Use of a brucea javanica oil in the manufacture of a medicament

Abstract

The application provides brucea javanica oil, a pharmaceutical composition containing active components of the brucea javanica oil, and a use of the pharmaceutical composition containing the brucea javanica oil in preparation of a medicine for preventing and / or treating abnormal thyroid function diseases. The medicine can regulate serum thyroid stimulating hormone level, regulate serum free triiodothyronine level, regulate serum free thyroxine level, inhibit thyroid follicle deformation and / or atrophy, promote thyroid follicle colloid secretion, inhibit interstitial fibrosis, and reduce thyroid tissue red blood cell extravasation, and has high clinical application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to the use of Brucea javanica oil in the preparation of medicines. Background Technology

[0002] The thyroid gland is a vital endocrine gland in the human body. Through the follicular epithelial cells, it produces, secretes, and stores thyroid hormones (TH), which act on the body to regulate metabolism and affect almost all oxygen-consuming tissues. Hypothyroidism is a clinical syndrome caused by insufficient synthesis, secretion, or biological effects of thyroid hormones due to various reasons. Based on whether it is caused by autoimmune disorders, hypothyroidism can be divided into autoimmune and non-autoimmune thyroid diseases. Autoimmune diseases include toxic diffuse goiter and chronic lymphocytic thyroiditis (Hashimoto's thyroiditis), both caused by autoimmune dysfunction. Non-autoimmune thyroid diseases include drug-induced hypothyroidism, post-radiotherapy hypothyroidism, and... 131 Hypothyroidism can occur after I-1 treatment, thyroid surgery, pituitary or hypothalamic tumor surgery, or is congenital. Clinically, different treatment methods are used for different types and pathogenesis of hypothyroidism.

[0003] Brucea Javanica (L.) Merr, a plant in the Simaroubaceae family, is a mature fruit of the plant. Also known as Kushenzi, it is extremely bitter and cold in nature, and poisonous. Its functions include clearing heat and detoxifying, corroding warts, softening and dispersing nodules, and stopping dysentery and malaria. The marketed drug, Brucea Javanica oil emulsion, is a sterile emulsion refined from the fat-soluble components of Brucea Javanica, with Brucea Javanica oil and soybean lecithin as its main components. Clinically, it is mainly used as an adjunct therapy for lung cancer, lung cancer brain metastases, gastrointestinal tumors, and liver cancer; it has not been used for the treatment of non-autoimmune thyroid diseases. Clinically, the main treatment for non-autoimmune thyroid diseases is thyroid hormone replacement therapy, but this generally requires lifelong medication, has strong dependence, and significant adverse reactions.

[0004] Therefore, searching for substances from known natural products or marketed drugs that can effectively prevent and / or treat thyroid dysfunction has important clinical application value. Summary of the Invention

[0005] The present invention aims to at least partially solve at least one of the technical problems existing in the prior art. To this end, the present invention provides the use of Brucea javanica oil, pharmaceutical compositions containing Brucea javanica oil as an active ingredient, pharmaceutical preparations containing Brucea javanica oil, and the use of these pharmaceutical preparations in the preparation of a medicament for the prevention and / or treatment of thyroid dysfunction.

[0006] This invention is based on the inventor's discoveries and understanding of the following problems:

[0007] Brucea javanica has the functions of clearing heat and detoxifying, eliminating masses and dissipating nodules. Thyroid dysfunction is accompanied by thyroid nodules. Based on long-term clinical experience, the inventor boldly speculated that Brucea javanica may have the effect of improving thyroid dysfunction.

[0008] Thyroid hormones (TH) include thyroxine (T4) and triiodothyronine (T3), most of which are bound to plasma proteins, with only their free forms possessing biological activity: free triiodothyronine (FT3) and free thyroxine (FT4). TH secretion is regulated by the hypothalamic-pituitary-thyroid axis. The hypothalamus produces thyrotropin-releasing hormone (TSH) which acts on the pituitary gland. The pituitary gland then releases thyroid-stimulating hormone (TSH) which acts on the thyroid gland, stimulating the thyroid to release TH, which then acts systemically. Similarly, TH can also negatively feedback into the pituitary gland, inhibiting TSH secretion. Clinically, TSH, FT3, and FT4 are often used to evaluate thyroid function. Therefore, the inventors constructed a propylthiouracil-induced rat hypothyroidism model using the antithyroid drug propylthiouracil. This is a non-autoimmune hypothyroidism model used to study the preventive or therapeutic effects of Brucea javanica oil on thyroid dysfunction.

