] (b2) Use in the preparation of a medicament for reducing bone marrow fat vacuoles;

[0017] (b3) Use in the preparation of drugs for improving bone microstructure;

[0018] The improvement of bone microstructure includes at least one of the following: (i) increasing the number of trabeculae; (ii) reducing the separation of trabeculae; (iii) reducing the trabecular structure model index; (iv) increasing the bone volume fraction;

[0019] Optionally, the use of the mulberry extract in preparing a medicament for treating and / or preventing diseases related to abnormal bone metabolism includes: the use of the mulberry extract in any of the following:

[0020] 1) Use in the preparation of drugs for increasing muscle mass;

[0021] 2) Use in the preparation of a drug for increasing muscle mass percentage;

[0022] 3) Use in the preparation of drugs for increasing muscle fat ratio;

[0023] 4) Use in the preparation of a drug for inhibiting muscle atrophy and / or edema; optionally, the inhibiting muscle atrophy and / or edema is specifically to increase the cross-sectional area, muscle fiber diameter and / or muscle fiber circumference of the muscle fiber;

[0024] 5) Use in the preparation of a drug for alleviating myenteric peripheral neuropathy and / or vascular lipid deposition;

[0025] 6) Use in the preparation of drugs for inhibiting muscle cell damage;

[0026] 7) Use in the preparation of drugs for increasing muscle strength;

[0027] In an optional embodiment of the present invention, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0028] Optionally, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight percentages of the components in the mulberry extract are:

[0029] Optionally, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight percentages of the components in the mulberry extract are:

[0030] Optionally, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight percentages of the components in the mulberry extract are:

[0031] Optionally, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight percentages of the components in the mulberry extract are:

[0032] Optionally, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight percentages of the components in the mulberry extract are:

[0033] Optionally, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight percentages of the components in the mulberry extract are:

[0034] Further optionally, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0035] Further optionally, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0036] Further optionally, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight contents of the components in the mulberry extract are:

[0037] Optionally, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight percentages of the components in the mulberry extract are:

[0038] Optionally, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight percentages of the components in the mulberry extract are:

[0039] Optionally, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight percentages of the components in the mulberry extract are:

[0040] Optionally, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight percentages of the components in the mulberry extract are:

[0041] Optionally, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight percentages of the components in the mulberry extract are:

[0042] Optionally, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight percentages of the components in the mulberry extract are:

[0043] Optionally, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight percentages of the components in the mulberry extract are:

[0044] Optionally, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight percentages of the components in the mulberry extract are:

[0045] Optionally, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight percentages of the components in the mulberry extract are:

[0046] Optionally, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight percentages of the components in the mulberry extract are:

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

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

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

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

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

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

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

[0054] Optionally, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight percentages of the components in the mulberry extract are:

[0055] Optionally, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight percentages of the components in the mulberry extract are:

[0056] Optionally, taking the sum of the weight percentages of the components of the mulberry extract as 100%, the weight percentages of the components in the mulberry extract are:

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

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

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

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

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

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

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

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

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

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

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

[0068] The weight content of each component can be any combination of the above values.

[0069] Optionally, the alkaloids include 1-deoxynojirimycin (DNJ), N-methyl-1-deoxynojirimycin (N-methly-1-deoxynojirimycin), fagomine (FAG), 3-epi-fagomine, 1,4-dideoxy-1,4-imino-D-arabinitol (DAB), calystegin B2, calystegin C1, 2-oxy-(α-D- One or more of 2-O-(α-D-galactopyranosyl)-1-deoxynojirimycin, 6-O-(β-D-glucopyranosyl)-1-deoxynojirimycin, and 1,4-dideoxy-1,4-imino-(2-O-β-D-glucopyranosyl)-D-arabinitol.

[0070] Wherein, optionally, the weight percentage of DNJ is not less than 50% (optionally 60-99%) of the total alkaloids.

[0071] Optionally, the heavy metal content of the mulberry extract does not exceed 10 ppm.

[0072] The drug is in the form of an oral dosage form; optionally, the drug is in the form of a tablet, a capsule, an oral solution, an oral emulsion, a pill, a granule, a syrup, and a powder.

[0073] In the present invention, the mulberry extract can be provided in the form of commercially available Morus alkaline tablets (National Medicine Standard No. Z20200002).

[0074] In another aspect, the present invention provides a method for preventing and / or treating diseases related to abnormal bone metabolism, comprising administering a therapeutically and / or preventively effective amount of mulberry extract to a patient in need thereof.

[0075] Optionally, the method for treating and / or preventing diseases related to abnormal bone metabolism comprises: administering a therapeutically and / or preventively effective amount of mulberry extract to a patient in need thereof to improve any one of the following (b1)-(b3):

[0076] (b1) Increase bone density;

[0077] (b2) reduce bone marrow fat vacuoles;

[0078] (b3) improve bone microstructure;

[0079] The improvement of bone microstructure includes at least one of the following: (i) increasing the number of trabeculae; (ii) reducing the separation of trabeculae; (iii) reducing the trabecular structure model index; (iv) increasing the bone volume fraction;

[0080] Optionally, the method for treating and / or preventing diseases related to abnormal bone metabolism comprises: administering a therapeutically and / or preventively effective amount of mulberry extract to a patient in need thereof to improve any one of the following 1)-7):

[0081] 1) Increase muscle mass;

[0082] 2) Increased muscle mass percentage;

[0083] 3) Increase muscle to fat ratio;

[0084] 4) inhibiting muscle atrophy and / or edema; optionally, the inhibiting muscle atrophy and / or edema is specifically increasing the muscle fiber cross-sectional area, muscle fiber diameter and / or muscle fiber circumference;

[0085] 5) Relieve intermuscular peripheral neuropathy and / or vascular lipid deposition;

[0086] 6) Inhibit muscle cell damage;

[0087] 7) Increase muscle strength.

[0088] Another aspect of the present invention provides use of the mulberry extract in treating and / or preventing diseases related to abnormal bone metabolism.

[0089] Optionally, the use of the mulberry extract in treating and / or preventing diseases related to abnormal bone metabolism includes: the use of the mulberry extract in any one of the following (b1) to (b3):

[0090] (b1) Use in increasing bone density;

[0091] (b2) Use in reducing bone marrow fat vacuoles;

[0092] (b3) Use in improving bone microstructure;

[0093] The improvement of bone microstructure includes at least one of the following: (i) increasing the number of trabeculae; (ii) reducing the separation of trabeculae; (iii) reducing the trabecular structure model index; (iv) increasing the bone volume fraction;

[0094] Optionally, the use of the mulberry extract in treating and / or preventing diseases related to abnormal bone metabolism includes: the use of the mulberry extract in any of the following:

[0095] 1) Use in increasing muscle mass;

[0096] 2) Use in increasing muscle mass percentage;

[0097] 3) Use in increasing muscle-to-fat ratio;

[0098] 4) Use in inhibiting muscle atrophy and / or edema; optionally, the inhibiting muscle atrophy and / or edema is specifically increasing the cross-sectional area, muscle fiber diameter and / or muscle fiber circumference;

[0099] 5) Use in alleviating intermuscular peripheral neuropathy and / or vascular lipid deposition;

[0100] 6) Use in inhibiting muscle cell damage;

[0101] 7) Use in increasing muscle strength;

[0102] Preferably, the bones include tibia and femur.

[0103] The muscles include skeletal muscles, such as gastrocnemius, tibialis anterior, quadriceps femoris and soleus, etc. The mulberry extract acts on humans or mammals.

[0104] Optionally, the treatment and / or prevention of diseases related to abnormal bone metabolism is reflected in improving any one of the patient's bone mass, bone quality, muscle mass, muscle strength, and body balance; optionally, the patient suffers from any one of osteoporosis, sarcopenia, sarcopenia-osteoporosis (musculoskeletal syndrom), muscular dystrophy, myasthenia, osteomalacia, rickets, primary hyperparathyroidism bone disease, fluorosis, osteoarthritis and bone displacement; further optionally, the patient suffers from osteoporosis, sarcopenia or sarcopenia-osteoporosis.

