Traditional Chinese medicine composition for treating toxic diffuse goiter and preparation method thereof

Abstract

The invention discloses a traditional Chinese medicine composition for treating toxic diffuse goiter and a preparation method of the traditional Chinese medicine composition, and relates to the technical field of traditional Chinese medicines. Comprising the following raw materials: 10-25 parts of oldenlandia diffusa, 10-25 parts of verbena, 8-20 parts of rhizoma pinellinae praeparata, 8-15 parts of amomum tsao-ko, 3-10 parts of rhizoma acori graminei, 8-20 parts of paris polyphylla, 8-15 parts of peach kernel, 8-15 parts of pogostemon cablin, 8-15 parts of eupatorium fortunei and 3-8 parts of liquorice. Through a specific ratio and a preparation process of monarch drugs, ministerial drugs, adjuvant drugs and conductant drugs, drug compatibility and dosage ratio are optimized, and the traditional Chinese medicine composition integrates functions of reducing phlegm and resolving masses, clearing away heat and toxic materials, promoting blood circulation and removing blood stasis, promoting diuresis and dissolving turbidity, and tonifying spleen and strengthening body resistance, and aims to dissipate goiter nodules caused by Graves diseases, eliminate pathological products, improve internal environment, relieve symptoms and recover viscera functions. The traditional Chinese medicine composition can effectively regulate the immune function and improve clinical symptoms, and has the advantages of improving the treatment effect and reducing adverse reactions.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, and in particular to a traditional Chinese medicine composition for treating toxic diffuse goiter and its preparation method. Background Technology

[0002] Toxic diffuse goiter, also known as Graves' disease (GD), is a common autoimmune thyroid disease. This disease primarily involves thyroid-stimulating antibodies acting on the thyroid-stimulating hormone receptor, leading to excessive thyroid hormone secretion and causing a hypermetabolic syndrome affecting the nervous, circulatory, and digestive systems. Typical clinical manifestations include hypermetabolic syndrome, diffuse goiter, and exophthalmos (protruding eye). With the accelerated pace of modern life and changing environmental factors, the incidence of this disease has been rising year by year, making it a common and frequently occurring disease of the endocrine system.

[0003] The pathogenesis of this disease is complex, involving multiple factors such as abnormal immune regulation and genetic susceptibility. Studies have shown that autophagy and apoptosis of thyroid cells play a key regulatory role in the development and progression of the disease, which provides new targets for the treatment of GD. Currently, the main clinical treatments are thionamide antithyroid drugs, radioactive iodine therapy, and surgery. However, these methods all have obvious limitations: thionamide drugs have long treatment courses, are prone to relapse, and may cause adverse reactions such as granulocytopenia; radiotherapy may lead to permanent hypothyroidism; and surgical treatment has problems such as surgical risks and postoperative complications.

[0004] Traditional Chinese medicine (TCM) has unique advantages in treating thyroid diseases. Through its holistic regulatory effects targeting multiple points and pathways, it can effectively improve clinical symptoms and regulate immune function. However, existing TCM treatment regimens still need optimization in terms of drug compatibility, dosage ratios, and preparation processes to better target the pathological characteristics of thyroid disease (GD), improve treatment efficacy, and reduce adverse reactions. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the present invention provides a traditional Chinese medicine composition for treating toxic diffuse goiter and its preparation method, aiming to optimize drug compatibility and dosage ratio, improve therapeutic effect, and reduce adverse reactions.

[0006] The first aspect of this invention provides a traditional Chinese medicine composition for treating toxic diffuse goiter, comprising the following raw materials in parts by weight: 10-25 parts of Oldenlandia diffusa, 10-25 parts of Verbena officinalis, 8-20 parts of Pinellia ternata, 8-15 parts of Amomum tsao-ko, 3-10 parts of Acorus tatarinowii, 8-20 parts of Paris polyphylla, 8-15 parts of Prunus persica, 8-15 parts of Pogostemon cablin, 8-15 parts of Eupatorium fortunei, and 3-8 parts of Glycyrrhiza uralensis.