[0009] Animal experiments showed that, compared with the disease model group, the Brucea javanica oil treatment group effectively regulated serum thyroid-stimulating hormone (TSH), serum free triiodothyronine (TTH), and serum free thyroxine (STH) levels in experimental animals, alleviated thyroid follicle deformation and / or atrophy, promoted thyroid follicle colloid secretion, inhibited interstitial fibrosis, and reduced erythrocyte exudation in thyroid tissue.

[0010] Therefore, the present invention proposes the use of Brucea javanica oil, pharmaceutical compositions containing Brucea javanica oil active ingredients, and pharmaceutical preparations containing Brucea javanica oil in the preparation of pharmaceuticals for the prevention and / or treatment of thyroid dysfunction.

[0011] According to an embodiment of the present invention, the thyroid dysfunction is a non-autoimmune thyroid dysfunction.

[0012] According to embodiments of the present invention, the above-mentioned uses may further include at least one of the following additional technical features:

[0013] According to embodiments of the present invention, the drug has at least one of the following uses:

[0014] 1) Regulate serum thyroid-stimulating hormone levels;

[0015] 2) Regulate serum free triiodothyronine levels;

[0016] 3) Regulate serum free thyroxine levels;

[0017] 4) Inhibits thyroid follicle deformation and / or atrophy;

[0018] 5) Promotes the secretion of thyroid follicular colloid;

[0019] 6) Inhibits the proliferation of interstitial fibrosis;

[0020] 7) Reduce the exudation of red blood cells from thyroid tissue.

[0021] According to an embodiment of the present invention, the non-autoimmune thyroid dysfunction is hypothyroidism.

[0022] According to an embodiment of the present invention, the hypothyroidism is caused by non-autoimmune thyroiditis.

[0023] According to embodiments of the present invention, the drug has at least one of the following uses:

[0024] 1) Lower serum thyroid-stimulating hormone levels;

[0025] 2) Increase serum free triiodothyronine levels;

[0026] 3) Increase serum free thyroxine levels;

[0027] 4) Inhibits thyroid follicle deformation and / or atrophy;

[0028] 5) Promotes the secretion of thyroid follicular colloid;

[0029] 6) Inhibits the proliferation of interstitial fibrosis;

[0030] 7) Reduce the exudation of red blood cells from thyroid tissue.

[0031] In some specific embodiments of the invention, the *Brucea javanica* oil or the pharmaceutical composition is provided in the form of an oral formulation, a spray formulation, or an injectable formulation. The *Brucea javanica* oil or pharmaceutical composition can be formulated into various dosage forms in combination with a pharmaceutically acceptable carrier. For example, for oral administration, the *Brucea javanica* oil or pharmaceutical composition can be prepared as a suspension, solution, emulsion, syrup, or elixir of hydrated oil, or as a dry product to be replenished with water or other suitable medium before use. Such liquid formulations may contain conventional additives such as suspending agents, sorbitol, cellulose methyl ether, glucose syrup, gels, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel, hydrogenated edible oils, emulsifiers such as lecithin, sorbitan monooleate, gum arabic; or non-aqueous carriers (possibly containing edible oils such as almond oil), oils such as glycerin, ethylene glycol, or ethanol; preservatives such as methylparaben or propylparaben, sorbic acid. Flavoring agents or coloring agents may be added if desired. For injectable formulations, Brucea javanica oil or pharmaceutical compositions can be prepared, for example, into ampoules for a single dose or into unit dosage forms such as multi-dose containers. Brucea javanica oil or pharmaceutical compositions can also be prepared into solutions or suspensions.

[0032] In some specific embodiments of the present invention, the pharmaceutical composition further comprises at least one of propylthiouracil and levothyroxine sodium. According to embodiments of the present invention, the pharmaceutical composition further comprises other drugs that can treat or prevent non-autoimmune thyroid dysfunction, particularly non-autoimmune hypothyroidism.

[0033] According to an embodiment of the present invention, the pharmaceutical composition further comprises: an emulsifier.

[0034] According to an embodiment of the present invention, the emulsifier contains phospholipids.

[0035] According to embodiments of the present invention, the emulsifier contains at least one of lecithin, egg yolk lecithin, and soybean lecithin. Using the above-mentioned emulsifier can improve the surface tension between the various constituent phases in the Brucea javanica oil emulsion, thereby forming a uniform and stable dispersion system or emulsion, which is beneficial for maintaining the stability and homogeneity of Brucea javanica oil.