[0105] Optionally, the disease related to abnormal bone metabolism is caused by aging, endocrine disorder, obesity and / or diabetes.

[0106] Alternatively, the mulberry extract can be prepared according to the method described in CN 110393738A. In one embodiment, the preparation of the mulberry extract comprises the following steps: preparing a crude extract of a moraceae plant; optionally, separating the extract with a cationic resin and / or an anionic resin; optionally, subjecting the resin effluent to alcohol precipitation and collecting the supernatant; and optionally, concentrating and / or drying the supernatant.

[0107] Optionally, in the present invention, the preparation of the mulberry extract comprises the following steps: 1) preparing a crude extract of a moraceae plant; 2) separating the crude extract through a cationic resin and / or an optional anionic resin to obtain a resin effluent; and optionally, step 3) subjecting the resin effluent of step 2) to alcohol precipitation and collecting the supernatant; and 4) concentrating and / or drying the supernatant. Optionally, the resin effluent of step 2) may be concentrated and / or dried before being subjected to alcohol precipitation.

[0108] Optionally, the plant of the Moraceae family 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, or a combination of one or more of the following:

[0109] Optionally, the moraceae plant is one or more selected from the group consisting of Guangdong mulberry, Shandong mulberry, white mulberry, fine-toothed mulberry, mountain mulberry, or hybrid mulberry, and the hybrid mulberry can be Yuesang No. 11, Guisang You No. 62, or Sang Te You No. 2. Any part of the moraceae plant, including leaves, roots, branches, bark, buds, stems, and fruits, can be used, and mulberry branches, mulberry leaves, or white mulberry bark can be used.

[0110] 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.

[0111] In one embodiment, the mulberry extract is prepared according to the following steps: crushing the moraceae plant, heating and refluxing the extract with water and / or alcohol solution or acid water, the amount of solvent is 3-20 times that of the original medicinal material, repeating the extraction 1-3 times, combining the extracts, concentrating, applying a cation exchange resin, eluting with 0.2-3N ammonia water, applying the eluate to an anion exchange resin, collecting the non-adsorbed portion, adding ethanol, precipitating to remove impurities, concentrating and / or drying to obtain the extract.

[0112] In one embodiment, the mulberry extract is prepared according to the following steps: crushing the moraceae plant, heating and refluxing the extract with water and / or alcohol solution or acid water, the amount of solvent is 3-20 times that of the original medicinal material, repeating the extraction 1-3 times, combining the extracts, concentrating, applying a cation exchange resin, eluting with 0.2-3N ammonia water, applying the eluate to an anion exchange resin, collecting the non-adsorbed portion, concentrating and / or drying to obtain the extract.

[0113] In one embodiment, the mulberry extract is prepared according to the following steps: crushing the moraceae plant, heating and refluxing the extract with water and / or alcohol solution or acid water, the amount of solvent is 3-20 times that of the original medicinal material, repeating the extraction 1-3 times, combining the extracts, concentrating, applying a cation exchange resin, eluting with 0.2-3N ammonia water, concentrating and / or drying the eluate to obtain the extract.

[0114] In one embodiment, the mulberry extract is prepared according to the following steps: crushing the moraceae plant, heating and refluxing the extract with water, the amount of solvent is 3-20 times (optionally 4-15 times, further optionally 4-12 times) of the original medicinal material, repeating the extraction 1-3 times (the extraction time can be 0.5-3 hours each time, further optionally 1-2 hours each time), combining the extracts, concentrating, applying a cation exchange resin, eluting with 0.2-3N ammonia water, applying the eluate to an anion exchange resin, collecting the non-adsorbed portion (i.e., the anion resin effluent), adding ethanol, precipitating to remove impurities, concentrating and / or drying to obtain an extract.

[0115] Optionally, the crude extract after the concentration treatment can also be subjected to alcohol precipitation treatment before the resin separation treatment in step 2). During the alcohol precipitation treatment, ethanol is added to the crude extract, stirred and mixed, and the stirring is stopped and allowed to stand for a certain time to precipitate the insoluble matter therein. Optionally, the volume mass ratio of the added ethanol to the plant raw material is 0.2-20 times, optionally 0.4-10 times, in terms of L / kg. Further optionally, an alcohol precipitation tank is used for alcohol precipitation treatment. Optionally, the stirring speed in the alcohol precipitation treatment is 10-600rpm, optionally 40-500rpm, further optionally 80-400rpm or 300rpm.

[0116] Optionally, after the cationic resin is loaded into the column, it is activated in the order of washing with an acidic solution, washing with an alkaline solution, and washing with an acidic solution. Optionally, the alkaline solution is washed until the pH of the eluate is 8.0-9.5, optionally 8.5-9.5; optionally, the alkaline solution is selected from aqueous ammonia solution, sodium hydroxide solution, potassium hydroxide solution, or sodium carbonate solution; optionally, the concentration of the alkaline solution is 0.5-4 mol / L, optionally 1-2 mol / L. Optionally, the acidic solution is washed until the pH of the eluate is 3.0-7.0, optionally 4.5-6.5. Optionally, the acidic solution is selected from hydrochloric acid solution, phosphoric acid solution, or sodium hydrogen phosphate-citrate buffer; optionally, the concentration of the acidic solution is 0.5-4 mol / L, optionally 1.5-2 mol / L. Optionally, after the final acidic solution wash, the cationic resin can be rinsed with 3-5 column volumes of deionized water.

[0117] 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.

[0118] 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).

[0119] After the crude plant extract is loaded onto the cationic resin, the loaded cationic resin is eluted with an eluent. Optionally, the eluent is a salt solution or alkaline solution containing cations, and can be selected from one or more of sodium chloride, ammonium chloride, ammonium sulfate, ammonium nitrate, ammonia water, potassium chloride, and sodium hydroxide.

[0120] 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).

[0121] Optionally, the eluent flow rate is 1-15 BV / h (optionally 5-10 BV / h, further optionally 5-6 BV / h).

[0122] Optionally, the weight of the eluent used for cationic resin separation is 0.1-30 times the weight of the plant raw material input. Optionally, the eluent is used in an amount of 0.5-10 times the weight of the plant raw material input for elution.

[0123] The collection starting point can be determined according to the pH of the cationic resin effluent. For example, when an alkaline solution such as aqueous ammonia is used for elution, the eluate is collected when the pH of the cationic column effluent is detected to be greater than 7, or the collection starting point of the effluent is determined based on a color development or precipitation reaction. Optionally, when the volume of the collected liquid reaches 0.1-10 times (further optionally, 0.2-5 times) the weight of the plant raw material fed, collection is stopped, and the collected liquid is optionally purified by an anion column.

[0124] When purifying by anion column, optionally, after the anion resin is loaded into the column, activation is performed in the order of washing with alkaline solution, washing with acidic solution, and washing with alkaline solution.

[0125] Optionally, washing with an alkaline solution until the pH of the eluate is 8.0-9.5, optionally 8.5-9.5;

[0126] 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.

[0127] Optionally, the acidic solution is washed until the pH of the eluate is 3.0-7.0, optionally 4.5-6.5. Optionally, the acidic solution is selected from hydrochloric acid solution, phosphoric acid solution, disodium hydrogen phosphate-citric acid buffer, and optionally, the concentration of the acidic solution is 0.5-4 mol / L, optionally 1-2 mol / L.

[0128] 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.

[0129] 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).

[0130] When the liquid flows out of the anion resin, collection begins. Optionally, collection is stopped when the volume of the collected liquid reaches 0.05-10 times (optionally, 0.1-5 times) the weight of the plant raw material input.

[0131] Optionally, the weight ratio of ethanol used in the alcohol precipitation treatment to the plant raw material is 1:4-600 (optionally 1:20-300, further optionally 1:20-50, 1:40, 1:80, 1:22). During the alcohol precipitation treatment, the stirring speed is 10-600 rpm (optionally 40-500 rpm, 80-400 rpm). The alcohol precipitation treatment time is 12-24 hours.

[0132] Furthermore, before the alcohol precipitation treatment, the anion resin effluent is subjected to a step of centrifugal impurity removal or microfiltration membrane filtration, followed by concentration by a reverse ion osmosis membrane. The specific gravity of the concentrated liquid can be 1.0-1.3, optionally 1.1-1.25.