[0007] Through the above technical solution, Hedyotis diffusa in the monarch drug has the effect of clearing heat and detoxifying, and Verbena officinalis can clear heat and detoxify, promote blood circulation to dredge the meridians, promote diuresis and reduce swelling. The two work synergistically to resolve the heat toxin transformed from the mutual binding of phlegm and stasis and stagnation over time, and assist in resolving blood stasis and phlegm-dampness, reducing local inflammation and swelling. If the dosage of the monarch drug is too small, the ability to clear heat and detoxify is weak, and if it is too high, it is likely to damage healthy qi. Pinellia ternata, Tsaoko fruit, Acorus tatarinowii, Paris polyphylla, and Prunus persica in the ministerial drugs regulate the PI3K / AKT / mTOR signaling pathway to affect autophagy and apoptosis of cells. Pinellia ternata and Tsaoko fruit dry dampness and resolve phlegm, Acorus tatarinowii opens the orifices and resolves phlegm, Paris polyphylla clears heat and detoxifies, and Prunus persica promotes blood circulation to remove blood stasis, jointly enhancing the effect of resolving phlegm and dissipating nodules. If the dosage of the ministerial drugs is too small, it is difficult to resolve phlegm and blood stasis, and if it is too large, it is likely to increase the burden on the spleen and stomach. Pogostemon cablin and Eupatorium fortunei, as adjuvant drugs, improve the body's metabolic environment. Pogostemon cablin and Eupatorium fortunei aromatize dampness and can regulate the functions of the spleen and stomach to assist drug absorption. Licorice, as the guiding drug, harmonizes all the drugs, enhances the stability of the overall drug effect, and at the same time plays an anti-inflammatory role. Each component is formulated in a specific ratio to form a coordinated effect of clearing heat and detoxifying, resolving phlegm and dissipating nodules, and promoting blood circulation to remove blood stasis.

[0008] Compared with the traditional western medicine that only inhibits the synthesis of thyroid hormones, this solution simultaneously regulates immune abnormalities, improves cell metabolism, and intervenes in the autophagy-apoptosis balance through the synergistic effect of multiple components, effectively improving goiter and related metabolic abnormalities, and reducing the continuous damage of the immune response to thyroid tissue. Compared with simply using thiourea drugs, this traditional Chinese medicine composition intervenes in the disease process through multiple pathways, maintains the homeostasis of thyroid function while controlling the development of the disease, not only reduces the recurrence risk, but also avoids side effects such as hypothyroidism. Its multi-target mechanism of action more conforms to the complex pathological characteristics of Graves' disease.

[0009] Optionally, the weight parts of Hedyotis diffusa and Verbena officinalis are 1:1.

[0010] Through the above technical solution, Hedyotis diffusa refers to the dried whole herb of the Compositae plant Hedyotis diffusa, which has the effects of clearing heat and detoxifying, dissipating carbuncles and nodules, and plays an anti-inflammatory and immune-regulating role after decoction; Verbena officinalis is the dried aerial part of the Verbena officinalis plant of the Verbenaceae family, which has the effects of promoting blood circulation to disperse stasis and promoting diuresis and reducing swelling. When the mass ratio of the two is 1:1, the liposoluble components and water-soluble components of the two can form a complex during the decoction process. Oleanolic acid in Hedyotis diffusa and verbenalin in Verbena officinalis produce synergistic effects, enhancing the inhibitory effect on the apoptosis of thyroid follicular epithelial cells. At the same time, it avoids gastrointestinal irritation caused by too high dosage of a single herb. Through the synergistic effect of the properties of the two herbs, it can not only inhibit excessive autophagy of thyroid cells, but also regulate the abnormal activation of the PI3K / AKT / mTOR signaling pathway, reduce the degree of goiter, and at the same time, by balancing immune regulation and cell protection, reduce the risk of abnormal liver function or leukopenia during the treatment process.

[0011] Optionally, the traditional Chinese medicine composition further includes cat's claw grass, wherein the mass ratio of cat's claw grass to white flower tongue grass and verbena is 1:3.

[0012] Through the above technical solution, cat's claw grass refers to the dried tuberous root of Ranunculum simonii, a plant of the Ranunculaceae family. It has the effects of resolving phlegm and dissipating nodules, detoxifying and reducing swelling. It enhances the efficacy of the principal drug in detoxification and dissipation. The combined use of Hedyotis diffusa and Verbena officinalis can exert the effect of clearing heat and detoxifying. Cat's claw grass further enhances the ability to intervene in immune abnormalities by regulating the autophagy and apoptosis levels of thyroid cells. The three are matched in a 1:3 ratio to establish a dual action pathway that takes into account both detoxification and dissipation and immune regulation. The quality ratio control avoids the problem of drug efficacy conflict caused by the imbalance of drug compatibility. It more accurately regulates the autophagy-related signaling pathways of thyroid cells. While maintaining the basic heat-clearing and detoxifying effects, the synergistic effect of cat's claw grass enhances the effect of dissipating goiter.