[0036] In some specific embodiments of the invention, the pharmaceutical composition may further include a pharmaceutically acceptable carrier, and the dosage form and route of administration of the pharmaceutical composition are not particularly limited. For oral administration, the pharmaceutically acceptable carrier may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, and fragrances. For injectable formulations, the pharmaceutically acceptable carrier may include buffers, preservatives, analgesics, solubilizers, isotonic agents, and stabilizers. For topical formulations, the pharmaceutically acceptable carrier may include bases, excipients, lubricants, and preservatives. Excipients and diluents in carriers suitable for pharmaceutical formulations may include: lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, rubber arabic, alginate, gel, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil. The pharmaceutical composition may also include fillers, anticoagulants, lubricants, humectants, fragrances, and preservatives.

[0037] According to embodiments of the present invention, the pharmaceutical composition is at least one of tablets, granules, powders, capsules, solutions, suspensions, and lyophilized formulations. Different dosage forms provide a variety of choices for clinical medication, facilitating patient medication in various scenarios.

[0038] In some specific embodiments of the present invention, the pharmaceutical preparation is in single-dose form. Single-dose formulations offer a simple administration method, eliminating the need for repeated daily administration, making them convenient for patients to take, thereby maximizing efficacy, and are also easy to carry and store.

[0039] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0040] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0041] Figure 1 The image shows the results of staining rat thyroid tissue using the hematoxylin-eosin staining method according to Example 1 of the present invention, where (A) is the blank group; (B) is the model group; and (C) is the positive group.

[0042] Figure 2 The image shows the results of staining rat thyroid tissue using the hematoxylin-eosin staining method according to Example 1 of the present invention. In the image, (A) represents the high-dose group; (B) represents the medium-dose group; and (C) represents the low-dose group. Detailed Implementation

[0043] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0044] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0045] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0046] In this document, the terms “comprising” or “including” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0047] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0048] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0049] In this document, the terms “treatment” and “relief” refer to the attainment of a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of disease or its symptoms, and / or therapeutic in terms of partial or complete cure of disease and / or adverse effects caused by disease. As used herein, “treatment” encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of disease in susceptible individuals who have not yet been diagnosed with the disease; (b) suppression of disease, such as inhibiting disease progression; or (c) relief of disease, such as reducing disease-related symptoms. As used herein, “treatment” encompasses any administration of a single-dose formulation, pharmaceutical composition, health product, food, feed, or medicament to an individual to treat, cure, relieve, improve, reduce, or suppress an individual’s disease, including but not limited to administering to an individual in need a strain, fermentation broth, bacterial suspension, or composition described herein.

[0050] In this article, the term "prevention" is used to refer to the attempt to stop the onset of a disease or symptom before it occurs.

[0051] In this article, the term "single-dose formulation" refers to a dosage form in which a single dose of a formulation is administered, and the dose is pharmaceutically acceptable and has potential therapeutic efficacy. "Single dose" refers to the dose taken by the subject in a single administration.

[0052] In this article, the term "drug combination" refers to two or more drugs existing alone or in combination, which can be administered to the subject simultaneously or separately as individual entities, without specific time restrictions. Such administration can provide two or more effective components in the body.

[0053] In this document, the term "pharmaceutical acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components of the formulation and / or the mammals to which it is treated. Preferably, "pharmaceutical acceptable" as used herein means approved by a federal regulatory agency or national government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, particularly in humans.

[0054] In this document, the term "pharmaceuticalally acceptable excipient" may include any solvent suitable for the specific target dosage form. The use of any conventional excipients that are incompatible with Brucea javanica oil, such as any adverse biological effects or harmful interactions with any other component of the pharmaceutically acceptable composition, is also within the scope of this disclosure.

[0055] In this article, the term "propylthiouracil" is equivalent to "PTU" or "Propylthiouracil," and depending on the context, it can refer to propylthiouracil solution. Propylthiouracil is an antithyroid drug commonly used to induce animal models of hypothyroidism. Specifically, PTU mimics the pathological state of hypothyroidism by inhibiting the activity of thyroid peroxidase, thereby blocking the synthesis of thyroid hormones.

[0056] In this article, "FT3" is equivalent to "free triiodothyronine," a form of thyroid hormone that primarily circulates in the blood. Clinically, decreased FT3 levels may indicate hypothyroidism.

