Use of mulberry extract in preparation of drug for alleviating and / or treating thyroid diseases

Abstract

A use of a mulberry extract in the preparation of a drug for alleviating and / or treating thyroid diseases. The use is a use of the mulberry extract in the preparation of a product for treating and / or alleviating thyroid diseases. Experiments prove that the mulberry extract can regulate the secretion levels of TSH, FT3 and FT4 of SD rats with hyperthyroidism or hypothyroidism, regulate the thyroid antibody levels of patients with Hashimoto's thyroiditis, and have a good alleviation effect on thyroid tissue lesions. As a component derived from natural plants, the mulberry extract has the advantages of small toxic and side effects, and mild and long-lasting effects.

Description

Application of mulberry extract in preparing medicine for improving and / or treating thyroid disease

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to the Chinese patent application with an application date of September 1, 2023 (application number: 202311122393.9) and the Chinese patent application with an application date of March 6, 2024 (application number: 202410254236.1). The entire contents of the above patent applications are hereby incorporated by reference into this document. Technical Field

[0003] The present invention belongs to the field of medicine, and particularly relates to the application of mulberry extract in the preparation of medicines for improving and / or treating thyroid diseases. Background Art

[0004] The thyroid gland is a very important endocrine gland in the human body. It produces, secretes, and stores thyroid hormone (TH) through thyroid follicular epithelial cells, which acts on the body, regulating metabolism or affecting almost all oxygen-consuming body tissues. TH includes thyroxine (T4) and triiodothyronine (T3). Most of them are bound to plasma proteins, and only the free forms are biologically active, namely free triiodothyronine (FT3) and free thyroxine (FT4). TH secretion is regulated by the hypothalamic-pituitary-thyroid axis. The hypothalamus can produce thyrotropin-releasing hormone to act on the pituitary gland, and the pituitary gland releases thyroid-stimulating hormone (TSH) to act on the thyroid gland, stimulating the thyroid gland to release TH to act on the whole body. Similarly, TH will also negatively feedback to the pituitary gland to inhibit TSH secretion. Therefore, TSH, FT3, and FT4 are often used in clinical evaluation of thyroid function.

[0005] Thyroid diseases are mainly divided into hyperthyroidism (hyperthyroidism), hypothyroidism (hypothyroidism), thyroid nodules, and thyroiditis. In hyperthyroidism, excessive thyroid hormone is secreted, while in hypothyroidism, a small amount of thyroid hormone is secreted. Thyroid nodules are caused by the abnormal proliferation of thyroid cells. Studies have shown that abnormal thyroid function has a serious impact on the body's growth, metabolism, and reproduction.

[0006] Hashimoto's thyroiditis (HT) is a common autoimmune thyroid disease characterized by the presence of thyroid-specific autoantibodies and thyroid lymphocyte infiltration, ultimately leading to structural destruction of the thyroid gland and hypothyroidism. In the early stages of the disease, antibodies secreted by the thyroid gland attack the thyroid gland, causing large amounts of pre-produced thyroid hormone to leak into the bloodstream, resulting in a state of hyperthyroidism. Later, due to depletion of early thyroid hormone production, the thyroid gland becomes damaged, and thyroid hormone production is restricted, leading to a state of hypothyroidism. Thyroid function in patients with HT often progresses through a progression from normal to hyperthyroidism to hypothyroidism. Clinical manifestations include generalized weakness, unilateral or bilateral diffuse goiter, particularly thickening of the isthmus, lymphocytic infiltration, fibrosis, interstitial atrophy, and eosinophilic changes in acinar cells within the thyroid gland. Patients also present with serum autoantibodies such as anti-thyroglobulin antibodies (TG-Ab) and anti-thyroid peroxidase antibodies (TPO-Ab). A small number of patients have elevated thyroid-stimulating hormone (TSH) and decreased free thyroid hormone (FT4).

[0007] Currently, clinical treatments for thyroid-related diseases primarily rely on thyroid hormone replacement therapy, antithyroid hormone drugs, and surgery. However, these medications are long-lasting, highly addictive, and have significant adverse reactions. While medications such as synthetic hormones have been used to treat both hyperthyroidism and hypothyroidism, these drugs also have side effects and limitations in their clinical effectiveness.

[0008] SUMMARY OF THE INVENTION

[0009] The present invention provides the use of a mulberry extract in the preparation of a drug for improving and / or treating thyroid diseases. Experiments have confirmed that the mulberry extract can regulate the secretion levels of TSH, FT3, and FT4 in SD rats with hyperthyroidism or hypothyroidism, regulate the levels of thyroid antibodies in patients with Hashimoto's thyroiditis, and significantly improve thyroid tissue lesions. As a natural plant-derived ingredient, the mulberry extract of the present invention has the unique advantage of having minimal toxic side effects and a mild, long-lasting effect. Therefore, the mulberry extract, with its greatly improved drug safety, offers significant advantages in the treatment of thyroid diseases. Technical Solutions

[0010] The purpose of the present invention is to provide a new medicinal use of a mulberry extract or its main active ingredient.

[0011] The new use of the mulberry extract or its main active ingredient provided by the present invention is any one of the following (a1)-(a4):

[0012] (a1) Use of mulberry extract in the preparation of products for preventing and / or treating thyroid diseases;

[0013] (a2) Use of mulberry extract in the preparation of products for improving thyroid diseases;

[0014] (a3) Use of mulberry extract in preventing and / or treating thyroid diseases;

[0015] (a4) Application of mulberry extract in improving thyroid diseases.

[0016] The thyroid disease includes at least one of the following: hypothyroidism, hyperthyroidism, thyroid nodules, and thyroiditis.

[0017] Preferably, the thyroiditis includes Hashimoto's thyroiditis.

[0018] The treatment and / or improvement of thyroid disease is embodied in at least one of the following aspects:

[0019] 1) Regulate the secretion level of serum thyroid-stimulating hormone (TSH) in patients with thyroid diseases;

[0020] 2) Regulate the secretion level of free triiodothyronine (FT3) in patients with thyroid disease;

[0021] 3) Regulate the secretion level of free thyroxine (FT4) in patients with thyroid disease;

[0022] 4) Improve thyroid tissue lesions in patients with thyroid diseases;

[0023] 5) Improve thyroid antibody levels in patients with thyroid diseases;

[0024] 6) Reduce ROS (reactive oxygen species) levels in patients with thyroid disease;

[0025] 7) Improve the levels of inflammatory factors in patients with thyroid diseases.

[0026] Furthermore, the thyroid antibodies are TPO-Ab antibodies and / or TG-Ab antibodies.

[0027] Furthermore, the inflammatory factor is at least one of the following: TNF-α, IL-1β, IL-6.

[0028] In the present invention, the thyroid disease is preferably hypothyroidism and / or hyperthyroidism.

[0029] Furthermore, the hypothyroidism may be an endocrine disease associated with hyperandrogenism or an endocrine disease associated with glucose and lipid metabolism disorders combined with hypothyroidism. Alternatively, the hypothyroidism may be caused by hyperandrogenism or an endocrine disease associated with hyperandrogenism, or the hypothyroidism may be caused by glucose and lipid metabolism disorders. The endocrine disease associated with hyperandrogenism may be hyperandrogenemia or PCOS.

[0030] The hypothyroidism is manifested by a disturbance in serum thyroid-stimulating hormone (TSH) levels, with or without decreased levels of free triiodothyronine (FT3) and / or free thyroxine (FT4). Alternatively, the hypothyroidism is manifested by a normal serum thyroid-stimulating hormone (TSH) level and decreased levels of free thyroxine (FT4), with or without decreased levels of free triiodothyronine (FT3). The disturbance in TSH levels includes an increase or decrease in TSH levels.

[0031] Furthermore, the hypothyroidism is manifested by an increase in serum thyroid-stimulating hormone (TSH) levels. Alternatively, the hypothyroidism is manifested by an increase in serum thyroid-stimulating hormone (TSH) levels and a decrease in free thyroxine (FT4) levels; or the hypothyroidism is manifested by an increase in serum thyroid-stimulating hormone (TSH) levels and a decrease in free triiodothyronine (FT3) and free thyroxine (FT4) levels.

[0032] Alternatively, the hypothyroidism is manifested in: decreased serum thyroid stimulating hormone (TSH) levels, decreased free triiodothyronine (FT3) and free thyroxine (FT4) levels.

[0033] Alternatively, the hypothyroidism is manifested by normal serum thyroid stimulating hormone (TSH) levels and decreased free thyroxine (FT4) levels. Alternatively, the hypothyroidism is manifested by normal serum thyroid stimulating hormone (TSH) levels and decreased free triiodothyronine (FT3) and free thyroxine (FT4) levels.

[0034] Furthermore, the prevention and / or treatment and / or improvement of hypothyroidism is embodied in: regulating TSH levels, and / or increasing FT3, and / or increasing FT4 levels. The term "regulating" means increasing, decreasing, or maintaining normal levels.

