Application of mulberry extract in preparation of medicine for improving and / or treating thyroid diseases

Abstract

The invention discloses an application of a mulberry extract in preparation of a medicine for improving and / or treating thyroid diseases. The application refers to application of the mulberry extract in preparation of products for treating and / or improving thyroid diseases. Experiments prove that the mulberry extract can regulate the secretion level of TSH, FT3 and FT4 of SD rats with hyperthyroidism or hypothyroidism and regulate the thyroid antibody level of patients with hashimoto thyroiditis, and has a very good improvement effect on thyroid tissue lesions. As a component derived from natural plants, the mulberry extract disclosed by the invention has the unique advantages of small toxic and side effects and mild and lasting action. Therefore, the mulberry extract with greatly improved medication safety has great advantages in the treatment of thyroid diseases.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to the Chinese patent application filed on September 1, 2023 (application number: 202311122393.9), the entire contents of which are hereby incorporated by reference into this document.

[0003] This application claims priority to a Chinese patent application filed on March 6, 2024 (application number: 202410254236.1). The contents of the patent application relating to Hashimoto's thyroiditis are incorporated herein by reference. Technical Field

[0004] The invention belongs to the field of medicine, and particularly relates to application of mulberry extract in preparing medicine for improving and / or treating thyroid diseases. Background Art

[0005] 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, regulates metabolism or affects 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 form is biologically active, namely free triiodothyronine (FT3) and free thyroxine (FT4). The secretion of TH is regulated by the hypothalamus-pituitary-thyroid axis. The hypothalamus can produce thyrotropin-releasing hormone to act on the pituitary, and the pituitary releases thyroid stimulating hormone (TSH) to act on the thyroid, stimulating the thyroid to release TH to act on the whole body. Similarly, TH will also negatively feedback the pituitary to inhibit the secretion of TSH. Therefore, TSH, FT3 and FT4 are often used to evaluate thyroid function in clinical practice.

[0006] Thyroid diseases are mainly divided into hyperthyroidism (hyperthyroidism), hypothyroidism (hypothyroidism), thyroid nodules, and thyroiditis. In hyperthyroidism, excessive thyroid hormones are secreted, and in hypothyroidism, a small amount of thyroid hormones are 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.

[0007] Hashimoto's thyroiditis (HT) is a common autoimmune thyroid disease in clinical practice. It is characterized by the presence of thyroid-specific autoantibodies and thyroid lymphocyte infiltration in the body, which eventually leads to the destruction of thyroid tissue structure and hypothyroidism. In the early stage of the disease, the antibodies secreted by the thyroid gland itself attack, causing a large amount of synthesized thyroid hormone to leak into the blood, resulting in a "hyperthyroidism" state. In the later stage, due to the exhaustion of early synthesized thyroid hormone, the thyroid gland is damaged and the synthesis of thyroid hormone is limited, which will enter the "hypothyroidism" state. In patients with HT, their thyroid function often changes according to the development law of "normal → hyperthyroidism → hypothyroidism". Clinical manifestations: general weakness, unilateral or bilateral diffuse goiter, especially thickening of the isthmus, lymphocyte infiltration, fibrosis, interstitial atrophy and eosinophilic changes of acinar cells in the thyroid gland. Autoantibodies such as anti-thyroglobulin antibodies (TG-Ab) and anti-thyroid peroxidase antibodies (TPO-Ab) appear in the patient's serum. A few patients have elevated thyroid stimulating hormone (TSH) and reduced free thyroid hormone (FT4).

[0008] At present, the main clinical treatments for thyroid-related diseases are thyroid hormone replacement therapy, antithyroid hormone drugs, surgery, etc., but the medication takes a long time, is highly dependent, and has a large number of adverse reactions. Drug treatments such as synthetic hormones have been used to treat hyperthyroidism and hypothyroidism, but these drugs have side effects and limitations in clinical symptoms. Summary of the invention

[0009] The purpose of the invention is to provide a new medicinal use of a mulberry extract or its main active ingredients.

[0010] 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):

[0011] (a1) Use of mulberry extract in the preparation of products for preventing and / or treating thyroid diseases;

[0012] (a2) Use of mulberry extract in the preparation of products for improving thyroid diseases;

[0013] (a3) Use of mulberry extract in preventing and / or treating thyroid diseases;

[0014] (a4) Application of mulberry extract in improving thyroid diseases.

[0015] The thyroid disease includes at least one of the following: hypothyroidism, hyperthyroidism, thyroid nodules, and thyroiditis.

[0016] Preferably, the thyroiditis includes Hashimoto's thyroiditis.

[0017] The treatment and / or improvement of thyroid disease is embodied in at least one of the following aspects:

[0018] 1) Regulate the secretion level of serum thyroid stimulating hormone (TSH) in patients with thyroid diseases;

[0019] 2) Regulate the secretion level of free triiodothyronine (FT3) in patients with thyroid disease;

[0020] 3) Regulate the secretion level of free thyroxine (FT4) in patients with thyroid disease;

[0021] 4) Improve thyroid tissue lesions in patients with thyroid diseases;

[0022] 5) Improve thyroid antibody levels in patients with thyroid diseases;

[0023] 6) Reduce ROS (reactive oxygen species) levels in patients with thyroid disease;

[0024] 7) Improve the levels of inflammatory factors in patients with thyroid diseases.

[0025] Furthermore, the thyroid antibodies are TPO-Ab antibodies and / or TG-Ab antibodies.

[0026] Furthermore, the inflammatory factor is at least one of the following: TNF-α, IL-1β, IL-6.

[0027] In the present invention, the thyroid disease is preferably hypothyroidism and / or hyperthyroidism.

[0028] Further, the hypothyroidism may be an endocrine disease related to high androgen or an endocrine disease related to glucose and lipid metabolism disorder with hypothyroidism. Or, the hypothyroidism may be caused by high androgen or its related endocrine disease, or the hypothyroidism may be caused by glucose and lipid metabolism disorder. The endocrine disease related to high androgen may be hyperandrogenemia or PCOS.

[0029] The hypothyroidism is manifested in: a disordered serum thyroid stimulating hormone (TSH) level, with or without a decrease in free triiodothyronine (FT3) and / or free thyroxine (FT4) levels. Or, the hypothyroidism is manifested in: a normal serum thyroid stimulating hormone (TSH) level, a decrease in free thyroxine (FT4) level, with or without a decrease in free triiodothyronine (FT3) level. The TSH level disorder includes an increase or decrease in the TSH level.

[0030] Furthermore, the hypothyroidism is manifested in: an increase in serum thyroid stimulating hormone (TSH) level. Or, the hypothyroidism is manifested in: an increase in serum thyroid stimulating hormone (TSH) level and a decrease in free thyroxine (FT4) level; or the hypothyroidism is manifested in: an increase in serum thyroid stimulating hormone (TSH) level and a decrease in free triiodothyronine (FT3) and free thyroxine (FT4) levels.

[0031] Alternatively, the hypothyroidism is manifested in: decreased serum thyroid stimulating hormone (TSH) levels, decreased free triiodothyronine (FT3) and free thyroxine (FT4) levels.

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

[0033] 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 "regulation" means: increasing, decreasing or maintaining normal levels.

[0034] Specifically, the prevention and / or treatment and / or improvement of hypothyroidism can be reflected in: reducing TSH levels and increasing FT3 and FT4 levels.

[0035] Further, the hyperthyroidism may be an endocrine disease related to high androgen or an endocrine disease related to glucose and lipid metabolism with hyperthyroidism. Alternatively, the hyperthyroidism may be caused by high androgen or an endocrine disease related to high androgen, or the hyperthyroidism may be caused by glucose and lipid metabolism disorder. Endocrine diseases related to high androgen may be hyperandrogenemia or PCOS.