[0057] In this article, "FT4" is equivalent to "free thyroxine," which is the unbound thyroxine (T4) in the blood and one of the active forms of thyroid hormones. Clinically, FT4 is an important indicator for diagnosing hyperthyroidism and hypothyroidism. Because FT4 levels are not affected by the concentration of thyroxine-binding globulin in the blood, it can specifically reflect the functional status of the thyroid gland. FT4 is usually tested together with FT3 and TSH to comprehensively assess thyroid function.

[0058] In this article, the term "TSH" is equivalent to "serum thyroid-stimulating hormone." TSH is a hormone secreted by the anterior pituitary gland, and its main functions are to regulate the proliferation of thyroid cells, thyroid blood supply, and the synthesis and secretion of thyroid hormones. Clinically, measuring serum TSH is one of the important indicators for diagnosing and treating hyperthyroidism and hypothyroidism. Elevated serum TSH levels are commonly seen in primary hypothyroidism, TSH-secreting tumors, and other conditions.

[0059] In this article, the term "levothyroxine sodium (Euthyrox)" refers to a synthetic thyroid hormone commonly used to treat hypothyroidism and other related conditions.

[0060] In this article, the term "hypothyroidism" is used interchangeably with "underactive thyroid," a common clinical endocrine disorder characterized by reduced synthesis and secretion of thyroid hormones, leading to a slowdown in overall metabolism. The etiology and pathogenesis of hypothyroidism are complex.

[0061] In this article, the term "thyroid follicle" is equivalent to "thyroid follicular cavity," which is the basic structural unit of the thyroid gland.

[0062] In this invention, by way of example, a pharmaceutical composition containing the active ingredient of Brucea javanica oil can be obtained by the following method: a) 700g to 900g of Brucea javanica is pulverized and soaked in 100kg to 300kg of petroleum ether at a temperature of 20°C to 30°C for 20 to 30 hours to obtain a first soaking solution; b) the first soaking solution is distilled at a temperature of 50°C to 70°C and condensed using a condenser to obtain a first condensate; c) Brucea javanica is soaked in the first condensate for 20 to 40 minutes to obtain a second soaking solution; d) Brucea javanica is repeatedly soaked in the soaking solution and the soaking solution is distilled and condensed. e) To obtain a fourth soaking solution, wherein each soaking time is 20 min to 40 min; f) To heat the fourth soaking solution to 70℃ to 90℃ to recover petroleum ether and obtain a first residual liquid; g) To recover the first residual liquid by vacuum distillation at 0.01 MPa and 80℃ to further recover petroleum ether and obtain crude Brucea javanica oil; g) To add 40 g to 50 g of activated carbon activated at 100℃ to 120℃ for 1 to 3 hours to crude Brucea javanica oil, and to adsorb and decolorize at 100℃ to 120℃ for 10 min to 30 min, and then filter to obtain Brucea javanica oil.

[0063] In this invention, by way of example, the Brucea javanica oil emulsion injection can be obtained by the following method: Take 15g of soybean lecithin, mix it with 25mL of warm glycerin and an appropriate amount of water for injection, transfer it into a high-speed tissue homogenizer (8000 rpm), stir twice, the first time for 5 minutes and the second time for 2 minutes, so that it is evenly dispersed, add 100mL of warm Brucea javanica oil (Brucea javanica oil obtained after crude extraction and removal of pigments, impurities and other components by adsorption, filtration and other methods), stir 3 times, 2 minutes each time, to form a primary emulsion, add water for injection to 1000mL, then transfer it into a high-pressure homogenizer (40MPa) for homogenization three times, filter, take the filtrate, fill and sterilize, and the product is obtained.

[0064] In this invention, by way of example, the oral emulsion of Brucea javanica oil can be obtained by the following method: 15g of soybean lecithin is mixed with water and transferred to a high-speed tissue homogenizer (8000 rpm), stirred three times for 3 minutes each time to disperse it evenly, 100mL of warm Brucea javanica oil is added and stirred for 9 minutes to form a pre-emulsion, and water is added to make 1000mL, then transferred to a high-pressure homogenizer and homogenized 1-2 times to make it uniform, then filled, sterilized and packaged to obtain the product.