[0035] Specifically, the prevention and / or treatment and / or improvement of hypothyroidism can be reflected in: reducing TSH levels and increasing FT3 and FT4 levels.

[0036] Furthermore, the hyperthyroidism may be a hyperandrogen-related endocrine disease or a glucose and lipid metabolism-related endocrine disease combined with hyperthyroidism. Alternatively, the hyperthyroidism may be caused by hyperandrogenism or a related endocrine disease, or the hyperthyroidism may be caused by a glucose and lipid metabolism disorder. A hyperandrogen-related endocrine disease may be hyperandrogenemia or PCOS.

[0037] Hyperthyroidism is manifested by abnormal serum thyroid-stimulating hormone (TSH) levels, with or without increased levels of free triiodothyronine (FT3) and / or free thyroxine (FT4). Alternatively, hyperthyroidism is manifested by normal serum thyroid-stimulating hormone (TSH) levels and increased levels of free triiodothyronine (FT3) and free thyroxine (FT4). The abnormal TSH levels include both increased and decreased TSH levels.

[0038] Furthermore, the hyperthyroidism is manifested by a decrease in serum thyroid-stimulating hormone (TSH) level. Alternatively, the hyperthyroidism is manifested by a decrease in serum thyroid-stimulating hormone (TSH) level and an increase in free thyroxine (FT4) level; or the hyperthyroidism is manifested by a decrease in serum thyroid-stimulating hormone (TSH) level and an increase in free triiodothyronine (FT3) and free thyroxine (FT4) levels; or the hyperthyroidism is manifested by a decrease in serum thyroid-stimulating hormone (TSH) level and an increase in free triiodothyronine (FT3) level.

[0039] Alternatively, the hyperthyroidism is manifested in: increased serum thyroid stimulating hormone (TSH) levels, increased free triiodothyronine (FT3) and free thyroxine (FT4) levels.

[0040] Alternatively, the hypothyroidism is manifested in: normal serum thyroid stimulating hormone (TSH) level, and increased free triiodothyronine (FT3) and free thyroxine (FT4) levels.

[0041] Furthermore, the prevention and / or treatment and / or improvement of hyperthyroidism is embodied in the following aspects: regulating TSH levels, and / or reducing FT3 levels, and / or reducing FT4 levels. The term "regulating" means increasing, reducing, or maintaining normal levels.

[0042] Specifically, the prevention and / or treatment and / or improvement of hyperthyroidism can be reflected in the following aspects: increasing TSH levels and reducing FT3 and FT4 levels.

[0043] Furthermore, the Hashimoto's thyroiditis is manifested in at least one of the following aspects: serum TPO-Ab and TG-Ab antibody levels are significantly increased.

[0044] Furthermore, the prevention and / or treatment and / or improvement of Hashimoto's thyroiditis is reflected in at least one of the following aspects: reducing serum TPO-Ab antibody levels and reducing serum TG-Ab antibody levels.

[0045] The present invention also protects the use of mulberry extract or its main active ingredient, which is at least one of the following (b1) to (b7):

[0046] (b1) preparing products for regulating serum thyroid stimulating hormone (TSH) secretion levels in patients with thyroid diseases;

[0047] (b2) preparing products for regulating the secretion level of free triiodothyronine (FT3) in patients with thyroid disease;

[0048] (b3) Preparation of products for regulating the secretion level of free thyroxine (FT4) in patients with thyroid disease

[0049] (b4) preparing a product that improves thyroid tissue lesions in patients with thyroid disease;

[0050] (b5) preparing and improving thyroid antibody levels in patients with thyroid diseases;

[0051] (b6) preparing a product that reduces ROS levels in patients with thyroid disease;

[0052] (b7) Prepare a product that improves the level of inflammatory factors in patients with thyroid diseases.

[0053] The product is a drug or a drug preparation.

[0054] The thyroid disease includes at least one of the following: hypothyroidism, hyperthyroidism, thyroid nodules, and thyroiditis.

[0055] The present invention also protects a method for preventing and / or treating and / or improving thyroid diseases.

[0056] The method comprises the following steps: administering the mulberry extract or its main active ingredients to a recipient animal or human to prevent and / or treat and / or improve thyroid disease.

[0057] In the present invention, the mulberry extract is mulberry branch extract, white mulberry bark extract and / or mulberry leaf extract.

[0058] The mulberry extract acts on humans or mammals.

[0059] In one embodiment of the present invention, the mulberry extract mainly includes alkaloids and also includes polysaccharides, flavonoids and amino acids.

[0060] Preferably, the alkaloids include 1-deoxynojirimycin (DNJ), N-methyl-1-deoxynojirimycin (N-methly-1-deoxynojirimycin), fagomine (FAG), 3-epi-fagomine, 1,4-dideoxy-1,4-imino-D-arabinitol (DAB), calystegin B2, calystegin C1, 2-oxy-(α-D At least one 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.

[0061] Wherein, preferably, the weight percentage of DNJ is not less than 50% (preferably 60-99%) of the total alkaloids.

[0062] Preferably, the heavy metal content of the mulberry extract does not exceed 10 ppm.

[0063] Preferably, the mulberry extract contains alkaloids in an amount of 3% or more by weight (preferably 3-99% by weight, more preferably 15-99% by weight, such as 15-90%, or 15-80%, or 15-75%, or 15-70%, further preferably 30-99% by weight, such as 30-90%, or 30-80%, or 30-75%, or 30-70%). , or 35-70%, or 60-75%), and / or contains no more than 70% by weight of polysaccharide (preferably contains 0.2-50% by weight of polysaccharide, more preferably contains 0.2-35%, 0.2-25%, 0.2-23%, 20-25%, 5-35%, 3-35%, 1-35% of polysaccharide, or preferably contains 20-70% by weight of polysaccharide, more preferably contains 20-50%, 20-40%, 20-35% , 20-30%, 20-23% polysaccharides), and / or contains flavonoids of not more than 10% by weight (preferably contains flavonoids of 0.05-5% by weight, more preferably contains 0-7%, 0-2%, 0.05-2%, 0.5-1.5%, 0-1%, 0.05-1% flavonoids), and / or contains amino acids of not more than 50% by weight (preferably contains amino acids of 0-30% by weight, more preferably contains 0-25%, 0-20%, 0-5%, 3-25% or 5-20% amino acids), and / or other components (weight content is preferably 0-25%, more preferably 0-20%, 0-15%, 0-11%, 2-20%, 4-8%), the sum of the contents of each component is 100%, wherein each component refers to all components in the plant extract including alkaloids, polysaccharides, flavonoids and amino acids, that is, the plant extract contains other components in addition to alkaloids, polysaccharides, flavonoids and amino acids.

[0064] The weight content of each component in the mulberry extract is:

[0065] Preferably, based on the mulberry extract, the weight content of each component is:

[0066] Preferably, based on the mulberry extract, the weight content of each component is:

[0067] Preferably, based on the mulberry extract, the weight content of each component is:

[0068] Preferably, based on the mulberry extract, the weight content of each component is:

[0069] Preferably, based on the mulberry extract, the weight content of each component is:

[0070] Preferably, based on the mulberry extract, the weight content of each component is:

[0071] Preferably, based on the mulberry extract, the weight content of each component is:

[0072] More preferably, based on the mulberry extract, the weight content of each component is:

[0073] More preferably, based on the mulberry extract, the weight content of each component is:

[0074] More preferably, based on the mulberry extract, the weight content of each component is:

[0075] Preferably, based on the mulberry extract, the weight content of each component is:

[0076] Preferably, based on the mulberry extract, the weight content of each component is:

[0077] Preferably, based on the mulberry extract, the weight content of each component is:

[0078] Preferably, based on the mulberry extract, the weight content of each component is:

[0079] Preferably, based on the mulberry extract, the weight content of each component is:

[0080] Preferably, based on the mulberry extract, the weight content of each component is:

[0081] Preferably, based on the mulberry extract, the weight content of each component is:

[0082] Preferably, based on the mulberry extract, the weight content of each component is:

[0083] Preferably, based on the mulberry extract, the weight content of each component is:

[0084] Preferably, based on the mulberry extract, the weight content of each component is:

[0085] Preferably, based on the mulberry extract, the weight content of each component is:

[0086] Preferably, based on the mulberry extract, the weight content of each component is:

[0087] Preferably, based on the mulberry extract, the weight content of each component is:

[0088] Preferably, based on the mulberry extract, the weight content of each component is:

[0089] Preferably, based on the mulberry extract, the weight content of each component is:

[0090] Preferably, based on the mulberry extract, the weight content of each component is:

[0091] Preferably, based on the mulberry extract, the weight content of each component is:

[0092] Preferably, based on the mulberry extract, the weight content of each component is:

[0093] The weight content of each component in the mulberry extract is:

[0094] The weight content of each component in the mulberry extract is:

[0095] The weight content of each component in the mulberry extract is:

[0096] The weight content of each component in the mulberry extract is:

[0097] The weight content of each component in the mulberry extract is:

[0098] The weight content of each component in the mulberry extract is:

[0099] The weight content of each component in the mulberry extract is:

[0100] The weight content of each component in the mulberry extract is:

[0101] The weight content of each component in the mulberry extract is:

[0102] The weight content of each component in the mulberry extract is:

[0103] The weight content of each component in the mulberry extract is:

[0104] The weight content of each component in the mulberry extract is:

[0105] In the present invention, the mulberry extract can be provided in the form of commercially available Morus alkaline tablets (National Medicine Standard No. Z20200002).