[0133] Optionally, the drug further comprises a pharmaceutically acceptable carrier. The carrier is an inactive ingredient that is non-toxic to the human body and is consistent with the route of administration or mode of administration. The carrier can be a solid or liquid excipient. Solid excipients include, for example, microcrystalline cellulose, mannitol, lactose, pregelatinized starch, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, sodium carboxymethyl starch, aspartame, calcium hydrogen phosphate, sodium lactate, poloxamer, sodium lauryl sulfate, sodium carboxymethyl cellulose, gelatin, xanthan gum, povidone, starch, magnesium stearate, sodium carboxymethyl starch, and talc; liquid excipients include, for example, water, ethanol, syrup, and glycerol.

[0134] In the present invention, the mulberry extract is referred to as SZ-A. Beneficial effects

[0135] Mulberry extract can increase bone density, promote osteoblast bone formation, inhibit osteoclast bone resorption, and at the same time improve muscle content and quality and promote myoblast proliferation and differentiation.

[0136] The present invention provides the use of mulberry extract in the preparation of medicines or health products for treating and / or preventing diseases with abnormal bone metabolism. The mulberry extract can significantly improve the bone density, bone calcium content, and muscle content of model mice, ultimately improving diseases related to abnormal bone metabolism. BRIEF DESCRIPTION OF THE DRAWINGS

[0137] FIG1 shows the results of bone density, muscle weight percentage, and muscle weight / fat weight determination of mice in each group of Experimental Example 1, where: *P<0.05, **P<0.01, ***P<0.001 vs. the model group.

[0138] Figure 2 is a graph showing the pathological sections of the gastrocnemius muscles of each group of mice in Experimental Example 1 (2A), as well as a bar graph showing the average cross-sections of the gastrocnemius muscle fibers of each group of mice (2B), a bar graph showing the average diameters of the gastrocnemius muscle fibers (2C), and a bar graph showing the average circumferences of the gastrocnemius muscle fibers (2D); and a graph showing the pathological sections of the tibia of mice (Figure 2E).

[0139] Figure 3 is a microCT scan of bone tissue of mice in each group of Experimental Example 2, showing (3A) trabeculae, (3B) trabecular bone, (3C) trabecular separation, (3D) bone density, (3E) bone volume fraction, (3F) bone structure model index, and (3G) trabecular thickness. *P<0.05, **P<0.01 vs. the model group.

[0140] FIG4 shows the results of bone density, muscle weight percentage, and muscle weight / fat weight determination of mice in each group of Experimental Example 2, where: *P<0.05, **P<0.01, ***P<0.001, vs. model group.

[0141] FIG5 shows the bone density measurement results of mice in each group in Experimental Example 2, where *P<0.05, **P<0.01, vs. the model group.

[0142] FIG6 is a photograph of the femoral pathological sections of mice in each group in Experimental Example 3.

[0143] FIG7 is the result of experimental example 4 on the effect of mulberry extract on muscle weight percentage in rats with endocrine disorder induced by LZ+HFD, *P<0.05, **P<0.01, vs. model group.

[0144] In Figure 8, A is the effect of mulberry extract from Experimental Example 5 on the pathological sections of the gastrocnemius muscle in the Apoe diabetic sexual dysfunction model induced by TZ combined with high-fat and high-cholesterol; BD are bar graphs of the effect of mulberry extract from Experimental Example 5 on the average area, average diameter and average circumference of the gastrocnemius muscle fibers in the Apoe diabetic sexual dysfunction model induced by TZ combined with high-fat and high-cholesterol, *P<0.05, **P<0.01, vs. model group.

[0145] FIG9 is a bar graph showing the bone density measurement results of mice in each group of Experimental Example 6, *P<0.05, **P<0.01, ***P<0.001, vs. the aging group.

[0146] FIG10 is a bar graph showing the grip strength of mice in each group in Experimental Example 7 (*P<0.05 vs. model group; ###P<0.001 vs. normal group).

[0147] FIG11 shows SA-β-galactosidase staining images (A) and statistical bar graphs (B) of the effects of mulberry extracts prepared in different examples on D-galactose-modeled C2C12 cells in Experimental Example 8, ***P<0.001, ****P<0.0001, vs. Model group.

[0148] 12 is a bar graph showing the distribution of (A) DNJ, (B) FA, and (C) DAB in bone marrow tissue of rats in Experimental Example 9 after oral administration of SZ-A (40 mk / kg). DETAILED DESCRIPTION

[0149] The present invention is further illustrated below through specific experimental examples. It should be understood that the following experiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention.

[0150] The mulberry extract of the present invention can be prepared by the following method:

[0151] 1. Preparation Example of Mulberry Extract

[0152] Preparation Example 1 of Mulberry Extract

[0153] 1000 kg of fresh mulberry branches (fine-toothed mulberry, Yuesang No. 11) were crushed and added to 4000 L of water. The extracts were then heated and refluxed for 2 hours. The combined extracts were filtered to remove insoluble matter to obtain a crude extract. The crude extract was heat-concentrated to a solids content of 4% by weight and then heated to 50°C to serve as the loading solution for a cationic resin column.

[0154] A 150 kg column of D113 macroporous weakly acidic phenyl propylene-based cationic resin was loaded and washed with 2 mol / L hydrochloric acid until the eluate pH reached 4.5; then with 1 mol / L sodium hydroxide until the eluate pH reached 8.5; and finally with 2 mol / L hydrochloric acid until the eluate pH reached 4.5. The column was then rinsed with 5 column volumes of deionized water to complete activation. The concentrated extract was loaded and then eluted with 1000 L of 2.5 mol / L ammonia at a rate of 6 BV / h. The eluate from the cation column was collected when the pH was >7. When the collected solution reached 900 L, the collection was stopped and the collected solution was directly passed through an anion column for purification.

[0155] Activate the column using 62.5 kg of D218 macroporous, strongly basic acrylic anion resin. Elute with 1.5 mol / L sodium hydroxide solution until the eluate has a pH of 9.0. Elute with 1.5 mol / L hydrochloric acid solution until the eluate has a pH of 3.5. Finally, elute with 1.5 mol / L sodium hydroxide solution until the eluate has a pH of 9.0. The collected eluate from the cationic resin is loaded onto the anionic resin, and the effluent is collected until 920 L of effluent is reached.

[0156] The collected liquid was centrifuged to remove impurities and then concentrated using a reverse ion osmosis membrane. The concentrated liquid had a specific gravity of 1.25 and was transferred to an alcohol precipitation tank. 25 L of anhydrous ethanol was added with a stirring paddle at 500 rpm. After the ethanol addition was complete, stirring was stopped and the mixture was allowed to settle for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain a mulberry branch extract (i.e., mulberry extract SZ-A).

[0157] The mulberry branch extract contains 52% by weight of alkaloids, 22% by weight of polysaccharides, 0.8% by weight of flavonoids, and 20% by weight of amino acids. Among the alkaloids, the content of 1-DNJ is 60%, FAG is 17%, and DAB is 15%.

[0158] Preparation Example 2 of Mulberry Extract

[0159] Take 10kg of fresh mulberry branches (Sang Teyou No. 2), crush them, add 150L of water, add them twice, extract them by decoction for 3h each time, combine the extracts, and filter to remove insoluble matter. The extract is hot concentrated until the solid content reaches 8%, then transferred to an alcohol precipitation tank, and 2367.9g of absolute ethanol (3L) is added under a stirring paddle at 300rpm. After the ethanol addition is complete, stop stirring, precipitate with alcohol for 24h, and take the supernatant as the loading liquid for a cationic resin column. Use 5kg of 002SC type strong acid styrene cationic resin to fill the column, and activate the cationic resin according to the method of Preparation Example 1. Load the extract after concentrated alcohol precipitation, then use 100L of 5mol / L potassium chloride to elute, with an elution rate of 5BV / h, and detect the effluent with 20% silicotungstic acid. Start collecting when a white precipitate is generated. Stop collecting when the collected liquid reaches 25L, and the collected liquid is directly purified by anion column.