[0036] The hyperthyroidism is manifested in: a disordered serum thyroid stimulating hormone (TSH) level, with or without an increase in free triiodothyronine (FT3) and / or free thyroxine (FT4) levels. Alternatively, the hyperthyroidism is manifested in: a normal serum thyroid stimulating hormone (TSH) level, with an increase in free triiodothyronine (FT3) and free thyroxine (FT4) levels. The disordered TSH level includes an increase and decrease in the TSH level.

[0037] Furthermore, the hyperthyroidism is manifested in: a decrease in serum thyroid stimulating hormone (TSH) level. Or, the hyperthyroidism is manifested in: a decrease in serum thyroid stimulating hormone (TSH) level and an increase in free thyroxine (FT4) level; or the hyperthyroidism is manifested in: 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 in: a decrease in serum thyroid stimulating hormone (TSH) level and an increase in free triiodothyronine (FT3) level.

[0038] Alternatively, the hyperthyroidism is manifested in: increased serum thyroid stimulating hormone (TSH) levels, increased free triiodothyronine (FT3) and free thyroxine (FT4) levels.

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

[0040] 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 "regulation" means: increasing, reducing or maintaining normal levels.

[0041] 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.

[0042] 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.

[0043] Furthermore, 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 antibody levels, lowering serum TG-Ab antibody levels.

[0044] The present invention also protects the use of mulberry extract or its main active ingredient, which is at least one of the following (b1)-(b7):

[0045] (b1) preparing products for regulating serum thyroid stimulating hormone (TSH) secretion levels in patients with thyroid diseases;

[0046] (b2) preparing products for regulating the secretion level of free triiodothyronine (FT3) in patients with thyroid disease;

[0047] (b3) Preparation of products for regulating the secretion level of free thyroxine (FT4) in patients with thyroid disease

[0048] (b4) preparing products for improving thyroid tissue lesions in patients with thyroid diseases;

[0049] (b5) preparing and improving thyroid antibody levels in patients with thyroid diseases;

[0050] (b6) preparing a product that reduces ROS levels in patients with thyroid disease;

[0051] (b7) Prepare products that improve the levels of inflammatory factors in patients with thyroid diseases.

[0052] The product is a drug or a drug preparation.

[0053] The thyroid disease includes at least one of the following: hypothyroidism, hyperthyroidism, thyroid nodules, and thyroiditis.

[0054] In the present invention, the mulberry extract is a mulberry branch extract, a mulberry bark extract and / or a mulberry leaf extract.

[0055] The mulberry extract acts on humans or mammals.

[0056] In one embodiment of the present invention, the mulberry extract mainly includes alkaloids and also includes polysaccharides, flavonoids and amino acids.

[0057] Preferably, the alkaloids include 1-deoxynojirimycin (DNJ), N-methly-1-deoxynojirimycin, fagomine (FAG), 3-epi-fagomine, 1,4-dideoxy-1,4-imino-D-arabinitol (DAB), calystegin B2, calystegin C1, 2-oxo-(α-D 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.

[0058] Wherein, preferably, the weight percentage of DNJ is not less than 50% (preferably 60-99%) of the total alkaloids.

[0059] Preferably, the heavy metal content of the mulberry extract does not exceed 10 ppm.

[0060] 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%, and 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 comprises a polysaccharide content of not more than 70% by weight (preferably comprises a polysaccharide content of 0.2-50% by weight, more preferably comprises 0.2-35%, 0.2-25%, 0.2-23%, 20-25%, 5-35%, 3-35%, 1-35% of polysaccharide, or preferably comprises a polysaccharide content of 20-70% by weight, more preferably comprises 20-50%, 20-40%, 20-35% , 20-30%, 20-23% polysaccharides), and / or contains flavonoids with a weight content of not more than 10% (preferably contains flavonoids with a weight content of 0.05-5%, more preferably contains 0-7%, 0-2%, 0.05-2%, 0.5-1.5%, 0-1%, 0.05-1%), and / or contains amino acids with a weight content of not more than 50% (preferably contains amino acids with a weight content of 0-30%, 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.

[0061] The weight content of each component in the mulberry extract is:

[0062]

[0063] Preferably, based on the mulberry extract, the weight content of each component is:

[0064]

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

[0066]

[0067]

[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] 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] More 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]

[0080]

[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]

[0087]

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

[0089]

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

[0091]

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

[0093]

[0094] Preferably, based on the mulberry extract, the weight content of each component is:

[0095] Preferably, based on the mulberry extract, the weight content of each component is:

[0096] Preferably, based on the mulberry extract, the weight content of each component is:

[0097] Preferably, based on the mulberry extract, the weight content of each component is:

[0098] Preferably, based on the mulberry extract, the weight content of each component is:

[0099] Preferably, based on the mulberry extract, the weight content of each component is:

[0100] Preferably, based on the mulberry extract, the weight content of each component is:

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

[0102]

[0103]

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

[0105]

[0106] The weight content of each component in the mulberry extract is:

[0107]

[0108] The weight content of each component in the mulberry extract is:

[0109]

[0110] The weight content of each component in the mulberry extract is:

[0111]

[0112] The weight content of each component in the mulberry extract is:

[0113]

[0114] The weight content of each component in the mulberry extract is:

[0115]

[0116] The weight content of each component in the mulberry extract is:

[0117]

[0118] The weight content of each component in the mulberry extract is:

[0119]

[0120] The weight content of each component in the mulberry extract is:

[0121]

[0122] The weight content of each component in the mulberry extract is:

[0123]

[0124] The weight content of each component in the mulberry extract is:

[0125]

[0126]

[0127] In the present invention, the mulberry extract can be provided in the form of commercially available mulberry branch total alkaloid tablets (National Medicine Standard No. Z20200002).

[0128] In one embodiment, the method for preparing the mulberry extract comprises the following steps: preparing a crude extract; optionally, separating through a cationic resin and / or an anionic resin; optionally, subjecting the resin effluent to alcohol precipitation; and optionally, concentrating and drying. Optionally, the mulberry extract can be prepared with reference 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 through a cationic resin and / or an optional anionic resin; optional step 3): subjecting the resin effluent of step 2) to alcohol precipitation; optional step 4) concentrating and drying.

[0129] In the present invention, 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. The method may further comprise the following steps: 3) subjecting the resin effluent of step 2) to an alcohol precipitation treatment and collecting the supernatant; 4) subjecting the supernatant to a concentration and drying treatment. The method may further comprise the following steps: subjecting the resin effluent of step 2) to a concentration and drying treatment. The moraceae plant may be selected from Guangdong mulberry, Shandong mulberry, white mulberry, fine-toothed mulberry, mountain mulberry or hybrid mulberry, and the hybrid mulberry is preferably Yue mulberry No. 11, Gui mulberry No. 62 or Sang Teyou No. 2. The leaves, roots, branches, skin, buds, stems, fruits and other parts of the plant may be used.

[0130] 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 part is collected, ethanol is added, impurities are removed by precipitation, and the extract is concentrated or dried to obtain an extract.

[0131] 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 part is collected, concentrated or dried to obtain an extract.

[0132] 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, and the eluate is concentrated or dried to obtain an extract.

[0133] 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 for extraction, the amount of solvent is 3-20 times (preferably 4-15 times, more preferably 4-12 times) of the original medicinal material, 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 part (i.e., the anion resin effluent) is collected, ethanol is added, impurities are removed by precipitation, and the extract is concentrated or dried to obtain an extract.