[0106] In one embodiment, the method for preparing the mulberry extract comprises the following steps: preparing a crude extract; optionally, separating the extract with a cationic resin and / or an anionic resin; optionally, subjecting the resin effluent to alcohol precipitation; and optionally, concentrating and drying. Alternatively, the mulberry extract can be prepared according to the method described in CN 110393738A. Preferably, the method for preparing the mulberry extract comprises the following steps: step 1) preparing a crude extract; step 2) separating the extract with a cationic resin and / or an anionic resin; optionally, step 3) subjecting the resin effluent from step 2) to alcohol precipitation; and optionally, step 4) concentrating and drying.

[0107] In the present invention, the method for preparing 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. The method may further comprise the following steps: 3) subjecting the resin effluent from step 2) to alcohol precipitation and collecting the supernatant; and 4) concentrating and drying the supernatant. The method may further comprise the following step: concentrating and drying the resin effluent from step 2). The Moraceae plant may be selected from Guangdong mulberry, Shandong mulberry, white mulberry, fine-toothed mulberry, mountain mulberry, or hybrid mulberry, with the hybrid mulberry preferably being Yuesang No. 11, Guisang You No. 62, or Sang Te You No. 2. Any part of the plant, including leaves, roots, branches, bark, buds, stems, and fruits, may be used.

[0108] In one embodiment, the mulberry extract is prepared according to the following steps: mulberry branches, mulberry leaves or mulberry bark are crushed, heated and refluxed with water and / or alcohol solution or acid water for extraction, the amount of solvent is 3-20 times that of the original medicinal material, the extraction is repeated 1-3 times, the extracts are combined, concentrated, applied to a cation exchange resin, eluted with 0.2-3N ammonia water, the eluate is concentrated and applied to an anion exchange resin, the non-adsorbed portion is collected, ethanol is added, precipitated to remove impurities, concentrated or dried to obtain an extract.

[0109] In one embodiment, the mulberry extract is prepared according to the following steps: mulberry branches, mulberry leaves or mulberry bark are crushed, and extracted by heating and refluxing with water and / or alcohol solution or acid water, the amount of solvent is 3-20 times that of the original medicinal material, the extraction is repeated 1-3 times, the extracts are combined, concentrated, applied to a cation exchange resin, eluted with 0.2-3N ammonia water, the eluate is concentrated and applied to an anion exchange resin, the non-adsorbed portion is collected, concentrated or dried to obtain an extract.

[0110] In one embodiment, the mulberry extract is prepared according to the following steps: mulberry branches, mulberry leaves or mulberry bark are crushed, and extracted by heating and refluxing with water and / or alcohol solution or acid water, with the amount of solvent being 3-20 times that of the original medicinal material, and the extraction is repeated 1-3 times, the extracts are combined, concentrated, applied to a cation exchange resin, eluted with 0.2-3N ammonia water, and the eluate is concentrated or dried to obtain an extract.

[0111] In one embodiment, the mulberry extract is prepared according to the following steps: mulberry branches, mulberry leaves or mulberry bark are crushed, and extracted with water by heating and refluxing, the amount of solvent is 3-20 times (preferably 4-15 times, more preferably 4-12 times) of the original medicinal material, and the extraction is repeated 1-3 times (the extraction time is preferably 0.5-3 hours each time, more preferably 1-2 hours each time), the extracts are combined, concentrated, applied to a cation exchange resin, eluted with 0.2-3N ammonia water, the eluate is applied to an anion exchange resin, the non-adsorbed portion (i.e., the anion resin effluent) is collected, ethanol is added, precipitated to remove impurities, and concentrated or dried to obtain an extract.

[0112] Optionally, the crude extract after the concentration treatment can also be subjected to an 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. Preferably, the volume mass ratio of the added ethanol to the plant raw material is 0.2-20, preferably 0.4-10 times, in terms of L / kg. More preferably, an alcohol precipitation tank is used for the alcohol precipitation treatment. Preferably, the stirring speed in the alcohol precipitation treatment is 10-600rpm, preferably 40-500rpm, more preferably 80-400rpm or 300rpm.

[0113] Preferably, 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. Preferably, the alkaline solution is washed until the pH of the eluate is 8.0-9.5, preferably 8.5-9.5; preferably, the alkaline solution is selected from aqueous ammonia solution, sodium hydroxide solution, potassium hydroxide solution, or sodium carbonate solution; preferably, the concentration of the alkaline solution is 0.5-4 mol / L, preferably 1-2 mol / L. Preferably, the acidic solution is washed until the pH of the eluate is 3.0-7.0, preferably 4.5-6.5. Preferably, the acidic solution is selected from hydrochloric acid solution, phosphoric acid solution, or sodium hydrogen phosphate-citrate buffer; preferably, the concentration of the acidic solution is 0.5-4 mol / L, preferably 1.5-2 mol / L. Optionally, after the final acidic solution wash, the cationic resin may be rinsed with 3-5 column volumes of deionized water.

[0114] Preferably, the cationic resin is 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.

[0115] Preferably, the weight ratio of the cationic resin to the plant raw material is 1:1-30 (preferably 1:1-25, 1:2-20, 1:2-15, 1:2-10, 1:2-7, 1:2-3). After the crude plant extract is loaded onto the cationic resin, the loaded cationic resin is eluted with an eluent. Preferably, the eluent is a salt solution or alkaline solution containing cations, preferably one or more of sodium chloride, ammonium chloride, ammonium sulfate, ammonium nitrate, ammonia water, potassium chloride and sodium hydroxide. Preferably, the concentration of the eluent is 0.04-5 mol / L (preferably 0.5-2.5 mol / L, 0.2-3 mol / L, more preferably 0.5-2.5 mol / L). Preferably, the eluent flow rate is 1-15 BV / h (preferably 5-10 BV / h, more preferably 5-6 BV / h). 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. Preferably, when the volume of the collected liquid reaches 0.1-10 times (more preferably, 0.2-5 times) the weight of the plant raw material, the collection is stopped, and the collected liquid is optionally purified by an anionic column.

[0116] When purifying by anion column, preferably, 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. Preferably, the alkaline solution is washed until the pH of the eluate is 8.0-9.5, preferably 8.5-9.5; preferably, the alkaline solution is selected from ammonia solution, sodium hydroxide solution, potassium hydroxide solution or sodium carbonate solution; preferably, the concentration of the alkaline solution is 0.5-4 mol / L, preferably 1-2 mol / L. Preferably, the acidic solution is washed until the pH of the eluate is 3.0-7.0, preferably 4.5-6.5. Preferably, the acidic solution is selected from hydrochloric acid solution, phosphoric acid solution, disodium hydrogen phosphate-citrate buffer solution; preferably, the concentration of the acidic solution is 0.5-4 mol / L, preferably 1-2 mol / L.

[0117] Preferably, the anion resin is a 711 strong base styrene anion resin, a 717 strong base styrene anion exchange resin, a D201 macroporous strong base styrene anion exchange resin, or a D218 macroporous strong base acrylic anion exchange resin. Preferably, the ratio of the amount of the anion resin to the weight of the plant raw material is 1:1-80 (preferably 1:1-64, 1:1-32, 1:1-24, 1:5-16, 1:3). Collection begins when the liquid flows out of the anion resin. Preferably, collection stops when the volume of the collected liquid reaches 0.05-10 times (preferably 0.1-5 times) the weight of the plant raw material.

[0118] Preferably, the weight ratio of ethanol used in the alcohol precipitation treatment to the plant raw material is 1:4-600 (preferably, 1:20-300, more preferably 1:20-50, 1:40, 1:80, 1:22). During the alcohol precipitation treatment, the stirring speed is 10-600 rpm (preferably 40-500 rpm, 80-400 rpm). The alcohol precipitation treatment time is 12-24 hours.

[0119] Furthermore, before the alcohol precipitation treatment, the anion resin effluent is subjected to a step of centrifugal impurity removal or microfiltration membrane filtration, and then concentrated by a reverse ion osmosis membrane. The specific gravity of the concentrated liquid can be 1.0-1.3, preferably 1.1-1.25.

[0120] Preferably, 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 glycerin.