[0013] Optionally, it may also include 24-40 servings of Job's tears; The mass ratio of the Job's tears to Hedyotis diffusa and Verbena officinalis is 1:1.

[0014] Through the above technical solution, coix seed refers to the dried mature seed kernel of the grass coix. After decoction, its diuretic and dampness-draining properties can work synergistically with the detoxification and nodule-dispersing functions of the principal drug. By regulating the body's water metabolism, it promotes the excretion of toxins. The precise control of the mass ratio allows coix seed to play an auxiliary therapeutic role while avoiding excessive use that could lead to spleen and stomach deficiency and cold. It effectively improves the edema state of thyroid tissue, enhances the efficiency of toxin metabolism, and at the same time, the precise ratio control avoids negative effects between drugs. While inhibiting the excessive proliferation of thyroid cells, it also regulates the body's immune balance.

[0015] Optionally, the mass ratio of the coix seed to licorice is 6:1.

[0016] Through the above technical solution, licorice refers to the rhizome of the legume plant licorice. The diuretic effect of coix seed can be balanced by the harmonizing properties of licorice, preventing excessive elimination of pathogens from damaging the body's vital energy. This maintains the ability of coix seed to clear abnormal thyroid metabolites, while the sustained-release effect of licorice reduces the stimulation of the gastrointestinal tract, reducing the generation of phlegm and dampness from the source. At the same time, it ensures that the composition has repeatable therapeutic stability when inhibiting the overactivation of the PI3K / AKT / mTOR signaling pathway, ensuring the sustainability and safety of the treatment.

[0017] Optionally, the mass ratio of patchouli to eupatorium is (1-1.5):1.

[0018] Through the above technical solution, the mass ratio of patchouli and eupatorium is controlled at (1-1.5):1. Patchouli alcohol and eupatorium lactone produce a synergistic effect. The concentration gradient of patchouli alcohol can enhance the penetration of thyroid follicular cells, ensuring that, on the basis of the principal drug's detoxification and the assistant drug's phlegm-resolving properties, the adjuvant drug can stably exert its auxiliary effects of removing dampness and turbidity. In the treatment of toxic diffuse goiter, it enhances the ability to clear damp-heat and phlegm, while avoiding the depletion of body fluids caused by excessive use of aromatic drugs. This ratio design makes the compatibility of the adjuvant drug with the principal and assistant drugs more distinct, which is conducive to improving the accumulation of dampness and turbidity in the thyroid microenvironment.

[0019] A second aspect of the present invention provides a traditional Chinese medicine preparation for treating toxic diffuse goiter, used to prepare the above-mentioned traditional Chinese medicine composition.

[0020] A third aspect of the present invention provides a method for preparing a traditional Chinese medicine composition for treating toxic diffuse goiter, used to prepare any of the above-described traditional Chinese medicine compositions, comprising the following steps: S1. Weigh all the raw materials according to the stated weight proportions, add cold water and soak for 2-3 hours, boil for 2-3 hours and then filter to obtain the first medicinal liquid and the dregs. S2. Add warm water to the dregs, boil for 1-2 hours, and then filter to obtain the second medicinal liquid; S3. After mixing the first and second medicinal solutions, filter and boil to obtain the traditional Chinese medicine composition.

[0021] The beneficial effects of this invention are as follows: by optimizing the drug compatibility and dosage ratio through specific proportions and manufacturing processes of the principal drug, assistant drug, adjuvant drug, and guiding drug, it integrates the functions of resolving phlegm and dissipating nodules, clearing heat and detoxifying, promoting blood circulation and removing blood stasis, promoting diuresis and eliminating turbidity, and strengthening the spleen and supporting the body's resistance. It aims to dissipate thyroid nodules caused by Graves' disease, clear pathological products, improve the internal environment, relieve symptoms, and restore organ function. It can effectively regulate immune function and improve clinical symptoms, and has the advantages of improving treatment effect and reducing adverse reactions. Attached Figure Description

[0022] Figure 1 This is a flowchart illustrating a method for preparing a traditional Chinese medicine composition for treating toxic diffuse goiter, as provided in an embodiment of the present invention.