[0160] A 10 kg column of 711 strong base styrene anion resin was loaded and activated according to the method of Preparation Example 1. The collected cationic resin eluate was loaded onto the anion resin, and the effluent was collected until the effluent reached 15 L. The collected eluate was reloaded onto the cationic resin and separated twice more using the cationic resin and anion resin, respectively, according to the above method.

[0161] The collected liquid obtained after three column separations was centrifuged for impurity removal and then concentrated using a reverse ion osmosis membrane. The concentrated liquid had a specific gravity of 1.25 and was transferred to an alcohol precipitation tank. 125 g of anhydrous ethanol was added under a stirring paddle at 1000 rpm. After the ethanol addition was complete, stirring was stopped and the mixture was allowed to precipitate with alcohol for 24 hours. The supernatant was then collected and concentrated under reduced pressure to obtain an extract. Separately, fresh Morus alba bark and mulberry leaf (Sang Teyou No. 2) were extracted using the same extraction method and parameters as described above.

[0162] The obtained mulberry branch extract has an alkaloid content of 98%, a polysaccharide content of 0.2%, a flavonoid content of 0.05%, and an amino acid content of 0. Among the alkaloids, the content of 1-DNJ is 99%, FAG is 0.5%, and DAB is 0.4%.

[0163] The obtained Morus alba bark extract contains 95% alkaloids, 2% polysaccharides, 0.1% flavonoids, and 1% amino acids. Among the alkaloids, the content of 1-DNJ is 96%, FAG is 1.5%, and DAB is 1.4%.

[0164] The mulberry leaf extract contains 90% alkaloids, 4% polysaccharides, 0.1% flavonoids, and 3% amino acids. Among the alkaloids, the content of 1-DNJ is 91%, FAG is 3.1%, and DAB is 2.8%.

[0165] Preparation Example 3 of Mulberry Extract

[0166] 1000 kg of fresh mulberry branches (Mulberry twigs) were crushed, added to 11,500 L of water, and heated under reflux for 2 hours. The extracts were combined and filtered to remove insoluble matter to obtain a crude extract. This crude extract was first centrifuged to remove impurities and then concentrated using a counter-ion permeation membrane to a solids content of 1% by weight. This was then used as the loading solution for the cationic resin column.

[0167] A column was loaded with 300 kg of D001 macroporous, strongly acidic styrene-based cationic resin, which was activated according to the method of Preparation Example 1. The concentrated crude extract was loaded and eluted with 5000 L of 0.04 mol / L ammonium nitrate at a rate of 5 BV / h. The effluent was tested with 20% silicotungstic acid. Collection began when a white precipitate formed and stopped when the collected solution reached 1000 L.

[0168] The collected liquid obtained after the cationic column separation is concentrated by nanofiltration membrane and concentrated under reduced pressure to obtain an extract concentrate.

[0169] The obtained mulberry branch extract has an alkaloid content of 15%, a polysaccharide content of 20%, a flavonoid content of 7%, and an amino acid content of 45%. Among the alkaloids, the content of 1-DNJ is 55%, FAG is 23%, and DAB is 10%.

[0170] Preparation Example 4 of Mulberry Extract

[0171] 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.

[0172] A 150 kg column of D113 macroporous weakly acidic phenyl propylene-based cationic resin was loaded and washed with 2 mol / L hydrochloric acid until the eluate pH reached 4.5; then with 1 mol / L sodium hydroxide until the eluate pH reached 8.5; and finally with 2 mol / L hydrochloric acid until the eluate pH reached 4.5. The column was then rinsed with 5 column volumes of deionized water to complete activation. The concentrated extract was loaded and then eluted with 1000 L of 2.5 mol / L ammonia at a rate of 6 BV / h. The eluate from the cation column was collected when the pH was >7. When the collected solution reached 900 L, the collection was stopped and the collected solution was directly passed through an anion column for purification.

[0173] Activate the column using 125 kg of D218 macroporous, strongly basic acrylic anion resin. Elute with 1.5 mol / L sodium hydroxide solution until the eluate has a pH of 9.0. Elute with 1.5 mol / L hydrochloric acid solution until the eluate has a pH of 3.5. Finally, elute with 1.5 mol / L sodium hydroxide solution until the eluate has a pH of 9.0. The collected eluate from the cationic resin is loaded onto the anionic resin. Collect the effluent with a pH greater than 8 until the effluent reaches 870 L.

[0174] The collected liquid after anion column separation was filtered through a microfiltration membrane to remove impurities and then concentrated using a counter-ion osmosis membrane. The concentrated liquid had a specific gravity of 1.1 and was transferred to an alcohol precipitation tank. 15 kg of anhydrous ethanol was added with a stirring paddle at 400 rpm. Stirring was stopped after the ethanol addition, and the mixture was allowed to settle for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain a mulberry branch extract. The sample contained 80% alkaloids by weight, 5% polysaccharides by weight, 0.1% flavonoids by weight, and 4% amino acids by weight. Among the alkaloids, 1-DNJ accounted for 75%, FAG for 12%, and DAB for 10%.

[0175] Preparation Example 5 of Mulberry Extract

[0176] 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.

[0177] Activate the column using 62.5 kg of D218 macroporous, strongly basic acrylic anion resin. Elute with 1.5 mol / L sodium hydroxide solution until the eluate has a pH of 9.0; then with 1.5 mol / L hydrochloric acid solution until the eluate has a pH of 3.5; and finally with 1.5 mol / L sodium hydroxide solution until the eluate has a pH of 9.0. The collected extract concentrate was loaded onto the anion resin, and the effluent was collected.

[0178] The collected solution after anion column separation was filtered through a microfiltration membrane to remove impurities, then concentrated using a counter-ion permeation membrane. Further vacuum concentration and drying yielded a mulberry twig extract. The sample contained 3% by weight of alkaloids, 70% by weight of polysaccharides, 10% by weight of flavonoids, and 10% by weight of amino acids. Among the alkaloids, the content of 1-DNJ was 68%, FAG was 17%, and DAB was 8%.

[0179] Preparation Example 6 of Mulberry Extract

[0180] 1500 kg of fresh mulberry branches (fine-toothed mulberry, Yuesang No. 11) were crushed and added to 6000 L of water. The extracts were heated and refluxed for 2 hours. The combined extracts were filtered to remove insoluble matter to obtain a crude extract. The crude extract was heat-concentrated to a solids content of 4% by weight and then heated to 50°C as the loading solution for a cationic resin column.

[0181] A 100 kg column of D113 macroporous weakly acidic phenyl propylene-based cationic resin was loaded and washed with 2 mol / L hydrochloric acid until the eluate pH reached 4.5; then with 1 mol / L sodium hydroxide until the eluate pH reached 8.5; and finally with 2 mol / L hydrochloric acid until the eluate pH reached 4.5. The column was then rinsed with 5 column volumes of deionized water to complete activation. The concentrated extract was loaded and then eluted with 1000 L of 2.5 mol / L ammonia at a rate of 6 BV / h. The eluate from the cation column was collected when the pH was >7. When the collected solution reached 900 L, the collection was stopped and the collected solution was directly passed through an anion column for purification.

[0182] A column was loaded with 62.5 kg of D218 macroporous, strongly alkaline acrylic anion resin. The column was washed with a 1.5 mol / L sodium hydroxide solution until the eluate had a pH of 9.0; then washed with a 1.5 mol / L hydrochloric acid solution until the eluate had a pH of 3.5; and finally washed with a 1.5 mol / L sodium hydroxide solution until the eluate had a pH of 9.0. Activation was completed. The collected cationic resin eluate was loaded onto an anionic resin, and the effluent was collected until the effluent reached 870 L. The effluent was concentrated under reduced pressure to obtain a mulberry branch extract concentrate, which contained 30% by weight alkaloids, 35% by weight polysaccharides, 2% by weight flavonoids, and 25% by weight amino acids. Among the alkaloids, the content of 1-DNJ was 62%, FAG was 20%, and DAB was 13%.

[0183] Preparation Example 7 of Mulberry Extract

[0184] 1000 kg of fresh mulberry branches (fine-toothed mulberry, Yuesang No. 11) were crushed and added to 4000 L of water. The extracts were then heated and refluxed for 2 hours. The combined extracts were filtered to remove insoluble matter to obtain a crude extract. The crude extract was heat-concentrated to a solids content of 4% by weight and then heated to 50°C to serve as the loading solution for a cationic resin column.