[0065] For example, a pharmaceutical preparation containing Brucea javanica oil can be obtained by the following method: each pharmaceutical preparation contains at least 10 mL of the aforementioned pharmaceutical composition, the pharmaceutical composition including Brucea javanica oil. The pharmaceutical preparation is in single-dose form. Each pharmaceutical preparation contains 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL, 50 mL, 55 mL, or 60 mL of the pharmaceutical composition. The single-dose form of the pharmaceutical preparation is convenient to take and easy to store. The single-dose form of the pharmaceutical preparation has a simple administration method, does not require repeated daily administration, is convenient for patients to take, thereby achieving maximum efficacy, and is easy to carry and store.

[0066] In this embodiment of the invention, a rat hypothyroidism model was constructed and evaluated in the following manner:

[0067] Establishment of a rat hypothyroidism model: A rat hypothyroidism model was established by administering propylthiouracil to rats via gavage. The specific dosage was: 0.1% propylthiouracil solution, 10 mL / kg, administered by gavage once daily for 4 weeks.

[0068] Model evaluation: The success of model construction was assessed by detecting serum FT3, FT4 and TSH levels in the model group and blank (control) group, as well as the body weight and wet weight of the thyroid gland, and by performing HE staining on the thyroid tissue.

[0069] The evaluation results of the hypothyroidism model in this invention showed that: 0.1% propylthiouracil solution was administered to rats by gavage at a rate of 10 mL / kg once a day for 4 weeks. (1) The experimental animals lost weight. Compared with the blank (control) group, the model group showed (2) a significant decrease in the wet weight of the rat thyroid gland, (3) a significant decrease in serum FT3 and FT4 levels, a significant increase in serum TSH level, and (4) a significant decrease in the number of thyroid follicles, deformation, significant atrophy, irregular size and shape with significant differences, reduced cavity size, enlarged and swollen epithelial cells, reduced follicular colloid, significant proliferation of interstitial fibers, and a significant increase in blood cell exudation. The above results indicate that the rat hypothyroidism model was successfully constructed.

[0070] In this embodiment of the invention, the model group rats were considered to have successfully established the hypothyroidism model by exhibiting weight loss, yellowish fur, and sluggish reactions. To prevent the model group rats from recovering on their own, PTU solution was administered by gavage after successful modeling to maintain the hypothyroid state, with the frequency changed from once / day to once every other day. In the specific experiment, the body weight (Table 1), serum FT3, FT4, and TSH levels (Tables 2 and 3) of the model group rats (with a dose of 0 of the oral emulsion of *Brucea javanica*) were further examined, as well as the physiological state of the thyroid tissue. Figure 1 The study aimed to assess the success of establishing a rat hypothyroidism model and the effectiveness of the drugs in the experimental group.

[0071] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0072] Example 1

[0073] 1. Experimental Design

[0074] ① Route of administration: Gavage administration.

[0075] ② Dosage:

[0076] Three groups were established for administering oral emulsion of *Brucea javanica*: a high-dose group, a medium-dose group, and a low-dose group. The clinical human daily dose of the oral emulsion (Shenyang Pharmaceutical University Lei Yunshang Pharmaceutical Co., Ltd., Z21021715) is 60 ml, which translates to a rat dose of 60 / 60 × 7 = 7 ml / kg. Therefore, the dosages for each group are as follows:

[0077] High-dose group: The high-dose administration group of Brucea javanica oral emulsion was administered to SD rats after successful modeling, with a dosage of 14 ml / kg.

[0078] Medium-dose group: The medium-dose group of oral administration of Brucea javanica emulsion was administered to SD rats at a dose of 7 ml / kg after successful modeling.

[0079] Low-dose group: Low-dose administration of Brucea javanica oral emulsion to SD rats after successful modeling, the dosage of the drug to rats was 3.5 ml / kg.

[0080] Positive control group: Levothyroxine sodium (Euthyrox) positive control group. After successful modeling in SD rats, the rats were given a dose of 10 μg / kg.

[0081] Blank group: The blank control group received no medication.

[0082] Model group: SD rat model control group. After successful modeling of SD rats, no drugs were administered.

[0083] ③ Administration method: SD rats were randomly divided into a blank control group, a model group, low-, medium-, and high-dose groups of Brucea javanica oral emulsion, and a positive control group of levothyroxine sodium 10 μg / kg, with 8 rats in each group. Except for the blank control group, all rats were administered 0.1% propylthiouracil solution (PTU) by gavage at a rate of 10 mL / kg once daily. After 4 weeks of gavage, the weight, coat color, and reaction behavior of the rats in each group were observed. Weight loss, yellowish coat color, and sluggish reaction were considered as successful model establishment. To prevent spontaneous recovery, PTU solution was continued by gavage after successful model establishment to maintain the hypothyroid state in the rats, with the frequency changed from once daily to once every other day.