[0134] Optionally, the crude extract after the concentration treatment can also be subjected to alcohol precipitation treatment before the resin separation treatment in step 2). During the alcohol precipitation treatment, ethanol is added to the crude extract, stirred and mixed, and the stirring is stopped and allowed to stand for a certain period of time to precipitate the insoluble matter therein. 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 alcohol precipitation treatment. Preferably, the stirring speed in the alcohol precipitation treatment is 10-600rpm, preferably 40-500rpm, and more preferably 80-400rpm or 300rpm.

[0135] 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 an aqueous ammonia solution, a sodium hydroxide solution, a potassium hydroxide solution, or a 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 a hydrochloric acid solution, a phosphoric acid solution, and a sodium hydrogen phosphate-citric acid buffer solution; preferably, the concentration of the acidic solution is 0.5-4 mol / L, preferably 1.5-2 mol / L. Optionally, the cationic resin can also be rinsed with 3-5 column volumes of deionized water after the last acidic solution wash.

[0136] Preferably, the cationic resin is 732 type strong acid styrene type cation exchange resin, 002SC type strong acid styrene type cation exchange resin, 734 type strong acid styrene type cation exchange resin, D001 type macroporous strong acid styrene type cation exchange resin or D113 type macroporous weak acid cation exchange resin.

[0137] 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 effluent from the cationic resin. For example, when an alkaline solution such as aqueous ammonia is used for elution, the eluate is collected when the pH of the effluent from the cationic column is detected to be greater than 7, or the collection starting point of the effluent is determined according to a color development or precipitation reaction. 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 input, the collection is stopped, and the collected liquid is optionally purified by an anion column.

[0138] 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, preferably, the concentration of the acidic solution is 0.5-4 mol / L, preferably 1-2 mol / L.

[0139] Preferably, the anion resin is a 711 type strong basic styrene anion resin, a 717 type strong basic styrene anion exchange resin, a D201 type macroporous strong basic styrene anion exchange resin or a D218 type macroporous strong basic acrylic anion exchange resin. Preferably, the amount of the anion resin is 1:1-80 (preferably, 1:1-64, 1:1-32, 1:1-24, 1:5-16, 1:3) to the weight of the plant raw material. When the liquid flows out of the anion resin, collection begins. Preferably, when the volume of the collected liquid reaches 0.05-10 times (preferably, 0.1-5 times) the weight of the plant raw material, collection is stopped.

[0140] 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.

[0141] Furthermore, before the alcohol precipitation treatment, the anion resin effluent is centrifuged or filtered through a microfiltration membrane to remove impurities, and then concentrated through a reverse ion osmosis membrane. The liquid after concentration has a specific gravity of 1.0-1.3, preferably 1.1-1.25.

[0142] Preferably, the drug further comprises a pharmaceutically acceptable carrier. The carrier is an inactive ingredient that is non-toxic to the human body and conforms to the route of administration or the mode of administration. The carrier can be a solid or liquid excipient. Solid excipients, for example, include microcrystalline cellulose, mannitol, lactose, pregelatinized starch, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, sodium carboxymethyl starch, aspartame, calcium hydrogen phosphate, sodium lactate, poloxamer, sodium lauryl sulfate, sodium carboxymethyl cellulose, gelatin, xanthan gum, polyvinylpyrrolidone, starch, magnesium stearate, sodium carboxymethyl starch and talc; liquid excipients, for example, include water, ethanol, syrup and glycerol.

[0143] Preferably, the drug is in the form of an oral dosage form; more preferably, the drug is in the form of a tablet, a capsule, an oral solution, an oral emulsion, a pill, a granule, a syrup and a powder.

[0144] 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

[0145] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0146] Figure 1 The contents of serum thyroid stimulating hormone (TSH), free triiodothyronine (FT3) and free thyroxine (FT4) in the blank group, model group and drug-treated group after 21 days of drug administration in Experimental Example 1, * indicates P<0.05 compared with the model group, ** indicates P<0.01;

[0147] Figure 2 HE staining pictures of thyroid tissues of rats in the blank group, model group, and drug-treated group after 21 days of drug administration in Experimental Example 1;

[0148] Figure 3 The contents of serum thyroid stimulating hormone (TSH), free triiodothyronine (FT3) and free thyroxine (FT4) in the blank group, model group and drug-treated group after 21 days of drug administration in Experimental Example 2, * indicates P < 0.05 compared with the model group, ** indicates P < 0.01;

[0149] Figure 4HE staining pictures of thyroid tissues of rats in the blank group, model group, and drug-treated group after 21 days of drug administration in Experimental Example 2;

[0150] Figure 5 The results of serum antibody determination of rats in the blank group, model group and drug-treated group after 21 days of drug administration in Experimental Example 3;

[0151] Figure 6 HE staining pictures of thyroid tissues of rats in the blank group, model group, and drug-treated group after 21 days of drug administration in Experimental Example 3;

[0152] Figure 7 The anti-thyroid peroxidase antibody and anti-thyroglobulin antibody levels of four patients with Hashimoto's thyroiditis before and after taking the medicine in the human experiment in Example 4;

[0153] Figure 8 The TNF-α content in the supernatant of each group in the thyroid inflammation cell modeling experiment in Example 4; * indicates P<0.05 compared with the model group, ** indicates P<0.01, and *** indicates P<0.001;

[0154] Fig. 9 The FT4 content in the supernatant of each group in the thyroid inflammation cell modeling experiment in Example 4; * indicates P<0.05 compared with the model group, ** indicates P<0.01, and *** indicates P<0.001;

[0155] Fig.10 The IL-6 content in the supernatant of each group in the thyroid inflammation cell modeling experiment in Example 4; * indicates P<0.05 compared with the model group, ** indicates P<0.01. DETAILED DESCRIPTION

[0156] The present invention is further described in detail by examples below. Through these exemplary descriptions, the characteristics and advantages of the present invention will become clearer and more specific. However, the present invention is not limited to the following examples. The methods are conventional methods unless otherwise specified. The raw materials can be obtained from public commercial channels unless otherwise specified.

[0157] The professional word “exemplary” used herein means “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.

[0158] In addition, the technical features involved in different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. In this specification, different component ranges can be combined arbitrarily; the upper and lower limits of the numerical ranges represented by "above", "below", "~" or "-" are values ​​that can be combined arbitrarily, and the values ​​of the embodiments (including preparation examples and experimental examples) can also be used as the upper and lower limits.

[0159] 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).

[0160] 1. Example of Preparation of Mulberry Extract

[0161] Preparation Example 1 of Mulberry Extract

[0162] Take 1000kg of fresh mulberry branches (fine-toothed mulberry branches, Yuesang 11), crush them, add 4000L of water, extract them by heating reflux for 2h, combine the extracts, filter and remove insoluble matter to obtain a crude extract. The crude extract is heat concentrated to a solid mass percentage of 4%, and kept at 50℃ as the loading liquid for the cationic resin column.

[0163] Use 150kg of D113 macroporous weakly acidic phenyl propylene cationic resin to load the column, wash with 2mol / L hydrochloric acid solution until the pH of the eluate is 4.5; wash with 1mol / L sodium hydroxide solution until the pH of the eluate is 8.5; wash with 2mol / L hydrochloric acid solution until the pH of the eluate is 4.5; then rinse with 5 times the column volume of deionized water to complete the activation. Load the concentrated extract, then elute with 1000L 2.5mol / L ammonia water at a rate of 6BV / h. Collect the eluate when the pH of the cation column effluent is >7. When the collected liquid reaches 900L, stop collecting and directly purify the collected liquid through the anion column.