[0185] A 100 kg column of D113 macroporous weakly acidic phenyl propylene-based cationic resin was loaded and washed with 2 mol / L hydrochloric acid until the eluate pH reached 4.5; then with 1 mol / L sodium hydroxide until the eluate pH reached 8.5; and finally with 2 mol / L hydrochloric acid until the eluate pH reached 4.5. The column was then rinsed with 5 column volumes of deionized water to complete activation. The concentrated extract was loaded and then eluted with 1000 L of 2.5 mol / L ammonia at a rate of 6 BV / h. The eluate from the cation column was collected when the pH was >7. When the collected solution reached 900 L, the collection was stopped and the collected solution was directly passed through an anion column for purification.

[0186] A 62.5 kg column of D218 macroporous, strongly alkaline acrylic anion resin was loaded and washed with a 1.5 mol / L sodium hydroxide solution until the eluate had a pH of 9.0; then with a 1.5 mol / L hydrochloric acid solution until the eluate had a pH of 3.5; and finally with a 1.5 mol / L sodium hydroxide solution until the eluate had a pH of 9.0. Activation was completed. The collected cationic resin eluate was loaded onto an anionic resin, and the effluent was collected until the effluent reached 870 L. The effluent was concentrated under reduced pressure to obtain a mulberry branch extract concentrate, which contained 40% by weight alkaloids, 25% by weight polysaccharides, 0.5% by weight flavonoids, and 25% by weight amino acids. Among the alkaloids, the content of 1-DNJ was 57%, FAG was 24%, and DAB was 16%.

[0187] Preparation Example 8 of Mulberry Extract

[0188] 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.

[0189] A 150 kg column of D113 macroporous weakly acidic phenyl propylene-based cationic resin was loaded and washed with 2 mol / L hydrochloric acid until the eluate pH reached 4.5; then with 1 mol / L sodium hydroxide until the eluate pH reached 8.5; and finally with 2 mol / L hydrochloric acid until the eluate pH reached 4.5. The column was then rinsed with 5 column volumes of deionized water to complete activation. The concentrated extract was loaded and then eluted with 1000 L of 2.5 mol / L ammonia at a rate of 6 BV / h. The eluate from the cation column was collected when the pH was >7. When the collected solution reached 900 L, the collection was stopped and the collected solution was directly passed through an anion column for purification.

[0190] Activate the column using 62.5 kg of D218 macroporous, strongly basic acrylic anion resin. Elute with 1.5 mol / L sodium hydroxide solution until the eluate has a pH of 9.0. Elute with 1.5 mol / L hydrochloric acid solution until the eluate has a pH of 3.5. Finally, elute with 1.5 mol / L sodium hydroxide solution until the eluate has a pH of 9.0. The collected cationic resin eluate is loaded onto the anion resin, and the effluent with a pH greater than 8 is collected until the effluent reaches 870 L.

[0191] The collected liquid after anion column separation was filtered through a microfiltration membrane to remove impurities and then concentrated using a counter-ion permeation membrane. The concentrated liquid had a specific gravity of 1.1 and was transferred to an alcohol precipitation tank. 15 kg of anhydrous ethanol was added with a stirring paddle at 400 rpm. After the ethanol addition was complete, stirring was stopped. The mixture was allowed to precipitate for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain a mulberry twig extract concentrate. The sample contained 63% alkaloids, 23% polysaccharides, 1% flavonoids, and 5% amino acids by weight.

[0192] Among the alkaloids, the content of 1-DNJ was 61.9%, FAG was 16.6%, and DAB was 11.1%.

[0193] 2. Efficacy test of mulberry extract

[0194] Experimental Example 1: High-fat diet (HFD) mouse model

[0195] Forty-five healthy male C57 mice aged 6 weeks were randomly divided into a normal group (NC), a high-fat model group (HFD), and different dose groups of mulberry extract (SZ-A) (divided into a low-dose group SZ-A100 (100 mg / kg / d, calculated as alkaloids), a medium-dose group SZ-A200 (200 mg / kg / d, calculated as alkaloids), and a high-dose group SZ-A300 (300 mg / kg / d, calculated as alkaloids)), with 9 mice in each group. The mulberry extract was the mulberry extract prepared in Preparation Example 8. The mice in the normal group were fed with a basal feed, and the model group and the SZ-A group were fed with a high-fat diet (Research Diet, D12492, 60 kcal% Fat); after 17 weeks of feeding, each experimental group was treated with the corresponding dose of the test drug for 6 consecutive weeks. During the treatment period, the high-fat diet was continued, and the body weight and food and water intake of the animals were monitored. The model group was given normal saline. The effect of SZ-A was evaluated by measuring bone density and other indicators before, 4 weeks after, and 6 weeks after administration. Body fat content, muscle content, etc. were measured before, 4 weeks after, and 6 weeks after the experiment. The experimental mice were anesthetized with Zotai and placed in a prone position in a FAXITRON ULTRAFOCUS100 bone density analyzer. The mice were scanned by X-rays to detect and analyze the fat, muscle content, and bone density in the mice. After the mice were collected, the tibia and gastrocnemius muscles were collected for HE staining. The femur was collected and fixed in 10% neutral buffered formalin for 24 hours, and excess soft tissue was removed. The femur was scanned using a Skyscan 1276 micro-CT instrument using the following settings, and the image was reconstructed using NRecon software.

[0196] (1) The results of the ratio of bone density, muscle content to body weight, i.e., muscle weight percentage and muscle fat ratio of mice are shown in Figure 1. The results showed that the bone density of mice in the model group was significantly reduced. After 6 weeks of treatment, the bone density of mice in each SZ-A dose group was significantly increased, indicating that mulberry extract can significantly increase bone density; the muscle weight percentage and muscle fat ratio of mice in the model group were significantly reduced. After 6 weeks of treatment, each SZ-A dose group could significantly increase the muscle weight percentage and muscle fat ratio of obese mice.

[0197] (2) Pathological examination.

[0198] The morphological changes of muscle fibers were observed by HE staining. The results are shown in Figure 2A, which is a pathological section of the gastrocnemius muscle of mice: the skeletal muscle structure of the normal group was clear, and there were no abnormalities in the muscle bundles and intermuscular fiber spaces, and no abnormalities in the intermuscular nerves and blood vessels. In the HFD group, a large number of muscle fibers were edematous, and muscle fiber edema and atrophy coexisted, with edema being the main symptom; the cross-sections of muscle fibers in the same muscle bundle were significantly different and of different sizes. Lipid deposits were visible under the intima of the intermuscular arterioles, and the arterial media was slightly thickened. The density of myelin sheath fibers in the intermuscular peripheral nerves was reduced (demyelinating changes). Compared with the model group, after administration of SZ-A300, the muscle fiber structure was clear, edema and muscle fiber atrophy were alleviated, the arteriolar structure was clear, no obvious lipid deposition was observed, and peripheral neuropathy was alleviated.

[0199] The results in Figures 2B-2D showed that compared with the model group, the average cross-section, average diameter and average circumference of the gastrocnemius muscle fibers increased significantly after SZ-A300 administration.

[0200] Tibiae were stained with H&E. Pathological results showed that SZ-A administration could reduce the number of fat vacuoles in bone tissue (Figure 2E).

[0201] (3) Bone tissue was scanned by microCT. The results are shown in Figure 3. In the model group, the number of trabeculae and bone density were significantly reduced, and the separation of trabeculae increased. SZ-A300 can improve the femoral microstructure of HFD mice, increase the number of trabeculae and bone density, and reduce the separation of trabeculae. In the model group, the bone volume fraction was significantly reduced, and the trabecular structure model index increased. SZ-A has a tendency to increase the bone volume fraction and reduce the trabecular structure model index.

[0202] Experimental Example 2: GAN diet-fed mouse model

[0203] The GAN diet is a modified, trans-fat-free, high-fat, high-cholesterol, high-fructose diet (containing 40% fat, 20% fructose, and 2% cholesterol).