[0084] ④ Serum collection: After the last treatment, all rats were fasted but allowed free access to water for 24 hours. They were anesthetized by intraperitoneal injection of 20% urethane (1.5 g / kg), and blood was collected from the abdominal aorta. The blood was centrifuged at 3000 r / min for 10 min, and the supernatant serum was collected and stored in a -80℃ refrigerator for testing.

[0085] ⑤ Tissue collection: After the rats were sacrificed, the skin of the rat's neck was quickly cut longitudinally on ice, the neck tissue was peeled off, the rat's thyroid tissue was cut off, weighed, and soaked in 10% paraformaldehyde solution for storage at room temperature until testing.

[0086] ⑥ Detection of free triiodothyronine (FT3), free thyroxine (FT4), and thyroid-stimulating hormone (TSH) in rats: A kit from ELISA was used, and the specific procedure is as follows:

[0087] (1) Take out the kit, let it equilibrate to room temperature for 30 minutes, then take out the required strips and seal the remaining strips and put them back in the 4℃ refrigerator.

[0088] (2) Design and label the standard wells and sample wells, and add 50 μL of standard with different concentration gradients to each standard well;

[0089] (3) Add 50 μL of the sample to be tested to each well, except for the blank well. If the sample needs to be diluted, use the sample diluent provided with the kit as required;

[0090] (4) Add 50 μL of biotin-labeled antibody to each well, seal the reaction well with sealing film, and incubate at 37°C in a water bath or incubator for 30 min. Do not add to the blank wells.

[0091] (5) Discard the liquid, pat dry on absorbent paper, fill each well with 350 μL of washing solution, let stand at room temperature for 1 min, remove the washing solution, pat dry on absorbent paper, and repeat this process 5 times.

[0092] (6) Except for the blank wells, add 100 μL of HRP-labeled detection antibody to both the standard wells and the sample wells, seal the well plate with sealing film, and incubate in a 37°C incubator for 30 min.

[0093] (7) Shake off the liquid, pat dry on absorbent paper, add 350 μL of washing solution to each well, let stand at room temperature for 1 min, discard the washing solution, pat dry with absorbent paper, and repeat this process 5 times.

[0094] (8) Add 50 μL of substrate A and B to each well and incubate in a constant temperature oven at 37°C for 15 min in the dark;

[0095] (9) Add 50 μL of stop solution to each well within 15 min, and use an ELISA reader to set the wavelength of 450 nm as the maximum absorption wavelength to measure the OD value of each well.

[0096] ⑦ Statistical Analysis: GraphPad Prism 5.01 statistical analysis software was used for data analysis. The data adopted... To indicate the statistical significance, the comparison between the two groups was performed using the independent groups test (ANOVA). A p-value < 0.05 was considered statistically significant.

[0097] ⑧ HE staining: Thyroid tissue sections were observed after HE staining.

[0098] 2. Test Results

[0099] Table 1: Changes in rat body weight (g)

[0100]

[0101]

[0102] Table 2: Wet weight of rat thyroid gland (mg)

[0103] Group Sample size Thyroid wet weight Blank group 8 36.90±6.06 Model group 8 94.66±8.23* positive group 8 49.66±8.62# High-dose group 8 58.21±12.55# medium dose group 8 71.38±12.38# low-dose group 8 85.95±11.99

[0104] Note: *: Significant difference between the model group and the blank group (p<0.01); #: Significant difference between the drug-treated groups (high, medium, and low dose groups) and the model group (p<0.01).

[0105] Observation results of thyroid tissue: Macroscopic observation of the thyroid appearance showed that the thyroid gland in the model group was significantly red and swollen and enlarged. The thyroid condition of the positive group and each drug administration group (high, medium and low dose groups) was improved to varying degrees compared with the model group.