[0023] Figure 2 These are pathological images of thyroid tissue from the control group, GD model group, traditional Chinese medicine group, and Western medicine group of this invention.

[0024] Figure 3 This is a schematic diagram showing the levels of T3, T4, FT3, FT4, and TSH in the serum of the control group, GD model group, JZJD group, and MMI group of this invention.

[0025] Figure 4This is a schematic diagram illustrating the expression of Caspase3, Beclin-1, P-P13K, P-AKT, P-mTOR, and P-TSC2 proteins in the control group, GD model group, JZJD group, and MMI group of this invention.

[0026] Figure 5 This is a schematic diagram showing the contents of Caspase3, Beclin-1, P-P13K, P-AKT, P-mTOR, and P-TSC2 in the control group, GD model group, JZJD group, and MMI group of this invention.

[0027] Figure 6 This is a schematic diagram showing the content of BAX, Beclin 1, BCL-2, LC3BI and Caspase 3 in the control group, GD model group, JZJD group and MMI group of this invention. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Figure 1 This is a flowchart of a traditional Chinese medicine composition for treating toxic diffuse goiter provided in an embodiment of the present invention, such as... Figure 1 As shown, the method includes the following steps: S1. Weigh all the raw materials according to the weight proportions, add cold water and soak for 2-3 hours, boil for 2-3 hours and then filter to obtain the first medicinal liquid and the dregs. S2. Add warm water to the dregs, boil for 1-2 hours, then filter to obtain the second medicinal liquid; S3. After mixing the first and second medicinal solutions, filter and boil to obtain the traditional Chinese medicine composition.

[0030] The raw material information involved in this invention is as follows: Healthy male BALB / c mice, 6-7 weeks old, were sourced from Hunan Slack Jingda Experimental Animal Co., Ltd. They were housed in an SPF animal facility, with room temperature controlled at 20-24℃ and humidity at 50%-70%. A 12-hour light-dark cycle was maintained, and standard feed and drinking water were provided regularly. Experiments were conducted one week after the mice had adapted to the new environment.

[0031] Cat's Claw Herb, Oldenlandia diffusa, Verbena officinalis, Pinellia ternata, Amomum tsao-ko, Acorus tatarinowii, Paris polyphylla, Prunus persica, Coix lacryma-jobi, Pogostemon cablin, Eupatorium fortunei, and Glycyrrhiza uralensis were purchased from the Traditional Chinese Medicine Pharmacy of Hunan Provincial Hospital of Integrated Traditional and Western Medicine; Methimazole tablets were purchased from Merck Pharmaceuticals Co., Ltd. (Jiangxi), specification: 10mg*50 tablets; Xylene P1110, anhydrous ethanol A8020, and hematoxylin-eosin (HE) staining kit G1120 were purchased from Wuhan Sewell Biotechnology Co., Ltd.; Neutral resin BKM-ZX-100 was purchased from Shenzhen Baikaimei Biotechnology Co., Ltd.

[0032] The preparation method of the important composition involved in this invention will be specifically described below with reference to specific embodiments and comparative examples: Example Example 1

[0033] A traditional Chinese medicine composition for treating toxic diffuse goiter is prepared through the following steps: S1. Add 10g of Oldenlandia diffusa, 10g of Verbena officinalis, 8g of Pinellia ternata, 8g of Amomum tsao-ko, 3g of Acorus tatarinowii, 8g of Paris polyphylla, 8g of Prunus persica and 3g of Glycyrrhiza uralensis to 500ml of cold water, soak for 2 hours, add water to 500ml, boil for 2 hours, reduce the liquid and filter to obtain 150ml of the first decoction and the dregs. S2. Add 300ml of warm water at 40℃ to the dregs, boil for 1 hour, reduce the liquid and filter to obtain 100ml of the second liquid.