[0164] Use 62.5 kg of D218 macroporous strongly alkaline acrylic anion resin to load the column, wash with 1.5 mol / L sodium hydroxide solution until the pH of the eluate is 9.0; wash with 1.5 mol / L hydrochloric acid solution until the pH of the eluate is 3.5; wash with 1.5 mol / L sodium hydroxide solution until the pH of the eluate is 9.0; complete activation. Load the collected cationic resin eluate onto the anion resin, and collect the effluent until the effluent reaches 920 L.

[0165] The collected liquid was centrifuged to remove impurities and then concentrated by reverse ion osmosis membrane. The liquid after concentration had a specific gravity of 1.25, and was transferred to an alcohol precipitation tank. 25L of anhydrous ethanol was added at 500rpm. After the addition of ethanol, stirring was stopped, and the alcohol precipitation was carried out for 24 hours. The supernatant was taken and concentrated under reduced pressure to obtain a mulberry branch extract (i.e., mulberry extract SZ-A).

[0166] 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%.

[0167] Preparation Example 2 of Mulberry Extract

[0168] Take 10kg of fresh mulberry branches (Sangteyou No. 2), crush them, add 150L of water, add them in 2 times, extract them by decoction for 3h each time, combine the extracts, and filter to remove insoluble matter. The extract is hot concentrated to 8% solid mass percentage, transferred to an alcohol precipitation tank, and 2367.9g of anhydrous ethanol (3L) is added under a stirring paddle of 300rpm. After the addition of ethanol, stop stirring, precipitate with alcohol for 24h, and take the supernatant as the sample liquid for the cationic resin column. Use 002SC type strong acid styrene cationic resin 5kg to load the column, and activate the cationic resin according to the method of Example 1. Load the extract treated with concentrated alcohol precipitation, then use 100L 5mol / L potassium chloride for elution, the elution rate is 5BV / h, and detect the effluent with 20% silicotungstic acid. Start collecting when a white precipitate is generated, and stop collecting when the collected liquid reaches 25L, and directly purify the collected liquid through an anion column.

[0169] A 711 type strong basic styrene anion resin (10 kg) was used to load the column, and the anion resin was activated according to the method of 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 liquid was reloaded onto the cationic resin, and the cationic resin and anion resin were used for separation twice in turn according to the above method.

[0170] The collected liquid obtained after three column separations was centrifuged to remove impurities and then concentrated by reverse ion permeation membrane. The concentrated liquid had a specific gravity of 1.25 and was transferred to an alcohol precipitation tank. 125 g of anhydrous ethanol was added under a stirring paddle of 1000 rpm. After the addition of ethanol, stirring was stopped, and the alcohol was precipitated for 24 hours. The supernatant was taken and concentrated under reduced pressure to obtain an extract. In addition, fresh mulberry bark and mulberry leaf (Sangteyou No. 2) were extracted, and the extraction method and parameters were the same as the above method.

[0171] In the obtained mulberry branch extract, the mass percentage of alkaloids is 98%, the mass percentage of polysaccharides is 0.2%, the mass percentage of flavonoids is 0.05%, and the mass percentage of amino acids is 0. Among the alkaloids, the content of 1-DNJ is 99%.

[0172] The obtained Morus alba extract has 95% by weight of alkaloids, 2% by weight of polysaccharides, 0.1% by weight of flavonoids, and 1% by weight of amino acids. Among the alkaloids, the content of 1-DNJ is 96%.

[0173] The obtained mulberry leaf extract has 90% by weight of alkaloids, 4% by weight of polysaccharides, 0.1% by weight of flavonoids, and 3% by weight of amino acids. Among the alkaloids, the content of 1-DNJ is 91% by weight.

[0174] Preparation Example 3 of Mulberry Extract

[0175] Take 1000kg of fresh mulberry branches (Guangdong mulberry), crush them, add 11500L of water, heat and reflux for 2h, combine the extracts, filter and remove insoluble matter to obtain a crude extract. The crude extract is first centrifuged to remove impurities, and then concentrated with a counter ion permeation membrane to a solid mass percentage of 1%, which is used as the loading solution for the cationic resin column.

[0176] 300 kg of D001 macroporous strong acid styrene cationic resin was loaded into the column, and the cationic resin was activated according to the method of Preparation Example 1. The concentrated crude extract was loaded, and eluted with 5000 L of 0.04 mol / L ammonium nitrate at a rate of 5 BV / h. The effluent was detected with 20% silicotungstic acid. When a white precipitate was generated, collection began, and when the collected liquid reached 1000 L, collection was stopped.

[0177] The collected liquid obtained after the cationic column separation is concentrated by nanofiltration membrane, and the extract is concentrated under reduced pressure to obtain an extract concentrate.

[0178] The obtained mulberry branch extract has 15% by weight of alkaloids, 20% by weight of polysaccharides, 7% by weight of flavonoids, and 45% by weight of amino acids. Among the alkaloids, the content of 1-DNJ is 55%.

[0179] Preparation Example 4 of Mulberry Extract

[0180] 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.

[0181] Use 150kg of D113 macroporous weakly acidic phenyl propylene cationic resin to load the column, wash with 2mol / L hydrochloric acid solution until the pH of the eluate is 4.5; wash with 1mol / L sodium hydroxide solution until the pH of the eluate is 8.5; wash with 2mol / L hydrochloric acid solution until the pH of the eluate is 4.5; then rinse with 5 times the column volume of deionized water to complete the activation. Load the concentrated extract, then elute with 1000L 2.5mol / L ammonia water at a rate of 6BV / h. Collect the eluate when the pH of the cation column effluent is >7. When the collected liquid reaches 900L, stop collecting and directly purify the collected liquid through the anion column.

[0182] Use 125kg of D218 macroporous strongly alkaline acrylic anion resin to load the column, wash with 1.5mol / L sodium hydroxide solution until the pH of the eluate is 9.0; wash with 1.5mol / L hydrochloric acid solution until the pH of the eluate is 3.5; wash with 1.5mol / L sodium hydroxide solution until the pH of the eluate is 9.0; complete activation. Load the collected cationic resin eluate onto the anion resin, and collect the effluent with a pH greater than 8 until the effluent reaches 870L.

[0183] The collected liquid obtained after the anion column separation is filtered through a microfiltration membrane to remove impurities and then concentrated with a counter ion permeation membrane. The concentrated liquid has a specific gravity of 1.1 and is transferred to an alcohol precipitation tank. 15 kg of anhydrous ethanol is added at a stirring paddle of 400 rpm. After the addition of ethanol, stirring is stopped, and the alcohol precipitation is carried out for 24 hours. The supernatant is taken and concentrated under reduced pressure to obtain a mulberry branch extract. Sample content: the mass percentage of alkaloids is 80%, the mass percentage of polysaccharides is 5%, the mass percentage of flavonoids is 0.1%, and the mass percentage of amino acids is 4%. Among the alkaloids, the content of 1-DNJ is 75%.

[0184] Preparation Example 5 of Mulberry Extract

[0185] 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.

[0186] Use 62.5 kg of D218 macroporous strongly alkaline acrylic anion resin to load the column, wash with 1.5 mol / L sodium hydroxide solution until the pH of the eluate is 9.0; wash with 1.5 mol / L hydrochloric acid solution until the pH of the eluate is 3.5; wash with 1.5 mol / L sodium hydroxide solution until the pH of the eluate is 9.0; complete activation. Load the collected extract concentrate onto the anion resin and collect the effluent.