[0204] Six-week-old healthy male C57BL / 6J mice were selected and divided into a normal group, a model group, and a mulberry extract (SZ-A) group. The mulberry extract components were divided into a low-dose group SZ-A100 (100 mg / kg / d, calculated as alkaloids), a medium-dose group SZ-A200 (200 mg / kg / d, calculated as alkaloids), and a high-dose group SZ-A300 (300 mg / kg / d, calculated as alkaloids). The mulberry extract was the mulberry extract prepared in Preparation Example 8. Among them, the normal group mice were fed with a normal feed group (normal saline), and the model group and the SZ-A group were fed with a GAN diet (Research Diet, D09100310). An animal model was established. After 20 weeks of induction, the mice were given drugs for six consecutive weeks. The mulberry extract group was given drugs, and the normal feed group and the model group were given normal saline. The corresponding feed continued to be fed during the treatment period. Specific animal grouping and drug treatment are shown in Table 1 below. Body fat content was detected after 4 and 6 weeks of administration. The experimental mice were anesthetized with Zotai and placed in a prone position in a FAXITRON ULTRAFOCUS100 bone density analyzer. The mice were scanned by X-rays to detect and analyze the body fat, muscle content, fat percentage and bone density.

[0205] Table 1 Animal groups and drug treatments

[0206] The results in Figure 4 show that while the bone density of mice in the model group was significantly reduced, after 6 weeks of treatment, the bone density of mice in all SZ-A dose groups increased significantly, indicating that mulberry extract can significantly increase bone density in mice. After 4 and 6 weeks of treatment, the percentage of muscle weight (the ratio of muscle to body weight) and the ratio of muscle weight to fat weight both increased significantly.

[0207] Test Example 3: HFD+CCl4 Model

[0208] Solution preparation: Shake carbon tetrachloride (CCl4) thoroughly, take CCl4 and olive oil (volume ratio = 1:19) in a glass bottle to prepare a 5% CCl4 solution.

[0209] C57BL / 6J mice were induced with a 60% HFD for 10 weeks. Mice of the same age in the blank control group were fed a normal diet for 10 weeks and then divided into a normal group, a model group, and a mulberry extract (SZ-A) group. The mulberry extract group was administered a dose of 200 mg / kg / d (calculated as alkaloids). The mulberry extract was the mulberry extract prepared in Preparation Example 8. Mice in the model and mulberry extract groups were intraperitoneally injected with 5% CCl4 solution at 2.5 mL / kg body weight twice a week, while the normal group was injected with the same volume of olive oil (e.g., 100 μL for a 40 g mouse) for 4 weeks. The normal group was gavaged with normal saline, the drug-treated group was administered according to Table 2 below, and the model group was given normal saline. Specific animal grouping and drug treatments are shown in Table 2 below. After the experiment, the femurs and tibias of the mice were harvested and bone density was measured using a bone densitometer (iNSiGHT VET DXA / OT20-2F7113-01). The bone density test results for each group are shown in Figure 5. The femurs were stained with H&E, and the results are shown in Figure 6.

[0210] Table 2 Experimental groups

[0211] The results in Figure 5 show that the bone density of mice in the model group was significantly reduced, while the bone density of the SZ-A200-treated group showed an increasing trend.

[0212] The results in Figure 6 show that in the model group, the trabecular edges of the femoral bone tissue were rough, the trabecular spaces were enlarged, the trabeculae were irregularly arranged with breakpoints, and the number of fat vacuoles in the bone marrow cavity increased. In the SZ-A200 group, the number of trabeculae increased and the number of fat vacuoles decreased.

[0213] Experimental Example 4 Pharmacodynamics of Mulberry Extract on LZ+HFD-Induced Endocrine Disorder Model

[0214] Mulberry extract of Preparation Example 1

[0215] 1. Drug preparation

[0216] The preparation concentration of letrozole (abbreviated as LZ) is: 0.25 mg / mL, and the administration volume is: 0.4 mL / 100 g.

[0217] 1% CMC: Weigh 1g of CMC and add it to 100mL of pure water. Place it on a magnetic stirrer at 60℃ and 500r / min, stirring until the CMC is completely dissolved and becomes transparent.

[0218] 2. Experimental Grouping

[0219] Table 3

[0220] 3. Experimental methods

[0221] Thirty healthy, 3-week-old female SD rats with lustrous fur were selected and acclimated for 3 days. The rats were then randomly divided into blank, model, and treatment groups. The model and treatment groups were fed a high-fat diet (HFD) (research diets D12492) for 3 weeks and then gavaged with LZ (1 mg / kg / d) for 6 weeks while on HFD to establish a rat endocrine disorder model. The treatment groups were administered the doses and routes listed in the table. During the treatment period, the blank and model groups received an equal amount of saline. After 21 days of treatment, the rats were weighed and the right calf gastrocnemius muscle was removed and weighed.

[0222] The results are shown in FIG7 , which show that compared with the model group, the muscle weight percentage (ratio of muscle to body weight) of the SZ-A administration groups at each dose increased significantly.

[0223] Experimental Example 5 STZ combined with high fat and high cholesterol induces a diabetic sexual dysfunction model in Apoe mice

[0224] Thirty 8-week-old Apoe mice were adaptively fed for one week and then fed a high-fat, high-cholesterol diet (HFHC) for 5 weeks. On the 6th week, they were intraperitoneally injected with STZ (100 mg / kg / d) to establish a mouse model of lipid metabolism disorder. At the same time, 10 8-week-old C57 mice were adaptively fed for one week and then fed a normal diet for 5 weeks. On the 6th week, they were intraperitoneally injected with an equal amount of solvent once to serve as the normal control group.

[0225] After successful modeling, the model group was randomly divided into three groups, including the model group and the low (150 mg / kg / d) and high (300 mg / kg / d) dose groups of SZ-A in Preparation Example 8. After 6 weeks of administration according to Table 4 below, the gastrocnemius muscles of the mice were removed, fixed in 4% paraformaldehyde solution, embedded in paraffin, and sectioned for H&E staining. A representative field of view of each sample was selected and photographed under a 200x microscope. The average circumference, average diameter, and average area of ​​the muscle fiber cross-section in the image were measured.

[0226] Table 4 Animal grouping and drug administration

[0227] The results are shown in Figure 8A. HE staining results showed that the gastrocnemius muscle structure in the normal group was clear, with no abnormalities in the muscle bundles or intermuscular spaces. In the model group, muscle fiber edema and atrophy coexisted, and the cross-sectional areas of the muscle fibers within the same muscle bundles varied significantly, with varying sizes. Compared with the model group, after SZ-A administration, the muscle fiber structure was clear, and edema and muscle fiber atrophy were alleviated. As shown in Figures 8B-D, the average cross-sectional area, average diameter, and average circumference of the gastrocnemius muscle fibers in the treatment group were significantly increased compared with the model group.

[0228] Experimental Example 6 Mouse Experiment

[0229] Animal grouping and treatment

[0230] Fifteen-month-old C57 mice were randomly divided into a natural aging group, an SZ-A-treated group, and a positive drug group, with 15 mice in each group. The drugs were administered orally once daily. Three-month-old C57 mice served as controls.

[0231] G1 (normal group): 3-month-old C57 mice, fed with normal diet and drinking water, and gavage with distilled water served as a control.

[0232] G2 (natural aging group): 15-month-old C57 mice, with normal diet and drinking water, and gavage with distilled water as a control.

[0233] G3: 15-month-old C57 mice were fed a normal diet and drank a normal amount of water. They were given 100 mg / kg / day (calculated as alkaloids) of the mulberry extract prepared in Preparation Example 1 for 5 months.

[0234] G4: 15-month-old C57 mice were fed a normal diet and drank a normal amount of water. At the same time, 200 mg / kg / day (calculated as alkaloids) of the mulberry extract prepared in Preparation Example 1 was administered for 5 months.

[0235] G5: 15-month-old C57 mice were fed a normal diet and drank a normal amount of water. They were given 100 mg / kg / day (calculated as alkaloids) of the mulberry extract prepared in Preparation Example 7 for 5 months.

[0236] G6: 15-month-old C57 mice were fed a normal diet and drank a normal amount of water. They were given 200 mg / kg / day (calculated as alkaloids) of the mulberry extract prepared in Preparation Example 7 for 5 months.