[0121] Preferably, the drug is in the form of an oral dosage form; further preferably, the drug is in the form of a tablet, capsule, oral solution, oral emulsion, pill, granule, syrup and powder. Beneficial effects

[0122] The present invention proves through pharmacodynamic experiments that mulberry extract can regulate the secretion levels of TSH, FT3 and FT4 in patients with thyroid diseases, regulate the thyroid antibody levels in patients with Hashimoto's thyroiditis, has a good improvement effect on thyroid tissue lesions, and can be used to treat thyroid diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0123] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0124] Figure 1 shows the levels of serum thyroid stimulating hormone (TSH), free triiodothyronine (FT3), and free thyroxine (FT4) in rats in the blank group, model group, and drug-treated group 21 days after administration in Experimental Example 1. * indicates P < 0.05 compared with the model group, and ** indicates P < 0.01.

[0125] Figure 2 is HE staining images of thyroid tissue of rats in the blank group, model group, and drug-treated group 21 days after drug administration in Experimental Example 1;

[0126] Figure 3 shows the levels of serum thyroid-stimulating hormone (TSH), free triiodothyronine (FT3), and free thyroxine (FT4) in rats in the blank group, model group, and drug-treated group 21 days after administration in Experimental Example 2. * indicates P < 0.05 compared with the model group, and ** indicates P < 0.01.

[0127] Figure 4 is HE staining images of thyroid tissues of rats in the blank group, model group, and drug-treated group 21 days after drug administration in Experimental Example 2;

[0128] Figure 5 shows the results of serum antibody determination in rats in the blank group, model group, and drug-treated group 21 days after administration in Experimental Example 3;

[0129] Figure 6 is HE staining images of thyroid tissues of rats in the blank group, model group, and drug-treated group 21 days after drug administration in Experimental Example 3;

[0130] FIG7 shows the levels of anti-thyroid peroxidase antibodies and anti-thyroglobulin antibodies in four patients with Hashimoto's thyroiditis before and after medication in the human experiment in Example 4;

[0131] Figure 8 shows the TNF-α content in the supernatant of each group in the thyroiditis cell modeling experiment in Example 4; * indicates P < 0.05 compared with the model group, ** indicates P < 0.01, and *** indicates P < 0.001;

[0132] Figure 9 shows the FT4 content in the supernatant of each group in the thyroiditis cell modeling experiment in Example 4; * indicates P < 0.05 compared with the model group, ** indicates P < 0.01, and *** indicates P < 0.001;

[0133] FIG10 shows the IL-6 content in the supernatant of each group in the thyroiditis cell modeling experiment in Example 4; * indicates P<0.05 compared with the model group, and ** indicates P<0.01.

[0134] Modes for carrying out the invention

[0135] The present invention is further described in detail below through the examples. These illustrative descriptions will make the features and advantages of the present invention more apparent. However, the present invention is not limited to the following examples. The methods described are conventional methods unless otherwise specified. The raw materials described can be obtained from publicly available commercial sources unless otherwise specified.

[0136] The professional word “exemplary” is used 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.

[0137] In addition, the technical features involved in the different embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other. In this specification, different component ranges can be combined arbitrarily; the upper and lower limits of the numerical ranges represented by "above," "below," "to," or "-" are values ​​that can be combined arbitrarily. In addition, the values ​​of the Examples (including Preparation Examples and Experimental Examples) can also be used as the upper and lower limits.

[0138] 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).

[0139] 1. Preparation Example of Mulberry Extract

[0140] Preparation Example 1 of Mulberry Extract

[0141] 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.

[0142] 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.

[0143] 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.

[0144] 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).

[0145] In the mulberry branch extract, the mass percentage of alkaloids is 52% (the mass percentage of DNJ in the alkaloids is 65%), the mass percentage of polysaccharides is 22%, the mass percentage of flavonoids is 0.8%, and the mass percentage of amino acids is 20%.

[0146] Preparation Example 2 of Mulberry Extract

[0147] Take 10kg of fresh mulberry branches (Sang Teyou No. 2), crush them, add 150L of water, and add them in two additions. Each time, use the decoction method to extract for 3 hours. The combined extracts are filtered to remove insoluble matter. The extract is heat-concentrated until the solid content reaches 8% by weight. It is 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, stirring is stopped. The alcohol precipitation is continued for 24 hours. The supernatant is taken as the loading liquid for the cationic resin column. 5kg of 002SC type strong acid styrene cationic resin is used to load the column, and the cationic resin is activated according to the method of Example 1. The extract treated with concentrated alcohol precipitation is loaded, and then eluted with 100L of 5mol / L potassium chloride at an elution rate of 5BV / h. The effluent is detected with 20% silicotungstic acid. When a white precipitate is generated, collection is started. When the collected liquid reaches 25L, collection is stopped and the collected liquid is directly passed through an anion column for purification.

[0148] A 10 kg column of 711 strong base styrene anion resin was loaded and activated according to the method in 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.

[0149] 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.

[0150] 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%.

[0151] The obtained Morus alba bark extract has a mass percentage of 95% alkaloids, 2% polysaccharides, 0.1% flavonoids, and 1% amino acids. Among the alkaloids, the content of 1-DNJ is 96%.

[0152] 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%.

[0153] Preparation Example 3 of Mulberry Extract

[0154] 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.

[0155] 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.

[0156] The collected liquid obtained after the cationic column separation is concentrated by nanofiltration membrane and concentrated under reduced pressure to obtain an extract concentrate.

[0157] The obtained mulberry branch extract has a mass percentage of alkaloids of 15%, a mass percentage of polysaccharides of 20%, a mass percentage of flavonoids of 7%, a mass percentage of amino acids of 45%, and a mass percentage of 1-DNJ of 55% among the alkaloids.

[0158] Preparation Example 4 of Mulberry Extract

[0159] 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.

[0160] 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.

[0161] 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.

[0162] The collected liquid after anion column separation was filtered through a microfiltration membrane to remove impurities and then concentrated using a counter-ion osmosis membrane. The concentrated liquid had a specific gravity of 1.1 and was transferred to an alcohol precipitation tank. 15 kg of anhydrous ethanol was added with a stirring paddle at 400 rpm. After the ethanol addition was complete, stirring was stopped. The alcohol precipitation was allowed to proceed for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain a mulberry twig 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, the content of 1-DNJ was 75%.

[0163] Preparation Example 5 of Mulberry Extract

[0164] 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.

[0165] 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.

[0166] The collected solution after anion column separation was filtered through a microfiltration membrane to remove impurities and then concentrated using a counter-ion permeation membrane. Further vacuum concentration and drying were performed to obtain a mulberry branch 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%.

[0167] Preparation Example 6 of Mulberry Extract

[0168] 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.

[0169] 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.

[0170] A column was loaded with 62.5 kg of D218 macroporous, strongly alkaline acrylic anion resin, washed with 1.5 mol / L sodium hydroxide solution until the eluate pH reached 9.0; then washed with 1.5 mol / L hydrochloric acid solution until the eluate pH reached 3.5; and finally washed with 1.5 mol / L sodium hydroxide solution until the eluate pH reached 9.0; activation was completed. The collected cationic resin eluate was loaded onto an anion resin, and the effluent was collected until the effluent reached 870 L. The effluent was concentrated under reduced pressure to obtain a mulberry branch extract, 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%.

[0171] Preparation Example 7 of Mulberry Extract

[0172] 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.

[0173] 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.

[0174] A column was loaded with 62.5 kg of D218 macroporous, strongly alkaline acrylic anion resin, washed with 1.5 mol / L sodium hydroxide solution until the eluate pH reached 9.0; then washed with 1.5 mol / L hydrochloric acid solution until the eluate pH reached 3.5; and finally washed with 1.5 mol / L sodium hydroxide solution until the eluate pH reached 9.0; activation was completed. The collected cationic resin eluate was loaded onto an anion resin, and the effluent was collected until the effluent reached 870 L. The effluent was concentrated under reduced pressure to obtain a mulberry branch extract, which contained 40% by weight alkaloids, 25% by weight polysaccharides, 0.5% by weight flavonoids, and 25% by weight amino acids. The alkaloid content of 1-DNJ was 57%.

[0175] Preparation Example 8 of Mulberry Extract

[0176] 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.

[0177] 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.

[0178] 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.

[0179] 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 settle for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain a mulberry twig extract. The sample contained 63% alkaloids by weight, 23% polysaccharides by weight, 1% flavonoids by weight, and 5% amino acids by weight. Among the alkaloids, the content of 1-DNJ was 61.9%.

[0180] Preparation Example 9 of Mulberry Extract

[0181] 1000 kg of fresh mulberry branches (Yue Sang No. 11) were crushed and added to 4000 L of water. The extracts were then heated and refluxed for 2 hours. The combined extracts were filtered to remove insoluble matter to obtain a crude extract. The crude extract was then heat-concentrated to a solids content of 4% and maintained at 50°C as the loading solution for a cationic resin column.