[0187] The collected solution obtained after the anion column separation was filtered through a microfiltration membrane to remove impurities and then concentrated through a counter ion permeation membrane, and further concentrated and dried under reduced pressure to obtain a mulberry branch extract. Sample content: 3% by mass of alkaloids, 70% by mass of polysaccharides, 10% by mass of flavonoids, and 10% by mass of amino acids. Among the alkaloids, the content of 1-DNJ was 68%.

[0188] Preparation Example 6 of Mulberry Extract

[0189] Take 1500kg of fresh mulberry branches (fine-toothed mulberry branches, Yuesang 11), crush them, add 6000L of water, extract them by heating reflux for 2h, combine the extracts, filter and remove insoluble matter to obtain a crude extract. The crude extract is heat concentrated to a solid mass percentage of 4%, and kept at 50℃ as the loading liquid for the cationic resin column.

[0190] Use 100kg of D113 macroporous weakly acidic phenyl propylene cationic resin to load the column, wash with 2mol / L hydrochloric acid solution until the pH of the eluate is 4.5; wash with 1mol / L sodium hydroxide solution until the pH of the eluate is 8.5; wash with 2mol / L hydrochloric acid solution until the pH of the eluate is 4.5; then rinse with 5 times the column volume of deionized water to complete the activation. Load the concentrated extract, then elute with 1000L 2.5mol / L ammonia water at a rate of 6BV / h. Collect the eluate when the pH of the cation column effluent is >7. Stop collecting when the collected liquid reaches 900L, and directly purify the collected liquid through the anion column.

[0191] Use 62.5kg of D218 macroporous strong alkaline acrylic anion resin to load the column, wash with 1.5mol / L sodium hydroxide solution until the pH of the eluate is 9.0; wash with 1.5mol / L hydrochloric acid solution until the pH of the eluate is 3.5; wash with 1.5mol / L sodium hydroxide solution until the pH of the eluate is 9.0; complete activation. Load the collected cationic resin eluate onto the anion resin, and collect the effluent until the effluent reaches 870L. Concentrate the effluent under reduced pressure to obtain a mulberry branch extract, in which the mass percentage of alkaloids is 30%, the mass percentage of polysaccharides is 35%, the mass percentage of flavonoids is 2%, and the mass percentage of amino acids is 25%. Among the alkaloids, the content of 1-DNJ is 62%.

[0192] Preparation Example 7 of Mulberry Extract

[0193] Take 1000kg of fresh mulberry branches (fine-toothed mulberry branches, Yuesang 11), crush them, add 4000L of water, extract them by heating reflux for 2h, combine the extracts, filter and remove insoluble matter to obtain a crude extract. The crude extract is heat concentrated to a solid mass percentage of 4%, and kept at 50℃ as the loading liquid for the cationic resin column.

[0194] Use 100kg of D113 macroporous weakly acidic phenyl propylene cationic resin to load the column, wash with 2mol / L hydrochloric acid solution until the pH of the eluate is 4.5; wash with 1mol / L sodium hydroxide solution until the pH of the eluate is 8.5; wash with 2mol / L hydrochloric acid solution until the pH of the eluate is 4.5; then rinse with 5 times the column volume of deionized water to complete the activation. Load the concentrated extract, then elute with 1000L 2.5mol / L ammonia water at a rate of 6BV / h. Collect the eluate when the pH of the cation column effluent is >7. Stop collecting when the collected liquid reaches 900L, and directly purify the collected liquid through the anion column.

[0195] Use 62.5kg of D218 macroporous strong alkaline acrylic anion resin to load the column, use 1.5mol / L sodium hydroxide solution to wash until the pH of the eluate is 9.0; use 1.5mol / L hydrochloric acid solution to wash until the pH of the eluate is 3.5; wash with 1.5mol / L sodium hydroxide solution until the pH of the eluate is 9.0; complete activation. Load the collected cationic resin eluate onto the anion resin, and collect the effluent until the effluent reaches 870L. Concentrate the effluent under reduced pressure to obtain a mulberry branch extract, in which the mass percentage of alkaloids is 40%, the mass percentage of polysaccharides is 25%, the mass percentage of flavonoids is 0.5%, and the mass percentage of amino acids is 25%. Among the alkaloids, the content of 1-DNJ is 57%.

[0196] Preparation Example 8 of Mulberry Extract

[0197] 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.

[0198] Use 150kg of D113 macroporous weakly acidic phenyl propylene cationic resin to load the column, wash with 2mol / L hydrochloric acid solution until the pH of the eluate is 4.5; wash with 1mol / L sodium hydroxide solution until the pH of the eluate is 8.5; wash with 2mol / L hydrochloric acid solution until the pH of the eluate is 4.5; then rinse with 5 times the column volume of deionized water to complete the activation. Load the concentrated extract, then elute with 1000L 2.5mol / L ammonia water at a rate of 6BV / h. Collect the eluate when the pH of the cation column effluent is >7. When the collected liquid reaches 900L, stop collecting and directly purify the collected liquid through the anion column.

[0199] Use 62.5kg of D218 macroporous strongly alkaline acrylic anion resin to load the column, wash with 1.5mol / L sodium hydroxide solution until the pH of the eluate is 9.0; wash with 1.5mol / L hydrochloric acid solution until the pH of the eluate is 3.5; wash with 1.5mol / L sodium hydroxide solution until the pH of the eluate is 9.0; complete activation. Load the collected cationic resin eluate onto the anion resin, and collect the effluent with a pH greater than 8 until the effluent reaches 870L.

[0200] The collected liquid obtained after the anion column separation is filtered through a microfiltration membrane to remove impurities and then concentrated with a counter ion permeation membrane. The concentrated liquid has a specific gravity of 1.1 and is transferred to an alcohol precipitation tank. 15 kg of anhydrous ethanol is added at a stirring paddle of 400 rpm. After the addition of ethanol, stirring is stopped, and the alcohol precipitation is carried out for 24 hours. The supernatant is taken and concentrated under reduced pressure to obtain a mulberry branch extract. Sample content: the mass percentage of alkaloids is 63%, the mass percentage of polysaccharides is 23%, the mass percentage of flavonoids is 1%, and the mass percentage of amino acids is 5%. Among the alkaloids, the content of 1-DNJ is 61.9%.

[0201] Preparation Example 9 of Mulberry Extract

[0202] Take 1000kg of fresh mulberry branches (Yue Sang No. 11), crush them, add 4000L of water, extract them by heating reflux method for 2h, combine the extracts, filter and remove insoluble matter to obtain crude extract. The crude extract is heat concentrated to a solid content of 4%, and kept at 50℃ as the loading liquid for the cationic resin column.

[0203] Use 120kg of D113 macroporous weakly acidic phenyl propylene series cationic resin to load the column, wash with 2mol / L hydrochloric acid solution until the pH of the eluate is 4.5; wash with 1mol / L sodium hydroxide solution until the pH of the eluate is 8.5; wash with 2mol / L hydrochloric acid solution until the pH of the eluate is 4.5; and then rinse with 5 times the column volume of deionized water to complete the activation. Load the concentrated extract, then elute with 1000L 2.5mol / L ammonia water at an elution rate of 6BV / h. Collect the eluate when the pH of the cation column effluent is >7. When the collected liquid reaches 900L, stop collecting and directly purify the collected liquid through the anion column.

[0204] Use 45kg of D218 macroporous strongly alkaline acrylic anion resin to load the column, wash with 1.5mol / L sodium hydroxide solution until the pH of the eluate is 9.0; wash with 1.5mol / L hydrochloric acid solution until the pH of the eluate is 3.5; wash with 1.5mol / L sodium hydroxide solution until the pH of the eluate is 9.0; complete activation. Load the collected cationic resin eluate onto the anion resin and collect the effluent until the effluent reaches 870L.