[0237] G7 (NMN group, i.e., positive drug group): 15-month-old C57 mice were fed a normal diet and drinking water and given 300 mg / kg / day of NMN for 5 months.

[0238] At the end of the experiment, the experimental mice were anesthetized intramuscularly with Zotai and placed in a prone position in a FAXITRON ULTRAFOCUS 100 bone density analyzer to detect and analyze the bone density of the mice.

[0239] The results are shown in Figure 9. The results showed that compared with the normal group, the bone density of the aging group mice was significantly reduced; compared with the aging group, the bone density of the mulberry extract group was significantly increased, and the effect was better than that of the NMN group.

[0240] Experimental Example 7: Feeding db / db diabetic mouse model with normal diet

[0241] The mulberry extract used in the experiment was the mulberry extract in Preparation Example 1.

[0242] Sixty five-week-old db / db male mice were purchased, and 20 db / m male mice served as a normal control group. The mice were housed at 22°C, 60% humidity, and a 12-hour light-dark cycle. After one week of adaptive feeding with a normal maintenance diet, 10 μL of blood was collected from the tail tip for random blood glucose (PBG) measurement, and the mice were weighed. Based on random blood glucose and body weight levels, the 60 male db / db mice were divided into a model group (db / db), a low-dose mulberry extract (SZ-A) group (SZ-A100 (100 mg / kg / d, calculated as alkaloids), and a medium-dose mulberry extract (SZ-A) group (SZ-A200 (200 mg / kg / d, calculated as alkaloids), with 20 mice in each group. Twenty five-week-old db / m mice served as a normal control group. All mice were fed with basal feed. After 2 weeks of adaptive feeding, each experimental group was given the corresponding dose of the test drug for 8 consecutive weeks. The basal feed was continued to be fed during the treatment period, and the body weight and blood sugar of the animals were monitored. The model group and the normal group were given normal saline. After 8 weeks of administration, a grip strength meter was used to evaluate the effect of SZ-A on the muscle strength of diabetic mice. The grip strength meter (Yuyan Instrument) consists of a gripping rod and a high-precision force measurement system. Place the forelimbs of the experimental mouse on the horizontal bar of the gripping rod, grab the tail of the mouse and gently pull it back. After the mouse grasps the gripping rod firmly, pull it back evenly to cause the mouse to loosen its claws. The instrument automatically records the maximum gripping force of the animal, and repeats the measurement many times to ensure the result, thereby indicating its muscle strength.

[0243] The results of mouse grip strength are shown in Figure 10. The results show that the grip strength of diabetic mice in the model group was significantly reduced. However, after 8 weeks of treatment, the grip strength of mice in the SZ-A 100 mg / kg / d and 200 mg / kg / d groups significantly increased. This indicates that mulberry extract can significantly improve muscle strength in db / db diabetic mice.

[0244] Test Example 8:

[0245] Inhibitory effect of mulberry extract on muscle cell damage

[0246] C2C12 cells were incubated in a 37°C, 5% CO2 incubator with growth medium (high glucose DMEM medium + 10% fetal bovine serum). After the cells reached approximately 85% viability, they were collected and grouped into a control group (NC), a model group (D-gal), and an SZ-A group (D-gal + SZ-A). The culture medium in each group was then replaced with a differentiation medium (high glucose DM EM medium + 2% horse serum). Depending on the purpose of the experiment, D-gal and SZ-A (SZ-A obtained in Preparation Examples 2, 6, and 8) were added to the differentiation medium before the end of the experiment. D-gal was added to the culture medium in the model group at a concentration of 100 mM, while SZ-A and 100 mM D-gal were added to the culture medium in the SZ-A group at a concentration of 100 μg / ml. The treatment lasted for 48 hours. SA-β galactosidase staining was used to detect the degree of cell damage in different groups.

[0247] Group 1: control group;

[0248] Group 2: Model group;

[0249] Group 3: SZ-A group, SZ-A is the mulberry branch extract in Preparation Example 2;

[0250] Group 4: SZ-A group, SZ-A is the mulberry branch extract in Preparation Example 6;

[0251] Group 5: SZ-A group, SZ-A is the mulberry branch extract in Preparation Example 8;

[0252] The results in Figure 11 show that SZ-A can inhibit the damage of C2C12 muscle cells caused by D-gal.

[0253] Test Example 9: Study on the distribution of total alkaloids from Morus alba in bone marrow tissue

[0254] 1. Medicines:

[0255] Miglitol reference substance (Lot: HRSLW-DM, content 100.1%) TCI (Shanghai) Chemical Industry Development Co., Ltd.

[0256] DNJ reference substance (Lot: 20171024, content: 99.29%) Beijing Wuhe Bio Pharmaceutical Co., Ltd.

[0257] FA reference (Lot: 16411, content: 100%) MedChemExpress

[0258] DAB control (Lot: 0000161756, content: 100%) Sigma

[0259] 2. Solution preparation

[0260] Preparation of dosing solution: 1.65 g of mulberry extract was accurately weighed and dissolved in an appropriate amount of ultrapure water, and then shaken to prepare a mulberry extract oral dosing solution with a concentration of 4 mg / ml (calculated as alkaloids).

[0261] Preparation of the assay solution: Preparation of the mobile phase: 0.1% ammonia water: Accurately pipette 4 ml of ammonia water (25%), add 1000 ml of water, shake well, filter, and degas by ultrasonication for 20 min.

[0262] Preparation of stock solution: Accurately weigh 17.0 mg of DNJ reference substance, 8.5 mg of FA reference substance, 8.5 mg of DAB reference substance, and 10.0 mg of miglitol reference substance, respectively, and place them in 10 ml volumetric flasks. Dissolve them with Wahaha water, make up to volume, and shake well to obtain stock solutions with DNJ concentration of 1.688 mg·ml-1, FA concentration of 0.850 mg·ml-1, DAB concentration of 0.850 mg·ml-1, and miglitol concentration of 1.0 mg·ml-1.

[0263] Preparation of mixed reference substance working solution for rat bone marrow tissue assay:

[0264] Accurately measure appropriate amounts of DNJ, FA, and DAB stock solutions, mix them in a 5 ml volumetric flask, and dilute to volume with methanol-water (4:1, v / v) to obtain a DNJ concentration of 25000 ng ml -1 , FA and DAB concentrations were 12500 ng·ml -1 The standard working solution was serially diluted to obtain DNJ concentrations of 125, 250, 625, 1250, 2500, 6250, and 12500 ng / ml, and FA and DAB concentrations of 62.5, 125, 312.5, 625, 1250, 3125, and 6250 ng·ml, respectively. -1 Mix the standard curve working solution in different concentration series.

[0265] Preparation of quality control solution for rat bone marrow tissue assay:

[0266] Accurately measure appropriate amounts of DNJ, FA, and DAB stock solutions, mix them in a 5 ml volumetric flask, and dilute to volume with methanol-water (4:1, v / v) to obtain a DNJ concentration of 25000 ng ml -1 , FA and DAB concentrations were 12500 ng·ml -1 The standard working solution was diluted serially to obtain DNJ concentrations of 50, 100, 1250, and 10,000 ng·ml. -1 , FA and DAB concentrations were 125, 625, and 5000 ng·ml -1 Low, medium and high concentration quality control solutions.

[0267] 3. Animal medication and material collection

[0268] Twenty-four 6- to 8-week-old Sprague-Dawley rats (180-200g), half male and half female, were enrolled. The rats were fasted for 12 hours prior to administration and had free access to water. The rats were orally gavaged with 40 mg / kg (calculated as alkaloids) of mulberry extract and then sacrificed 5 minutes, 30 minutes, 4 hours, and 12 hours later. Six rats (half male and half female) were sacrificed at each time point. Bone marrow (femur) was harvested and stored at -80°C.

[0269] Bone marrow tissue sample processing: The rat bone marrow tissue sample was placed in a freezing grinder for homogenization. The freezing grinder was turned on 30 minutes in advance and refrigerated to 4°C. The homogenization parameters were: frequency 60 Hz, running time 60 seconds, 2 times, and interruption time 5 seconds. The tissue homogenate was prepared and set aside.