[0182] A 120 kg column of D113 macroporous weakly acidic phenyl propylene-based cationic resin was loaded and washed with 2 mol / L hydrochloric acid until the eluate pH reached 4.5; then with 1 mol / L sodium hydroxide solution until the eluate pH reached 8.5; then with 2 mol / L hydrochloric acid until the eluate pH reached 4.5; and finally with 5 column volumes of deionized water to complete the activation. The concentrated extract was loaded and then eluted with 1000 L of 2.5 mol / L ammonia at a rate of 6 BV / h. The eluate from the cation column was collected when the pH was > 7. Collection was stopped when 900 L of the collected solution reached and the collected solution was directly passed through an anion column for purification.

[0183] Activate the column using a 45 kg column of D218 macroporous, strongly basic acrylic anion resin. Elute with 1.5 mol / L sodium hydroxide solution until the eluate has a pH of 9.0. Elute with 1.5 mol / L hydrochloric acid solution until the eluate has a pH of 3.5. Elute with 1.5 mol / L sodium hydroxide solution until the eluate has a pH of 9.0. The collected cation resin eluate is loaded onto the anion resin, and the effluent is collected until 870 L of effluent are reached.

[0184] After anion column separation, the collected liquid 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 300 rpm. After the ethanol addition was complete, stirring was stopped. The mixture was allowed to settle for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain an extract. The sample contained 70% by weight of alkaloids, 20% by weight of polysaccharides, 0.6% by weight of flavonoids, and 5% by weight of amino acids.

[0185] Among the alkaloids, the content of 1-DNJ is 70%, FAG is 13%, and DAB is 10%.

[0186] Preparation Example 10 of Mulberry Extract

[0187] 80 kg of fresh mulberry branches (Guisangyou No. 62) were crushed and added to 1000 L of water. The mixture was extracted by heating and refluxing for 2 h. The extracts were combined and filtered to remove insoluble matter to obtain a crude extract. The crude extract was heat-concentrated to a solids content of 4% and then heated at 50°C to serve as the loading solution for a cationic resin column.

[0188] A 50 kg column of D113 macroporous weakly acidic phenyl propylene-based cationic resin was loaded and washed with 2 mol / L hydrochloric acid until the eluate pH reached 4.5; then with 1 mol / L sodium hydroxide until the eluate pH reached 8.5; then with 2 mol / L hydrochloric acid until the eluate pH reached 4.5; and finally with 5 column volumes of deionized water to complete the activation. The concentrated extract was loaded and then eluted with 600 L of 2.5 mol / L ammonia at a rate of 6 BV / h. The eluate was collected when the pH of the cation column effluent was >7. Collection was stopped when 800 L of the collected solution reached and the collected solution was directly passed through an anion column for purification.

[0189] Activate the column using a 65 kg column of D218 macroporous, strongly basic acrylic anion resin. Elute with 1.5 mol / L sodium hydroxide solution until the eluate has a pH of 9.0; then with 1.5 mol / L hydrochloric acid solution until the eluate has a pH of 3.5; and finally with 1.5 mol / L sodium hydroxide solution until the eluate has a pH of 9.0. The collected eluate from the cationic resin is loaded onto the anionic resin, and the effluent is collected until 750 L of effluent is reached. Reload the collected eluate onto the cationic resin and repeat the separation using the cationic and anionic resins, sequentially.

[0190] The collected liquid from these two separations was filtered through a microfiltration membrane to remove impurities and then concentrated using a counter-ion osmosis membrane. The concentrated liquid had a specific gravity of 1.1 and was transferred to an alcohol precipitation tank. 400 g of anhydrous ethanol was added with a stirring paddle at 350 rpm. Stirring was stopped after the ethanol addition, and the mixture was allowed to settle for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain an extract. Sample content: 90% by weight of alkaloids, 3% by weight of polysaccharides, 0.5% by weight of flavonoids, and 2% by weight of amino acids.

[0191] Among the alkaloids, the content of 1-DNJ is 94%, FAG is 2%, and DAB is 1%.

[0192] 2. Verification of the effect of mulberry extract

[0193] Experimental Example 1

[0194] 1. Experimental Animals

[0195] Thirty 3-week-old female SD rats, which were newly weaned, in good health and had shiny fur, were selected and raised in an SPF-grade facility.

[0196] 2. Experimental drugs

[0197] 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.

[0198] Letrozole-1% CMC solution (abbreviated as LZ): 0.25 mg / mL. Weigh a certain amount of LZ, dissolve it in 1% CMC solution, and stir to mix.

[0199] The mulberry extract SZ-A prepared in Preparation Example 1 was prepared at different concentrations and dissolved in an aqueous solution.

[0200] 3. Modeling and drug administration

[0201] The hypothalamic-pituitary-thyroid axis and the hypothalamic-pituitary-ovarian axis are bidirectional circuits. Abnormalities in either axis will affect the normal function of the other. When excessive exogenous androgens or certain inducers are used to inhibit androgen metabolism, resulting in excessive androgen levels in the body, the hypothalamic-pituitary-ovarian axis will be damaged, leading to hyperandrogenism or PCOS. This further affects the pituitary and thyroid function regulated by the hypothalamus, leading to abnormal levels of thyroid-stimulating hormone (TSH) secreted by the pituitary or FT3 and FT4 secreted by the thyroid, and then the body will develop hyperthyroidism or hypothyroidism, as well as thyroid inflammation or nodules and other symptoms.

[0202] Thirty healthy, glossy, 3-week-old female SD rats were selected and acclimated for 3 days. The rats were then randomly divided into a blank group, a model group, and a drug group. The blank group received a basal chow diet; the model and drug groups were fed a high-fat diet (HFD) (research diets D12492) for 3 weeks and then orally administered LZ (1 mg / kg / d) for 6 weeks, concurrently with a HFD (research diets D12492) to establish a rat model. After successful model evaluation (serum thyroid-stimulating hormone (TSH) levels were significantly decreased and free triiodothyronine (FT3) and free thyroxine (FT4) levels were significantly increased in the model group compared to the blank group), the drug groups were administered the doses and routes listed in Table 1. During the drug administration period, the blank and model groups received an equal volume of normal saline. After 21 days of treatment, samples were collected for drug efficacy evaluation.

[0203] Table 1

[0204] 4. Observation indicators and detection methods

[0205] (1) Expression of related hormones in serum: On the 21st day after administration, the serum FT3, FT4 and TSH levels of rats in each group were measured.

[0206] Serum FT3, FT4, and TSH were assayed using ELISA. The following kits were purchased: 1) Free thyroxine (FT4) ELISA kit, manufacturer: Elabscience, catalog number: E-EL-0122C; 2) Free thyroxine (FT3) ELISA kit, manufacturer: Elabscience, catalog number: E-EL-0079C; 3) Rat thyrotropin (TSH) ELISA kit, manufacturer: Elabscience, catalog number: E-EL-0079C.

[0207] (2) After 21 days of administration, the rats were killed and thyroid tissues were collected for HE staining.

[0208] 5. Experimental results

[0209] As shown in Figure 1, compared with the Blank group, the Model group showed a significant decrease in serum thyroid-stimulating hormone (TSH), while free triiodothyronine (FT3) and free thyroxine (FT4) levels increased significantly, suggesting hyperthyroidism in the Model group. After treatment with SZ-A, the low, medium, and high doses of SZ-A significantly increased TSH expression and decreased FT3 and FT4 expression. Furthermore, the improvement of TSH, FT3, and FT4 levels by SZ-A was dose-dependent.

[0210] The results of HE staining of thyroid tissue are shown in Figure 2. The thyroid lobe tissue structure of the Blank group was clear, with follicles of varying sizes and shapes. The follicles were surrounded by a single layer of upright epithelial cells, and the follicular cavities were filled with darkly stained transparent gelatin. The thyroid tissue structure of the Model group was abnormal, with follicular epithelial cells significantly thinner than those in the Blank group, enlarged follicular cavities, and obvious fusion of follicles.

[0211] The size and structure of thyroid follicles in the SZ-AL group were significantly improved. Follicular epithelial cells were thicker than those in the model group, with less fusion. Thyroid tissue in the SZ-AM and SZ-AH groups was structurally similar to that in the control group. Follicles varied in size and were composed of a single layer of cuboidal epithelial cells. Darkly stained transparent gelatin was visible within the follicle lumen. This suggests that SZ-A has a significant ameliorative effect on thyroid tissue lesions.

[0212] Experimental Example 2

[0213] 1. Experimental drugs

[0214] DHEA (dehydroepiandrosterone) oil solution: 30 mg / mL, 30 mg DHEA is dissolved in 1 mL castor oil, placed on a magnetic stirrer, 50°C, speed 500 r / min, and stirred until DHEA is completely dissolved.

[0215] The mulberry extract SZ-A prepared in Preparation Example 1 was prepared at different concentrations and dissolved in an aqueous solution.

[0216] 2. Experimental animals are the same as those in Experimental Example 1.