[0205] The collected liquid obtained after separation by anion column is filtered through a microfiltration membrane to remove impurities and then concentrated by a counter ion permeation membrane. The concentrated liquid has a specific gravity of 1.1 and is transferred to an alcohol precipitation tank. 15 kg of anhydrous ethanol is added at 300 rpm. After the addition of ethanol, stirring is stopped, and the alcohol precipitation is carried out for 24 hours. The supernatant is taken and concentrated under reduced pressure to obtain an extract. Sample content: the mass percentage of alkaloids is 70%, the mass percentage of polysaccharides is 20%, the mass percentage of flavonoids is 0.6%, and the mass percentage of amino acids is 5%.

[0206] Among the alkaloids, the content of 1-DNJ is 70%, FAG is 13%, and DAB is 10%.

[0207] Preparation Example 10 of Mulberry Extract

[0208] Take 80kg of fresh mulberry branches (Guisangyou No. 62), crush them, add 1000L of water, extract them by heating reflux method for 2h, combine the extracts, filter and remove insoluble matter to obtain crude extract. The crude extract is heat concentrated to a solid content of 4%, and kept at 50℃ as the loading liquid for the cationic resin column.

[0209] Use 50kg of D113 macroporous weakly acidic phenyl propylene cationic resin to load the column, wash with 2mol / L hydrochloric acid solution until the pH of the eluate is 4.5; wash with 1mol / L sodium hydroxide solution until the pH of the eluate is 8.5; wash with 2mol / L hydrochloric acid solution until the pH of the eluate is 4.5; and then rinse with 5 times the column volume of deionized water to complete the activation. Load the concentrated extract, then elute with 600L 2.5mol / L ammonia water at an elution rate of 6BV / h. Collect the eluate when the pH of the cation column effluent is >7. When the collected liquid reaches 800L, stop collecting and directly purify the collected liquid through the anion column.

[0210] Use 65kg of D218 macroporous strongly alkaline acrylic anion resin to load the column, wash with 1.5mol / L sodium hydroxide solution until the pH of the eluate is 9.0; wash with 1.5mol / L hydrochloric acid solution until the pH of the eluate is 3.5; wash with 1.5mol / L sodium hydroxide solution until the pH of the eluate is 9.0; complete activation. Load the collected cationic resin eluate onto the anion resin and collect the effluent until the effluent reaches 750L. Reload the collected liquid onto the cationic resin, and separate it again using the cationic resin and anionic resin in turn according to the above method.

[0211] The collected liquid obtained after the two separations was filtered through a microfiltration membrane to remove impurities and then concentrated using a counter ion permeation membrane. The concentrated liquid had a specific gravity of 1.1 and was transferred to an alcohol precipitation tank. 400 g of anhydrous ethanol was added under a stirring paddle of 350 rpm. After the addition of ethanol, stirring was stopped, and the alcohol precipitation was continued for 24 hours. The supernatant was taken and concentrated under reduced pressure to obtain an extract. Sample content: the mass percentage of alkaloids was 90%, the mass percentage of polysaccharides was 3%, the mass percentage of flavonoids was 0.5%, and the mass percentage of amino acids was 2%.

[0212] Among the alkaloids, the content of 1-DNJ was 94%, FAG was 2%, and DAB was 1%.

[0213] 2. Verification of the effect of mulberry extract

[0214] Experimental Example 1

[0215] 1. Experimental Animals

[0216] Thirty 3-week-old female SD rats that were just weaned, in good health, and had shiny fur were selected and raised in SPF grade facilities.

[0217] 2. Experimental drugs

[0218] 1% CMC: Weigh 1g CMC, add it into 100mL pure water, place it on a magnetic stirrer, set the temperature at 60℃, and the speed at 500r / min, and stir until the CMC is completely dissolved and becomes transparent.

[0219] 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.

[0220] The mulberry extract SZ-A prepared in Preparation Example 1 was prepared in different concentrations and dissolved in aqueous solution.

[0221] 3. Modeling and drug administration

[0222] The hypothalamus-pituitary-thyroid axis and the hypothalamus-pituitary-ovarian axis are bidirectional circuits. Any abnormality in any axis will affect the normal function of the other axis. When excessive exogenous androgens or certain inducers are given to inhibit androgen metabolism, resulting in excessive androgen levels in the body, the hypothalamus-pituitary-ovarian axis will be destroyed, leading to hyperandrogenemia or PCOS, etc. It will further affect 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 experience hyperthyroidism or hypothyroidism, as well as thyroid inflammation or nodules and other symptoms.

[0223] Thirty 3-week-old female SD rats in good health and shiny fur were selected and adaptively fed for 3 days, and then randomly divided into a blank group, a model group, and a drug group. The blank group rats were fed with a basic diet (chow); the model group and the drug group were given HFD (high-fat diet research diets D12492) for 3 weeks, and then LZ (1 mg / kg / d) was given by gavage for 6 weeks, and HFD (high-fat diet research diets D12492) was given at the same time to construct a rat model. After the model evaluation was successful (compared with the Blank group, the serum thyroid stimulating hormone (TSH) in the Model group was significantly decreased, and free triiodothyronine (FT3) and free thyroxine (FT4) were significantly increased), the drug group was administered according to the dose and method in Table 1. During the drug administration period, the blank group and the model group were given an equal amount of normal saline. After 21 days of treatment, the samples were collected for efficacy evaluation.

[0224] Table 1

[0225]

[0226] 4. Observation indicators and detection methods

[0227] (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.

[0228] Serum FT3, FT4 and TSH were detected by Elisa. The kits were purchased from: 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 thyroid gonadotropin (TSH) ELISA kit, manufacturer: Elabscience, catalog number: E-EL-0079C.

[0229] (2) 21 days after administration, the rats were killed and thyroid tissues were obtained for HE staining.

[0230] 5. Experimental results

[0231] from Figure 1 It can be seen that compared with the Blank group, the serum thyroid stimulating hormone (TSH) in the Model group decreased significantly, and free triiodothyronine (FT3) and free thyroxine (FT4) increased significantly, indicating hyperthyroidism in the Model group. After treatment with SZ-A, the low, medium, and high dose groups of SZ-A could significantly increase TSH expression and significantly reduce FT3 and FT4 expression. And the improvement effect of SZ-A on TSH, FT3, and FT4 was dose-dependent.

[0232] The results of HE staining of thyroid tissue are shown in Figure 2 The tissue structure of the thyroid gland lobe in 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 follicle cavity was filled with dark-stained transparent colloid. The thyroid tissue structure in the Model group was abnormal, the thickness of the follicular epithelial cells was significantly thinner than that in the Blank group, the follicular cavity was enlarged, and the follicles had obvious fusion.

[0233] The size and structure of thyroid follicles in the SZ-AL group were significantly improved, and the thickness of follicular epithelial cells was thicker than that in the model group, with less fusion. The structure of thyroid tissue in the SZ-AM and SZ-AH dose groups was similar to that in the blank group. The follicles were of different sizes and were surrounded by a single layer of cubic epithelial cells. Deeply stained transparent colloid was visible in the follicle cavity. This suggests that SZ-A has a good improvement effect on thyroid tissue lesions.

[0234] Experimental Example 2

[0235] 1. Experimental drugs

[0236] DHEA (dehydroepiandrosterone) oil solution: 30 mg / mL, 30 mg DHEA is dissolved in 1 mL castor oil, placed on a magnetic stirrer, 50°C, 500 r / min, and stirred until DHEA is completely dissolved.