[0270] Take 50 μl of rat bone marrow homogenate, add 10 μl of methanol-water (4:1, v:v) solution and 200 μl of 100 ng·ml -1 The internal standard protein precipitation solvent was vortexed for 2 minutes, placed at 4°C for 10 minutes, centrifuged at 14000 rpm × 10 minutes, and 100 μl of the supernatant was taken and diluted to 200 μl with 75% acetonitrile (0.1% formic acid), vortexed for 1 minute, centrifuged at 14000 rpm for 5 minutes, and the supernatant was taken for sampling and determination.

[0271] 4. LC-MS / MS chromatography and mass spectrometry conditions

[0272] 4.1 Chromatographic conditions

[0273] The chromatographic column was an XBridge™ Amide column (3.5 μm, 4.6 mm × 150 mm, Waters, USA); mobile phase A was water (0.1% ammonia water), phase B was acetonitrile, and the gradient elution was: 0.0-4.0 min 65% mobile phase B, 4.0 min-7.0 min 43% mobile phase B, 7.0 min-13.0 min 65% mobile phase B; the flow rate was 0.5 ml / min; the column temperature was 35°C; the injector temperature was 15°C; and the injection volume was 5 μl.

[0274] 4.2 Mass spectrometry conditions

[0275] Electrospray ion source (ESI), Curtain Gas (CUR): 35.0, Collision Gas (CAD): 9, IonSprayVoltage (IS): 5500, Temperature (TEM): 500, IonSource Gas1 (GS1): 55.0, IonSource Gas2 (GS2) 50.0, Entrance Potential (EP): 10.0; Collision Cell Exit Potential (CXP): 6.0. The ionization scanning mode was adopted, and the scanning mode was multiple reaction monitoring (MRM). The reaction ions used for quantitative analysis of DNJ, FA, DAB and miglitol (internal standard, IS) were: m / z 164.3→69.0 (DP 70ev, CE 27ev), m / z 148.2→112.1 (DP 56ev, CE 18ev), m / z 134.3→68.1 (DP 55ev, CE 25ev), m / z 208.3→146.1 (DP 70ev, CE 27ev).

[0276] 5. Results statistics and analysis

[0277] The drug concentration in bone marrow tissue was calculated as shown in Table 4, and a histogram of the drug tissue distribution in bone marrow was drawn.

[0278] Table 4 Drug concentrations in bone marrow tissue of rats given 40 mg / kg of total alkaloids from Morus alba by oral administration (ng / g, n=6)

[0279] 6. Results and Discussion

[0280] The results showed that after rats were orally gavaged with mulberry extract, alkaloids could be detected in the bone marrow tissue within 5 minutes. The distribution trends of DNJ, FA and DAB in the bone marrow tissue were consistent, reaching the maximum drug concentration in 0.5 hours and being basically eliminated from the bone marrow tissue after 12 hours of administration, indicating that there was no risk of accumulation of SZ-A in the bone marrow tissue and it was relatively safe.

Claims

1. Use of mulberry extract in the preparation of a medicament for treating and / or preventing diseases associated with abnormal bone metabolism, characterized in that, Based on the sum of the weight percentages of each component of the mulberry extract being 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 contains polysaccharides with a weight content not higher than 70% (optionally containing polysaccharides with a weight content of 0.2 - 70%, further optionally containing polysaccharides with a weight content of 0.2 - 50%, further optionally containing polysaccharides with a weight content of 0.2 - 35%, further optionally containing polysaccharides with a weight content of 0.2 - 25%, further optionally containing polysaccharides with a weight content of 0.2 - 23%, further optionally containing polysaccharides with a weight content of 20 - 25%), and / or contains flavonoids with a weight content not higher than 10% (optionally containing flavonoids with a weight content of 0.05 - 5%, further optionally containing flavonoids with a weight content of 0 - 2%, further optionally containing flavonoids with a weight content of 0.05 - 2%, further optionally containing flavonoids with a weight content of 0.5 - 1.5%, further optionally containing flavonoids with a weight content of 0 - 1%, further optionally containing flavonoids with a weight content of 0.05 - 1%), and / or contains amino acids with a weight content not higher than 50% (optionally containing amino acids with a weight content of 0 - 30%, further optionally containing amino acids with a weight content of 0 - 25%, further optionally containing amino acids with a weight content of 0 - 20%, further optionally containing amino acids with a weight content of 0 - 5%, further optionally containing amino acids with a weight content of 3 - 25% or further optionally containing amino acids with a weight content of 5 - 20%), 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, wherein The bone metabolism disorder - related disease is any one of osteoporosis, sarcopenia, sarcopenia - osteoporosis, muscular atrophy, myasthenia, osteomalacia, rickets, primary hyperparathyroidism - related bone disease, skeletal fluorosis, osteoarthritis and bone displacement; optionally osteoporosis, sarcopenia or sarcopenia - osteoporosis.

3. The use according to claim 1 or 2, characterized in that, The use of the mulberry extract in the preparation of a drug for treating and / or preventing bone metabolism disorder - related diseases includes: the use of the mulberry extract in any one of the following (b1) - (b3): (b1) The use in the preparation of a drug for increasing bone density; (b2) The use in the preparation of a drug for reducing bone marrow fat vacuoles; (b3) The use in the preparation of a drug with improved bone microstructure; The improved bone microstructure includes at least one of the following: (i) increasing the number of trabeculae; (ii) reducing the trabecular separation; (iii) reducing the trabecular structure model index; (iv) increasing the bone volume fraction; and / or the use as described in any one of the following (1)-(7): 1) The use in the preparation of a drug for increasing muscle mass; 2) The use in the preparation of a drug for increasing the percentage of muscle weight; 3) The use in the preparation of a drug for increasing the muscle fat ratio; 4) The use in the preparation of a drug for inhibiting muscle atrophy and / or edema; optionally, the inhibition of muscle atrophy and / or edema specifically refers to increasing the cross-sectional area, diameter, and / or perimeter of muscle fibers; 5) The use in the preparation of a drug for relieving intermuscular peripheral neuropathy and / or vascular lipid deposition; 6) The use in the preparation of a drug for inhibiting muscle cell damage; 7) The use in the preparation of a drug for increasing muscle strength.

4. The use according to any one of claims 1 to 3, characterized in that, Based on the total weight percentage of each component of the mulberry extract being 100%, the weight content of each component in the mulberry extract is as follows: Optionally, based on the total weight percentage of the components of the mulberry extract being 100%, the weight content of each component in the mulberry extract is as follows: Optionally, based on the sum of the weight percentages of the components of the mulberry extract being 100%, the weight contents of the components in the mulberry extract are as follows: Optionally, based on the sum of the weight percentages of the components of the mulberry extract being 100%, the weight content of each component in the mulberry extract is as follows: Optionally, based on the total weight percentage of the components of the mulberry extract being 100%, the weight content of each component in the mulberry extract is as follows: Optionally, based on the sum of the weight percentages of the components of the mulberry extract being 100%, the weight content of each component in the mulberry extract is as follows: Optionally, based on the sum of the weight percentages of the components of the mulberry extract being 100%, the weight contents of the components in the mulberry extract are as follows: Further optionally, based on the total weight percentage of the components of the mulberry extract being 100%, the weight contents of the components in the mulberry extract are as follows: Further optionally, based on the total weight percentage of the components of the mulberry extract being 100%, the weight contents of the components in the mulberry extract are as follows: Further optionally, based on the sum of the weight percentages of the components of the mulberry extract being 100%, the weight contents of the components in the mulberry extract are as follows:

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

6. The use according to any one of claims 1-5, 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 cation resin and / or an optional anion resin to obtain the mulberry extract.

7. The use according to claim 6, characterized in that: The method further comprises the following steps: 3) Performing alcohol precipitation on the resin effluent of step 2), and collecting the supernatant; 4) Performing concentration and / or drying on the supernatant; Optionally, it further comprises the following step: performing concentration and / or drying on the resin effluent of step 2).

8. The use according to any one of claims 1-7, characterized in that, The mulberry extract acts on humans or mammals.

9. Use according to any one of claims 1-8, characterized in that, The drug is an oral dosage form; optionally, the drug is tablets, capsules, oral solutions, oral emulsions, pills, granules, syrups and powders.

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