[0217] 3. Modeling and drug administration

[0218] The hypothalamic-pituitary-thyroid axis and the hypothalamic-pituitary-ovarian axis are bidirectional circuits. Abnormalities in either axis will affect the normal function of the other. When excessive exogenous androgens or certain inducers are used to inhibit androgen metabolism, resulting in excessive androgen levels in the body, the hypothalamic-pituitary-ovarian axis will be damaged, leading to hyperandrogenism or PCOS. This further affects the pituitary and thyroid function regulated by the hypothalamus, leading to abnormal levels of thyroid-stimulating hormone (TSH) secreted by the pituitary or FT3 and FT4 secreted by the thyroid, and then the body will develop hyperthyroidism or hypothyroidism, as well as thyroid inflammation or nodules and other symptoms.

[0219] Table 2

[0220] 21-day-old, newly weaned, healthy, and glossy female SD rats were selected. After three days of adaptive feeding, they were randomly divided into a blank group, a model group, a SZ-AL group, a SZ-AM group, and a SZ-AH group, with six rats in each group. The blank group received a basal diet and a subcutaneous injection of castor oil (60 mg / kg / d). The model and treatment groups received a high-fat diet (research diets D12492) and a subcutaneous injection of DHEA oil solution (60 mg / kg / d). The induction was continued for 7 weeks to establish the rat model.

[0221] After successful model evaluation (compared with the Blank group, serum thyroid-stimulating hormone (TSH) in the Model group was significantly increased, and free triiodothyronine (FT3) and free thyroxine (FT4) were significantly decreased), the blank group rats continued to receive a basal diet without treatment; the Model and Treatment groups continued to receive a high-fat diet. Simultaneously, the Model group received oral gavage of normal saline, the SZ-AL group received oral gavage of SZ-A (67 mg / kg / d), the SZ-AM group received oral gavage of SZ-A (100 mg / kg / d), and the SZ-AH group received oral gavage of SZ-A (150 mg / kg / d). After 21 days of treatment, samples were collected for efficacy testing. Specific testing and analysis methods were the same as those in Experimental Example 1.

[0222] 4. Experimental results

[0223] The results are shown in Figure 3. Compared with the Blank group, the Model group showed a significant increase in serum thyroid-stimulating hormone (TSH) and a significant decrease in free triiodothyronine (FT3) and free thyroxine (FT4), suggesting hypothyroidism in the Model group. After treatment with SZ-A, the low, medium, and high doses of SZ-A significantly reduced TSH expression and increased FT3 and FT4 expression. The improvement of TSH, FT3, and FT4 levels by SZ-A was dose-dependent.

[0224] The results of HE staining of thyroid tissue are shown in Figure 4. HE staining of thyroid tissue in the Blank group showed clear thyroid tissue structure, with follicles of varying sizes surrounded by a single layer of upright epithelial cells, and the follicle cavities filled with darkly stained hyaline colloid. In the Model group, thyroid acinar proliferation was diffuse, nodular, and the majority of the follicles were small cells. Hyaline colloid was absent or minimally present in the follicle cavities, with some lymphocytic infiltration, suggesting hypothyroidism with mild inflammation.

[0225] In the SZ-AL group, thyroid acinar nodular hyperplasia decreased, with some small and some medium-sized follicles. The epithelial cells were single-layered and upright, secretory production was observed in the alveolar lumen, and there was no lymphocyte infiltration, suggesting that SZ-AL has a certain effect on thyroid tissue lesions.

[0226] The proliferation of thyroid acinar nodules in the SZ-AM group was significantly reduced, and the size of the follicles was similar to that in the Blank group. Light red transparent gelatin was seen in the follicle cavity, and there was no lymphocyte infiltration.

[0227] The SZ-AH group showed almost no acinar nodules in the thyroid tissue. Instead, the glands contained irregularly shaped and variably sized follicles with abundant dark-stained transparent colloid within the follicles. This structure was similar to that of the blank group, with no lymphocyte infiltration. This suggests that SZ-A has a dose-dependent effect on thyroid tissue improvement.

[0228] Experimental Example 3

[0229] 1. Experimental drugs

[0230] DHEA (dehydroepiandrosterone) oil solution: 30 mg / mL, 30 mg DHEA is dissolved in 1 mL castor oil, placed on a magnetic stirrer, 50°C, speed 500 r / min, and stirred until DHEA is completely dissolved.

[0231] The mulberry extract SZ-A prepared in Preparation Example 1 was prepared at different concentrations and dissolved in an aqueous solution.

[0232] 2. Experimental Animals

[0233] Twenty-four 3-week-old, newly weaned female SD rats with good health and shiny fur were selected and raised in SPF-grade facilities.

[0234] 3. Modeling and drug administration

[0235] Table 3

[0236] 21-day-old, newly weaned, healthy, and lustrous female SD rats were selected. After three days of adaptive feeding, they were randomly divided into a blank group, a model group, a SZ-AL group, and a SZ-AH group, with six rats in each group. The blank group received a basal diet and a subcutaneous injection of castor oil (60 mg / kg / d). The model and treatment groups received a high-fat diet (research diets D12492) and a subcutaneous injection of DHEA oil solution (60 mg / kg / d). The induction was continued for 7 weeks to establish the rat model.

[0237] After successful model evaluation (TPO-Ab and TG-Ab antibodies increased significantly compared to the Blank group), the blank group continued to receive a basal diet without treatment; the model and treatment groups continued to receive a high-fat diet. The model group received oral administration of normal saline, the SZ-AL group received oral administration of SZ-A (67 mg / kg / d), and the SZ-AH group received oral administration of SZ-A (150 mg / kg / d). After 21 days of treatment, samples were collected for efficacy testing.

[0238] 4. Experimental results

[0239] Serum was collected and assayed for TPO-Ab and TG-Ab antibody levels using ELISA. The results are shown in Figure 5. Compared with the Blank group, TPO-Ab and TG-Ab levels were significantly increased in the Model group, suggesting an increase in thyroid-specific autoantibodies in the Model group. Treatment with SZ-A reduced TPO-Ab and TG-Ab expression in both the low- and high-dose SZ-A groups.

[0240] The results of HE staining of thyroid tissue are shown in Figure 6. The results of HE staining of thyroid tissue showed that in the Blank group, the thyroid tissue structure was clear, with follicles of varying sizes surrounded by a single layer of upright epithelial cells, and the alveolar cavity was filled with dark-stained transparent gelatin. In the Model group, the thyroid tissue acinar proliferation was severe, mostly small cell follicles, with a large number of lymphocyte infiltration, indicating that the thyroid tissue structure was severely damaged. In the SZ-AL group, the acinar proliferation of thyroid tissue was reduced, and some acinar cells returned to normal structure with a single layer of upright epithelial cells. Secretion was observed in the alveolar cavity, and there was no lymphocyte infiltration, indicating that SZ-AL had a certain improvement on thyroid tissue lesions. In the SZ-AH group, there was almost no acinar nodular hyperplasia of thyroid tissue, and acinar of varying sizes and irregular shapes were observed. A large amount of dark-stained transparent gelatin was observed in the alveolar cavity, similar to the thyroid tissue structure of the Blank group, with no lymphocyte infiltration, indicating that SZ-AH had a good improvement effect on thyroid tissue.

[0241] The above research results suggest that SZ-A has a good therapeutic effect on Hashimoto's thyroiditis.

[0242] Experimental Example 4: Pharmacodynamics of Mulberry Extract in Treating Hashimoto's Thyroiditis

[0243] 1. Animal Experimental Protocol

[0244] 1.1 Experimental animals

[0245] Lewis rats, 5-6 weeks old, weighing 120-140 g, were placed in the animal room for 3 days of adaptive feeding. After weighing, they were randomly divided into 6 groups (counted as the first day of the first week), with 10 rats in each group.

[0246] The experimental groups were: 1. normal control group, 2. model group, 3. positive control group, 4-6. SZ-A groups with different dosages (Preparation Example 1, calculated as alkaloids, low, medium, high, dosages 67 mg / ml, 100 mg / ml, 150 mg / ml).

[0247] 1.2 Experimental drugs

[0248] Sodium iodide

[0249] Porcine thyroglobulin

[0250] Freund's complete adjuvant

[0251] Freund's incomplete adjuvant

[0252] Levothyroxine sodium

[0253] Mulberry extract in Preparation Example 8

[0254] 1.3 Modeling and drug administration

[0255] On the day of grouping, all rats except the normal control group were established into a HT model. On the first day of the first week, rats were injected with 0.2 mL of porcine thyroglobulin (4 mg / mL) emulsified in complete Freund's adjuvant into the footpad twice weekly for primary immunization. Immunizations were boosted with a single injection of the same dose of porcine thyroglobulin emulsified in incomplete Freund's adjuvant at weeks 2 and 4. Model success was determined by changes in TPO-Ab and TG-Ab levels. During modeling, rats in all other groups, except the normal control group, drank double-distilled water. All rats drank 0.05% sodium iodide solution until the end of the modeling period.