[0237] The mulberry extract SZ-A prepared in Preparation Example 1 was prepared in different concentrations and dissolved in aqueous solution.

[0238] 2. Experimental animals are the same as those in Experimental Example 1.

[0239] 3. Modeling and drug administration

[0240] The hypothalamus-pituitary-thyroid axis and the hypothalamus-pituitary-ovarian axis are bidirectional circuits. Any abnormality in any axis will affect the normal function of the other axis. When excessive exogenous androgens or certain inducers are given to inhibit androgen metabolism, resulting in excessive androgen levels in the body, the hypothalamus-pituitary-ovarian axis will be destroyed, leading to hyperandrogenemia or PCOS, etc. It will further affect 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 experience hyperthyroidism or hypothyroidism, as well as thyroid inflammation or nodules and other symptoms.

[0241] Table 2

[0242]

[0243]

[0244] Female SD rats that were just weaned at 21 days old, in good health and with shiny fur were selected. After 3 days of adaptive feeding, they were randomly divided into blank group, model group, SZ-AL group, SZ-AM group and SZ-AH group, with 6 rats in each group. Rats in the blank group were given a basic diet and subcutaneously injected with solvent castor oil (60 mg / kg / d). Rats in the model group and the drug-treated group were given a HFD (high-fat diet (research diets D12492)) and subcutaneously injected with DHEA oil solution (60 mg / kg / d). The model was induced continuously for 7 weeks to establish the rat model.

[0245] After the model evaluation was successful (compared with the Blank group, the serum thyroid stimulating hormone (TSH) in the Model group increased significantly, and free triiodothyronine (FT3) and free thyroxine (FT4) decreased significantly), the blank group rats continued to be fed with basal feed without treatment; the model and drug-treated groups continued to be fed with high-fat feed, and at the same time, the model group was orally gavaged with normal saline, the SZ-AL group was orally gavaged with SZ-A (67 mg / kg / d), the SZ-AM group was orally gavaged with SZ-A (100 mg / kg / d), and the SZ-AH group was orally gavaged with SZ-A (150 mg / kg / d). After 21 days of continuous treatment, the drug efficacy test was performed, and the specific detection and analysis methods were the same as those in Experimental Example 1.

[0246] 4. Experimental results

[0247] Results Figure 3 Compared with the Blank group, the serum thyroid stimulating hormone (TSH) in the Model group increased significantly, and free triiodothyronine (FT3) and free thyroxine (FT4) decreased significantly, indicating hypothyroidism in the Model group. After treatment with SZ-A, the low, medium, and high dose groups of SZ-A could significantly reduce TSH expression, and significantly increase FT3 and FT4 expression. And the improvement effect of SZ-A on TSH, FT3, and FT4 was dose-dependent.

[0248] The results of HE staining of thyroid tissue are shown in Figure 4 . The results of HE staining of thyroid tissue showed that the thyroid tissue structure of the Blank group was clear, with follicles of varying sizes, surrounded by a single layer of upright epithelial cells, and the follicle cavity was filled with dark-stained transparent colloid. The thyroid tissue alveoli of the Model group showed diffuse nodular hyperplasia, most of which were small cell follicles, with no or little transparent colloid in the follicle cavity, and some lymphocyte infiltration, indicating hypothyroidism with mild inflammation.

[0249] In the SZ-AL group, the proliferation of thyroid acinar nodules was reduced, some of them were small cell follicles, some were medium-sized follicles, the epithelial cells were single-layered, secretion was observed in the follicle cavity, and there was no lymphocyte infiltration, which indicated that SZ-AL had a certain improvement on thyroid tissue lesions.

[0250] The proliferation of acinar nodules in the thyroid tissue of the SZ-AM group was significantly reduced, and the size of the follicles was similar to that of the Blank group. Light red transparent colloid was seen in the follicle cavity, and there was no lymphocyte infiltration.

[0251] There were almost no acinar nodules in the thyroid tissue of the SZ-AH group, and irregular cell follicles of varying sizes were seen in the glands. A large amount of dark-stained transparent colloid was seen in the follicle cavity, which was similar to the thyroid tissue structure of the blank group, and there was no lymphocyte infiltration. This suggests that SZ-A has a certain dose-related effect on the improvement of thyroid tissue.

[0252] Experimental Example 3

[0253] 1. Experimental drugs

[0254] DHEA (dehydroepiandrosterone) oil solution: 30 mg / mL, 30 mg DHEA is dissolved in 1 mL castor oil, placed on a magnetic stirrer, 50°C, 500 r / min, and stirred until DHEA is completely dissolved.

[0255] The mulberry extract SZ-A prepared in Preparation Example 1 was prepared in different concentrations and dissolved in aqueous solution.

[0256] 2. Experimental Animals

[0257] Twenty-four 3-week-old female SD rats, which were just weaned, in good health and had shiny fur, were selected and raised in SPF conditions.

[0258] 3. Modeling and drug administration

[0259] Table 3

[0260]

[0261] Female SD rats that were just weaned at 21 days old, in good health and with shiny fur were selected. After 3 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 6 rats in each group. The blank group: rats were given a basic diet and subcutaneously injected with a solvent castor oil (60 mg / kg / d). The model group and the drug-treated group were given a HFD (high-fat diet (researchdiets D12492)) and subcutaneously injected with DHEA oil solution (60 mg / kg / d). The model was induced continuously for 7 weeks to establish the rat model.

[0262] After the model evaluation was successful (TPO-Ab and TG-Ab antibodies increased significantly compared with the Blank group), the blank group rats continued to be fed with basal feed without treatment; the model and drug-treated groups continued to be fed with high-fat feed, and the model group was orally gavaged with normal saline, the SZ-AL group was orally gavaged with SZ-A (67 mg / kg / d), and the SZ-AH group was orally gavaged with SZ-A (150 mg / kg / d). After 21 days of continuous treatment, the samples were collected for drug efficacy testing.

[0263] 4. Experimental results

[0264] Serum was collected and tested for TPO-Ab and TG-Ab antibody levels using Elisa. Figure 5 Compared with the Blank group, the TPO-Ab and TG-Ab in the Model group increased significantly, indicating that the thyroid-specific autoantibodies in the Model group increased. After SZ-A treatment, both the low-dose and high-dose SZ-A groups could reduce the expression of TPO-Ab and TG-Ab.

[0265] The results of HE staining of thyroid tissue are shown in Figure 6 . The results of HE staining of thyroid tissue showed that the thyroid tissue structure of the Blank group was clear, the follicles were of different sizes, surrounded by a single layer of upright epithelial cells, and the follicle cavity was filled with dark-stained transparent colloid. In the Model group, the thyroid tissue acinar proliferation was severe, most of which were smaller 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 seen in the follicle 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 different sizes and irregular shapes were seen. A large amount of dark-stained transparent colloid was seen in the follicle cavity, which was similar to the thyroid tissue structure of the blank group, without lymphocyte infiltration, indicating that SZ-AH had a good improvement effect on thyroid tissue.

[0266] The above research results suggest that SZ-A has a good therapeutic effect on Hashimoto's thyroiditis.

[0267] Experimental Example 4: Pharmacodynamics of Mulberry Extract in the Treatment of Hashimoto's Thyroiditis

[0268] 1. Animal Experimental Protocol

[0269] 1.1 Experimental animals

[0270] Lewis rats, 5-6 weeks old, weighing 120-140 g, were fed for 3 days after entering the animal room for 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.