[0106] Table 3: Comparison of serum FT3, FT4 and TSH levels in rats

[0107] Group FT3 (pmol / L) FT4 (pmol / L) TSH (μIU / mL) Blank group 9.00±0.81 31.27±2.66 5.15±0.45 Model group 6.47±1.17* 22.71±1.87* 13.36±2.67* positive group 8.95±0.72# 30.50±3.95# 6.21±0.69# High-dose group 9.35±1.06# 33.27±2.40# 6.66±0.52# medium dose group 8.85±1.12# 33.77±2.92# 6.77±1.14# low-dose group 7.39±1.61 26.46±2.95# 8.13±2.00#

[0108] Note: *: The model group was significantly different from the blank group (p<0.01); #: The drug administration group (high, medium and low dose group) was significantly different from the model group (p<0.01).

[0109] Observation of HE-stained sections revealed ( Figure 1 ):

[0110] The control group showed normal thyroid tissue morphology, clear staining, no reduction or atrophy of follicles, regular size and shape, and normal follicular colloid.

[0111] Model group: The number of thyroid follicles was significantly reduced, deformed, and atrophied significantly. Their size and shape were irregular and varied significantly. The cavity was reduced, the epithelial cells were swollen and enlarged, the follicular colloid was reduced, the interstitial fibrosis was significantly proliferated, and the exudation of blood cells was significantly increased.

[0112] Positive group: Thyroid follicles are similar in morphology to those in the high-dose group, with significant recovery in both number and morphology. Follicular colloid has recovered considerably, with a small amount of interstitial fibrosis and a large number of blood cells distributed in the interstitial area.

[0113] High-dose group: Thyroid tissue morphology tends to be normal, follicle deformation and atrophy are reduced compared with the model group, cavity size is restored, shape is more regular, follicular colloid is significantly increased, and blood cell exudation is rare.

[0114] Medium-dose group: Thyroid follicle deformation was alleviated, but follicle atrophy was still obvious, with irregular size and shape and significant differences in some areas. Follicular colloid reduction was not significantly different from the L group, and follicular epithelial swelling did not improve.

[0115] Low-dose group: Thyroid follicles decreased, deformed, and atrophied, with irregular size and shape. Follicular colloid decreased slightly but recovered compared to the model group. There was a small amount of interstitial fibrosis and blood cell exudation.

[0116] The results showed that the high-dose group had a therapeutic effect comparable to that of the positive group. Different doses of oral administration of Brucea javanica emulsion improved symptoms of non-autoimmune hypothyroidism induced by propylthiouracil.

[0117] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. Use of Brucea javanica oil, pharmaceutical compositions containing Brucea javanica oil as an active ingredient, and pharmaceutical preparations containing Brucea javanica oil in the preparation of a medicament for the prevention and / or treatment of thyroid dysfunction.

2. The application according to claim 1, characterized in that: The thyroid dysfunction mentioned is a non-autoimmune thyroid dysfunction.

3. The use according to claim 1, characterized in that, The drug has at least one of the following uses: 1) Regulate serum thyroid-stimulating hormone levels; 2) Regulate serum free triiodothyronine levels; 3) Regulate serum free thyroxine levels; 4) Inhibits thyroid follicle deformation and / or atrophy; 5) Promotes the secretion of thyroid follicular colloid; 6) Inhibits the proliferation of interstitial fibrosis; 7) Reduce the exudation of red blood cells from thyroid tissue.

4. The use according to claim 2, characterized in that, The non-autoimmune thyroid dysfunction is hypothyroidism.

5. The use according to claim 4, characterized in that, The hypothyroidism mentioned is caused by non-autoimmune thyroiditis.

6. The use according to claim 4 or 5, characterized in that, The drug has at least one of the following uses: 1) Lower serum thyroid-stimulating hormone levels; 2) Increase serum free triiodothyronine levels; 3) Increase serum free thyroxine levels; 4) Inhibits thyroid follicle deformation and / or atrophy; 5) Promotes the secretion of thyroid follicular colloid; 6) Inhibits the proliferation of interstitial fibrosis; 7) Reduce the exudation of red blood cells from thyroid tissue.

7. The use according to any one of claims 1 to 6, characterized in that, The Brucea javanica oil or the pharmaceutical composition is provided in the form of an oral formulation, a spray formulation or an injectable formulation.

8. The use according to any one of claims 1 to 6, characterized in that, The pharmaceutical composition further comprises: an emulsifier; optionally, the emulsifier contains phospholipids; optionally, the emulsifier contains at least one of lecithin, egg yolk lecithin, and soybean lecithin.

9. The use according to claim 8, characterized in that, The pharmaceutical composition is in the form of at least one of tablets, granules, powders, capsules, solutions, suspensions, or lyophilized preparations.

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