[0256] After the modeling was completed, drug treatment was carried out. The rats in group 3 were orally gavaged with levothyroxine sodium, and the rats in groups 4-6 were orally gavaged with mulberry extract SZ-A. The remaining groups were orally gavaged with the same dose of normal saline according to their body weight. The drug administration lasted for 4 weeks, once a day.

[0257] 1.4 Sampling and testing

[0258] Twenty-four hours after the final treatment, 3 mL of blood was collected from each group and centrifuged to obtain serum for the determination of FT3, FT4, TSH, TPO-Ab, and TG-Ab levels. Following blood collection, the rats were anesthetized and sacrificed, and their thyroid tissues were collected and divided into two portions, each containing thyroid tissue from 10 rats per group. One portion was fixed in 4% paraformaldehyde for HE staining, and the other portion was frozen at -80°C for Western blotting.

[0259] The levels of TPO-Ab and TG-Ab in rat serum were detected by ELISA;

[0260] HE staining was used to detect the pathological changes of rat thyroid tissue;

[0261] The experimental results showed that SZ-A could reduce the diffuse hyperplasia of thyroid tissue in rats and improve the levels of serum TPO-Ab and TG-Ab.

[0262] 2. Cell Experiment Protocol

[0263] 2.1 Experimental cells

[0264] Thyroid follicular epithelial cells (Nthy-ori3-1)

[0265] 2.2 Experimental drugs

[0266] Sodium iodide

[0267] Interferon gamma

[0268] Fetal bovine serum

[0269] RPMI 1640 medium

[0270] Preparation of the mulberry branch extract in Example 1-8

[0271] 2.3 Cell culture and modeling

[0272] 2.3.1 Nthy-ori3-1 cell culture

[0273] Immortalized human thyroid follicular epithelial cell line (Nthy-ori3-1) was cultured in RPMI 1640 medium (containing 100 IU / mL penicillin and 100 μg / mL streptomycin) containing 10% FBS in a constant temperature cell culture incubator at 37°C and 5% CO2.

[0274] When Nthy-ori3-1 cells have grown to 85%-90% of the culture dish, discard the culture medium and wash twice with 1×PBS, then add 1mL of 0.25% trypsin containing EDTA to the culture dish, place it in a 37℃ constant temperature cell culture incubator for digestion, and process for about 1 minute. Observe under an inverted optical microscope. When the Nthy-ori3-1 cells slowly begin to round, the gaps between cells gradually become larger, and some cells float up, add 3mL of culture medium to terminate the trypsin digestion. Use a pipette to gently blow the attached Nthy-ori3-1 cells from top to bottom to make them fall off the culture dish. Then transfer the cell suspension to a sterile 15mL centrifuge tube, centrifuge at 1000rpm for 5 minutes at room temperature, discard the supernatant, add 4mL of culture medium to resuspend, use a pipette to draw 10μL to fill the pool in the cell counting plate, use a microscope to observe whether the pool is evenly filled and count, and use 3×10 5 The cells were added into a 6-well plate at a concentration of 100 cells / well and 2 mL of culture medium was added into the 6-well plate.

[0275] 2.3.2 Thyroiditis cell model

[0276] After the thyroid follicular epithelial cells (Nthy-ori3-1) entered the logarithmic growth phase, the control group cells were cultured with normal culture medium, and the model group cells were cultured with culture medium containing IFN-γ. After further culture for 24 hours, the mulberry branch extracts of the above-mentioned different preparation examples (Preparation Examples 1-8) were respectively given and cultured for another 24 hours.

[0277] Detection indicators: ROS detection, inflammatory factor detection: TNF-α, IL-1β, IL-6.

[0278] The mulberry branch extracts in Preparation Examples 1-8 can reduce the increase of intracellular ROS induced by IFN-γ to varying degrees, and reduce the levels of TNF-α, IL-1β, and IL-6 in the cell supernatant.

[0279] 3. Human Experiments

[0280] Take the mulberry branch extract of Preparation Example 1, add appropriate amount of auxiliary materials, mix evenly, add water to make a soft material, granulate and dry, add magnesium stearate, mix evenly, and tablet to obtain a mulberry branch extract preparation, each tablet containing 50 mg of total alkaloids.

[0281] Clinical cases and treatment:

[0282] (1) Case 1: A 50-year-old Chinese female, 65 kg, 160 cm tall, had Hashimoto's thyroiditis for 6 years. She took tablets containing the mulberry extract of Preparation Example 1 orally, three times a day, two tablets at a time. After 16 weeks, the anti-thyroid peroxidase antibody TPO-Ab decreased to the normal range, and the anti-thyroglobulin antibody TG-Ab decreased by 17.7%. After 28 weeks of oral administration, the anti-thyroid peroxidase antibody remained stable in the normal range, and the anti-thyroglobulin antibody decreased by 43.5% (see Figure 7), and the thyroid pain was significantly relieved.

[0283] (2) Case 2: A 50-year-old Chinese female, weighing 71 kg and 158 cm in height, had Hashimoto's thyroiditis for 4 years. She took tablets containing the mulberry extract from Preparation Example 1 orally, two tablets at a time, three times a day, for 16 weeks. Her anti-thyroid peroxidase antibodies and anti-thyroglobulin antibodies decreased by 71.0% and 51.2%, respectively (see Figure 7).

[0284] (3) Case 3: A 35-year-old Chinese female, weighing 60 kg and 162 cm in height, had Hashimoto's thyroiditis for 3 years. She took tablets containing the mulberry extract of Preparation Example 1 orally, two tablets at a time, three times a day, for 8 weeks. Her anti-thyroglobulin antibody level decreased by 18.4%, and her anti-thyroid peroxidase antibody level, which was originally normal, stabilized within the normal range after taking the medicine (see Figure 7).

[0285] (4) Case 4: A 52-year-old Chinese female, weighing 62 kg and 163 cm in height, had Hashimoto's thyroiditis for 7 years. She took tablets containing the mulberry extract from Preparation Example 1 orally, two tablets at a time, three times a day, for 24 weeks. Her anti-thyroglobulin antibodies decreased to the normal range, and her anti-thyroid peroxidase antibodies decreased by 76.6% (see Figure 7).

[0286] The above research results suggest that mulberry branch extract has a good therapeutic effect on Hashimoto's thyroiditis.

[0287] IV. Cell Experiment Protocol

[0288] 4.1 Experimental cells

[0289] thyroid follicular epithelial cells

[0290] 4.2 Experimental drugs

[0291] Interferon gamma

[0292] Fetal bovine serum

[0293] RPMI 1640 medium

[0294] The mulberry branch extracts of Preparation Examples 2-4, Preparation Example 6, Preparation Example 8 and Preparation Example 10.

[0295] 4.3 Cell culture and modeling

[0296] 4.3.1 Nthy-ori-3.1 Cell Culture

[0297] Immortalized human thyroid follicular epithelial cell line (Nthy-ori3-1) was cultured in RPMI 1640 medium (containing 100 IU / mL penicillin and 100 μg / mL streptomycin) containing 10% FBS in a constant temperature cell culture incubator at 37°C and 5% CO2.

[0298] When Nthy-ori3-1 cells have grown to 85%-90% of the culture dish, discard the culture medium and wash twice with 1×PBS, then add 1mL of 0.25% trypsin containing EDTA to the culture dish, place it in a 37℃ constant temperature cell culture incubator for digestion, and process for about 1 minute. Observe under an inverted optical microscope. When the Nthy-ori3-1 cells slowly begin to round, the cracks between cells gradually become larger, and some cells float up, add 3mL of culture medium to terminate the trypsin digestion. Use a pipette to gently blow the attached Nthy-ori3-1 cells from top to bottom to make them fall off the culture dish. Then transfer the cell suspension to a sterile 15mL centrifuge tube, centrifuge at 1000rpm for 5min at room temperature, discard the supernatant, add 4mL of culture medium to resuspend, use a pipette to draw 10μL to fill the pool in the cell counting plate, use a microscope to observe whether the pool is evenly filled and count, and use 1×10 4 The cells were added into a 48-well plate and 250 μl of culture medium was added into the 48-well plate.

[0299] 4.3.2 Thyroiditis cell model

[0300] After thyroid follicular epithelial cells (Nthy-ori3-1) entered the logarithmic growth phase, the control group cells were cultured in normal culture medium, and the model group cells were cultured in culture medium containing 150 ng / mL IFN-γ. Simultaneously, the drug-treated groups were given mulberry branch extracts from different preparations (preparations 2-4, 6, 8, and 10) at a concentration of 150 μg / mL. The cells were cultured for 48 hours. The supernatant was collected and centrifuged at 1000 × g for 20 minutes at 4°C to remove impurities and cell debris. The supernatant was then collected for analysis.