[0271] 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, based on alkaloids, low, medium, high, dosages 67 mg / ml, 100 mg / ml, 150 mg / ml).

[0272] 1.2 Experimental drugs

[0273] Sodium iodide

[0274] Porcine thyroglobulin

[0275] Freund's complete adjuvant

[0276] Freund's incomplete adjuvant

[0277] Levothyroxine Sodium

[0278] Mulberry extract in Preparation Example 8

[0279] 1.3 Modeling and drug administration

[0280] On the day of grouping, except for the normal control group, the HT model was established in all groups. On the first day of the first week, 0.2 mL of porcine thyroglobulin (4 mg / mL) emulsified with Freund's complete adjuvant was injected into the foot pad of the rats twice a week for primary immunization, and the same dose of porcine thyroglobulin emulsified with incomplete Freund's adjuvant was injected once in the second and fourth weeks for booster immunization. The success of the model was determined by the changes in TPO-Ab and TG-Ab levels. During the modeling period, except for the rats in the normal control group who drank double distilled water, the rats in other groups drank 0.05% sodium iodide solution until the end of the modeling.

[0281] 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.

[0282] 1.4 Sampling and testing

[0283] 24 hours after the last treatment, 3 mL of blood was collected from each group of rats, and serum was obtained by centrifugation for the detection of FT3, FT4, TSH, TPO-Ab, and TG-Ab levels. After blood collection, the rats were anesthetized and killed, and the rat thyroid tissues were collected and divided into two parts, each containing thyroid tissues of 10 rats in each group. One part was fixed in 4% paraformaldehyde for HE staining, and the other part was frozen in a -80°C refrigerator for Western blotting experiments.

[0284] ELISA method was used to detect the levels of TPO-Ab and TG-Ab in rat serum;

[0285] HE staining was used to detect the pathological changes of thyroid tissue in rats;

[0286] The experimental results showed that SZ-A could reduce diffuse hyperplasia of thyroid tissue in rats and improve serum TPO-Ab and TG-Ab levels.

[0287] II. Cell Experiment Protocol

[0288] 2.1 Experimental cells

[0289] Thyroid follicular epithelial cells (Nthy-ori3-1)

[0290] 2.2 Experimental drugs

[0291] Sodium iodide

[0292] Interferon gamma

[0293] Fetal bovine serum

[0294] RPMI 1640 medium

[0295] Preparation of the mulberry branch extract in Example 1-8

[0296] 2.3 Cell culture and modeling

[0297] 2.3.1 Nthy-ori3-1 cell culture

[0298] 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 at 37°C and 5% CO. 2 Cultured in a constant temperature cell culture incubator.

[0299] 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 treat for about 1 minute. Observe under an inverted optical microscope. When the Nthy-ori3-1 cells slowly begin to become 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 gun 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 gun 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 the same number of cells / well and 2 mL of culture medium was added into the 6-well plate.

[0300] 2.3.2 Thyroid inflammation cell model

[0301] 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 continuing to 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.

[0302] Detection indicators: ROS detection, inflammatory factor detection: TNF-α, IL-1β, IL-6.

[0303] 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.

[0304] 3. Human Experiments

[0305] 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.

[0306] Clinical cases and treatment:

[0307] (1) Case 1: A 50-year-old Chinese female, 65 kg, 160 cm tall, had Hashimoto's thyroiditis for 6 years. She took the tablets containing the mulberry extract of Preparation Example 1 orally, three times a day, two tablets each time. After 16 weeks of oral administration, 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 stabilized in the normal range, and the anti-thyroglobulin antibody decreased by 43.5% (see Figure 7 ), thyroid pain felt significantly relieved.

[0308] (2) Case 2: A 50-year-old Chinese female, 71 kg, 158 cm tall, had Hashimoto's thyroiditis for 4 years. She took the tablets containing the mulberry extract of Preparation Example 1 orally, two tablets 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 ).

[0309] (3) Case 3: A 35-year-old Chinese female, 60 kg, 162 cm tall, had Hashimoto's thyroiditis for 3 years. She took the tablets containing the mulberry extract from Preparation Example 1 orally, 3 times a day, 2 tablets each time, for 8 weeks. Her anti-thyroglobulin antibody decreased by 18.4%, and her anti-thyroid peroxidase antibody was originally normal and stabilized within the normal range after taking the medicine (see Figure 7 ).

[0310] (4) Case 4: A 52-year-old Chinese female, 62 kg, 163 cm tall, had Hashimoto's thyroiditis for 7 years. She took the tablet containing the mulberry extract of Preparation Example 1 orally, two tablets 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 ).

[0311] The above research results suggest that mulberry branch extract has a good therapeutic effect on Hashimoto's thyroiditis.

[0312] IV. Cell Experiment Protocol

[0313] 4.1 Experimental cells

[0314] Thyroid follicular epithelial cells

[0315] 4.2 Experimental drugs

[0316] Interferon gamma

[0317] Fetal bovine serum

[0318] RPMI 1640 medium

[0319] The mulberry branch extracts of Preparation Examples 2-4, Preparation Example 6, Preparation Example 8 and Preparation Example 10.

[0320] 4.3 Cell culture and modeling

[0321] 4.3.1 Nthy-ori-3.1 cell culture

[0322] 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 at 37°C and 5% CO. 2 Cultured in a constant temperature cell culture incubator.

[0323] 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 treat for about 1 minute. Observe under an inverted optical microscope. When the Nthy-ori3-1 cells slowly begin to become 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 gun 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 gun to draw 10μL to fill the pool in the cell counting plate, use a microscope to observe whether the pool is filled evenly and count, and use 1×10 4 The cells were added into a 48-well plate and 250ul of culture medium was added into the 48-well plate.

[0324] 4.3.2 Thyroid inflammation cell model

[0325] After the thyroid follicular epithelial cells (Nthy-ori3-1) enter the logarithmic growth phase, the cells in the control group are cultured in normal culture medium, and the cells in the model group are cultured in culture medium containing 150ng / mL IFN-γ. At the same time, the drug administration group is given mulberry branch extracts of different preparation examples (mulberry branch extracts of Preparation Examples 2-4, Preparation Example 6, Preparation Example 8 and Preparation Example 10), the concentration of mulberry branch extracts is 150ug / ml, and cultured for 48h. Collect the supernatant, centrifuge at 4°C, 1000×g for 20min, remove impurities and cell debris, and take the supernatant for testing.

[0326] According to the instructions of the ELISA test kits for TNF-α, FT4 and IL-6, after processing the supernatant samples after centrifugation, TNF-α and FT4 were detected with the original solution, and IL-6 was detected after the original solution was diluted 40 times. The absorbance (OD) of each well was detected with an ELISA reader at a wavelength of 450nm, and the content of TNF-α, FT4 and IL-6 in the supernatant of each group was calculated based on the OD value. 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; Human interleukin 6 (IL-6) ELISA kit manufacturer: elabscience catalog number: E-EL-H6156.

[0327] Results Figure 8 , Fig. 9 and Fig.10 .

[0328] 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 to varying degrees and increase FT4 in the supernatant. The mulberry branch extracts in Preparation Examples 2 and 4 could reduce IL-6 in the cell supernatant to varying degrees. The above cell level experimental results proved that: (1) the mulberry branch extract could relieve Hashimoto's thyroiditis; (2) the mulberry branch extract could relieve hypothyroidism.

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 and decreased free thyroxine level; 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: reducing serum TPO-Ab, reducing 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 mulberry branch extract, mulberry bark extract and / or mulberry leaf extract.

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

10. The use according to any one of claims 1 to 9, 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.

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