[0301] Following the instructions for the ELISA assay kits for TNF-α, FT4, and IL-6, centrifuged supernatant samples were processed. TNF-α and FT4 were assayed using the stock solution, while IL-6 was assayed using a 40-fold dilution of the stock solution. The absorbance (OD) of each well was measured at 450 nm using a microplate reader. The OD values ​​were used to calculate the TNF-α, FT4, and IL-6 levels in the supernatant of each group. Human tumor necrosis factor α (TNF-α) ELISA kit (manufacturer: Elabscience, catalog number: E-EL-H0109). Free thyroxine (fT4) ELISA kit (manufacturer: Elabscience, catalog number: E-EL-0122); and human interleukin 6 (IL-6) ELISA kit (manufacturer: Elabscience, catalog number: E-EL-H6156).

[0302] The results are shown in Figures 8, 9 and 10.

[0303] The results showed that the mulberry branch extracts of Preparation Examples 2-4, Preparation Example 6, Preparation Example 8, and Preparation Example 10 could reduce TNF-α in the cell supernatant and increase FT4 in the supernatant to varying degrees. The mulberry branch extracts of Preparation Examples 2 and 4 could reduce IL-6 in the cell supernatant to varying degrees. The above cell-level experimental results demonstrated that: (1) the mulberry branch extract could alleviate Hashimoto's thyroiditis; (2) the mulberry branch extract could alleviate hypothyroidism. Industrial Applicability

[0304] The present invention discloses the use of mulberry extract in the preparation of a drug for improving and / or treating thyroid diseases. Experiments have confirmed that the mulberry extract can regulate the secretion levels of TSH, FT3, and FT4 in SD rats with hyperthyroidism or hypothyroidism, regulate the levels of thyroid antibodies in patients with Hashimoto's thyroiditis, and significantly improve thyroid tissue lesions. As a natural plant-derived ingredient, the mulberry extract of the present invention has the unique advantage of having minimal toxic side effects and a mild, long-lasting effect. Therefore, the mulberry extract, with its greatly improved drug safety, offers significant advantages in the treatment of thyroid diseases.

Claims

1. The use of mulberry extract or its main active ingredient as described in any one of the following (a1)-(a4): (a1) Use of mulberry extract in the preparation of products for preventing and / or treating thyroid diseases; (a2) Use of mulberry extract in the preparation of products for improving thyroid diseases; (a3) Use of mulberry extract in preventing and / or treating thyroid diseases; (a4) Application of mulberry extract in improving thyroid diseases.

2. The use according to claim 1, characterized in that: The thyroid disease includes at least one of the following: hypothyroidism, hyperthyroidism, thyroid nodules, thyroiditis; Preferably, the thyroiditis is Hashimoto's thyroiditis.

3. The use according to claim 1 or 2, characterized in that: The treatment or improvement of thyroid disease is embodied in at least one of the following aspects: 1) Regulate the secretion level of serum thyroid-stimulating hormone in patients with thyroid diseases; 2) Regulate the secretion level of free triiodothyronine in patients with thyroid disease; 3) Regulate the secretion level of free thyroxine in patients with thyroid disease; 4) Improve thyroid tissue lesions in patients with thyroid diseases; 5) Improve thyroid antibody levels in patients with thyroid diseases; 6) Reduce ROS levels in patients with thyroid disease; 7) Improve the levels of inflammatory factors in patients with thyroid diseases.

4. The use according to claim 1 or 2, characterized in that: The thyroid disease is hypothyroidism; Or, the hypothyroidism is manifested by: increased serum thyroid stimulating hormone levels; Or, the hypothyroidism is manifested by: increased serum thyroid stimulating hormone levels and decreased free thyroxine levels; Or, the hypothyroidism is manifested in: increased serum thyroid stimulating hormone levels, decreased free triiodothyronine and free thyroxine levels; Or, the prevention and / or treatment and / or improvement of hypothyroidism is embodied in at least one of the following aspects: reducing serum thyroid stimulating hormone levels, increasing free triiodothyronine and free thyroxine levels; Or, the hypothyroidism is manifested in: disordered serum thyroid stimulating hormone levels, with or without decreased free triiodothyronine and / or free thyroxine levels; the disordered serum thyroid stimulating hormone levels include increased and decreased serum thyroid stimulating hormone levels; Or, the hypothyroidism is manifested by: normal serum thyroid stimulating hormone level, decreased free thyroxine level, with or without decreased free triiodothyronine level; Or, the hypothyroidism is manifested in: decreased serum thyroid stimulating hormone level, decreased free triiodothyronine and free thyroxine levels; Or, the hypothyroidism is manifested in: normal serum thyroid stimulating hormone level, Decreased levels of prostate hormone; Or, the hypothyroidism is manifested in: normal serum thyroid stimulating hormone level, decreased free triiodothyronine and free thyroxine levels; Alternatively, the prevention and / or treatment and / or improvement of hypothyroidism is embodied in: regulating serum thyroid stimulating hormone levels, and / or increasing free triiodothyronine, and / or increasing free thyroxine levels; the regulation is: increasing, decreasing or maintaining normal levels.

5. The use according to claim 1 or 2, characterized in that: The thyroid disease is hyperthyroidism; Or, the hyperthyroidism is manifested in: decreased serum thyroid stimulating hormone level; Or, the hyperthyroidism is manifested in: decreased serum thyroid stimulating hormone level and increased free thyroxine level; Or, the hyperthyroidism is manifested in: decreased serum thyroid stimulating hormone level, increased free triiodothyronine and free thyroxine levels; Or, the prevention and / or treatment and / or improvement of hyperthyroidism is embodied in at least one of the following aspects: increasing serum thyroid stimulating hormone levels, and reducing levels of triiodothyronine and free thyroxine; Or, the hyperthyroidism is manifested in: disordered serum thyroid stimulating hormone levels, with or without increased free triiodothyronine and / or free thyroxine levels; the disordered TSH levels include increased and decreased TSH levels; Or, the hyperthyroidism is manifested in: normal serum thyroid stimulating hormone level, increased free triiodothyronine and free thyroxine levels; Or, the hyperthyroidism is manifested by: decreased serum thyroid stimulating hormone level and increased free triiodothyronine level; Or, the hyperthyroidism is manifested in: increased serum thyroid stimulating hormone levels, increased free triiodothyronine and free thyroxine levels; Or, the hypothyroidism is manifested by: normal serum thyroid stimulating hormone level, increased free triiodothyronine and free thyroxine levels; Alternatively, the prevention and / or treatment and / or improvement of hyperthyroidism is embodied in the following aspects: regulating serum thyroid stimulating hormone levels, and / or reducing free triiodothyronine, and / or free thyroxine levels; the regulation is: increasing, reducing or maintaining normal levels.

6. The use according to claim 1 or 2, characterized in that: The thyroid diseases include Hashimoto’s thyroiditis; Or, the Hashimoto's thyroiditis is manifested in at least one of the following aspects: serum TPO-Ab and TG-Ab antibody levels are significantly increased; Alternatively, the prevention and / or treatment and / or improvement of Hashimoto's thyroiditis is embodied in at least one of the following aspects: lowering serum TPO-Ab, lowering serum TG-Ab antibody levels.

7. The use of mulberry extract or its main active ingredient is at least one of the following (b1)-(b7): (b1) preparing products for regulating serum thyroid stimulating hormone secretion levels in patients with thyroid diseases; (b2) preparing products for regulating the level of free triiodothyronine secretion in patients with thyroid disease; (b3) preparing products for regulating the secretion level of free thyroxine in patients with thyroid disease; (b4) preparing products for improving thyroid tissue lesions in patients with thyroid diseases; (b5) preparing and improving thyroid antibody levels in patients with thyroid diseases; (b6) preparing a product that reduces ROS levels in patients with thyroid disease; (b7) Prepare products that improve the levels of inflammatory factors in patients with thyroid diseases.

8. The use according to any one of claims 1 to 7, characterized in that: The mulberry extract is a mulberry branch extract, a mulberry bark extract and / or a mulberry leaf extract; Furthermore, 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; Furthermore, the method further comprises the following steps: 3) performing alcohol precipitation on the resin effluent of step 2) and collecting the supernatant; 4) concentrating and drying the supernatant; Or, the method further comprises the following step: concentrating and drying the resin effluent from step 2).

9. The use according to any one of claims 1 to 8, characterized in that: The main active ingredients of the mulberry extract include: at least one of 1-deoxynojirimycin, N-methyl-1-deoxynojirimycin, buckwheat alkaloids, 3-epi-bubbheat alkaloids, 1,4-dideoxy-1,4-imino-D-ribitol, calendula officinalis B2, calendula officinalis C1, 2-oxy-(α-D-galactopyranosyl)-1-deoxynojirimycin, 6-oxy-(β-D-pyranose glucose)-1-deoxynojirimycin, and 1,4-dideoxy-1,4-imino-(2-oxy-β-D-pyranose glucose)-D-arabinitol; Alternatively, the mulberry extract acts on humans or mammals.

10. A method for preventing and / or treating and / or improving thyroid diseases, comprising the steps of administering the mulberry extract or its main active ingredient according to any one of claims 1 to 9 to a recipient animal or human to prevent and / or treat and / or improve thyroid diseases.

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