[0034] S3. After mixing the first and second medicinal solutions, filter and boil to obtain the traditional Chinese medicine composition. Example 2

[0035] A traditional Chinese medicine composition for treating toxic diffuse goiter is prepared through the following steps: S1. Add 25g of Oldenlandia diffusa, 25g of Verbena officinalis, 20g of Pinellia ternata, 15g of Amomum tsao-ko, 10g of Acorus tatarinowii, 20g of Paris polyphylla, 15g of Prunus persica and 8g of Glycyrrhiza uralensis to 1500ml of cold water, soak for 2 hours, add water to 1500ml, boil for 2 hours, reduce the liquid and filter to obtain 150ml of the first decoction and the dregs. S2. Add 800ml of warm water at 40℃ to the dregs, boil for 1 hour, reduce the liquid and filter to obtain 100ml of the second medicinal liquid.

[0036] S3. After mixing the first and second medicinal solutions, filter and boil to obtain the traditional Chinese medicine composition. Example 3

[0037] A traditional Chinese medicine composition for treating toxic diffuse goiter is prepared through the following steps: S1. Add 15g of Oldenlandia diffusa, 15g of Verbena officinalis, 10g of Pinellia ternata, 10g of Amomum tsao-ko, 6g of Acorus tatarinowii, 10g of Paris polyphylla, 10g of Prunus persica and 5g of Glycyrrhiza uralensis to 800ml of cold water, soak for 2 hours, add water to 800ml, boil for 2 hours, reduce the liquid and filter to obtain 150ml of the first decoction and the dregs. S2. Add 500ml of warm water at 40℃ to the dregs, boil for 1 hour, reduce the liquid and filter to obtain 100ml of the second liquid.

[0038] S3. After mixing the first and second medicinal solutions, filter and boil to obtain the traditional Chinese medicine composition. Example 4

[0039] A traditional Chinese medicine composition for treating toxic diffuse goiter is prepared through the following steps: S1. Add 10g of cat's claw grass, 15g of white flower tongue grass, 15g of verbena, 10g of pine needle grass, 10g of cardamom, 6g of calamus, 10g of Paris polyphylla, 10g of peach kernel, 30g of coix seed, 12g of patchouli, 10g of agastache rugosa, and 5g of licorice to 1700ml of cold water and soak for 2 hours. Add water to 1700ml, boil for 2 hours, reduce the liquid, and filter to obtain 150ml of the first decoction and the dregs. S2. Add 1000ml of warm water at 40℃ to the dregs, boil for 1 hour, reduce the liquid and filter to obtain 100ml of the second liquid.

[0040] S3. After mixing the first and second medicinal solutions, filter and boil to obtain the traditional Chinese medicine composition. Example 5

[0041] A traditional Chinese medicine composition for treating toxic diffuse goiter is prepared through the following steps: S1. Add 8g of cat's claw grass, 12g of white flower tongue grass, 12g of verbena, 10g of pine needle grass, 10g of cardamom, 6g of calamus, 10g of Paris polyphylla, 10g of peach kernel, 24g of coix seed, 12g of patchouli, 10g of agastache rugosa, and 4g of licorice to 1400ml of cold water and soak for 2 hours. Add water to 1400ml, boil for 2 hours, reduce the liquid, and filter to obtain 150ml of the first decoction and the dregs. S2. Add 700ml of warm water at 40℃ to the dregs, boil for 1 hour, reduce the liquid and filter to obtain 100ml of the second liquid.

[0042] S3. After mixing the first and second medicinal solutions, filter and boil to obtain the traditional Chinese medicine composition. Example 6

[0043] A traditional Chinese medicine composition for treating toxic diffuse goiter is prepared through the following steps: S1. Add 10g of cat's claw grass, 15g of white flower tongue grass, 15g of verbena, 10g of pine needle grass, 10g of cardamom, 6g of calamus, 10g of Paris polyphylla, 10g of peach kernel, 30g of coix seed, 11g of patchouli, 11g of agastache rugosa, and 5g of licorice to 1700ml of cold water and soak for 2 hours. Add water to 1700ml, boil for 2 hours, reduce the liquid, and filter to obtain 150ml of the first decoction and the dregs. S2. Add 1000ml of warm water at 40℃ to the dregs, boil for 1 hour, reduce the liquid and filter to obtain 100ml of the second liquid.

[0044] S3. After mixing the first and second medicinal solutions, filter and boil to obtain the traditional Chinese medicine composition. Example 7

[0045] A traditional Chinese medicine composition for treating toxic diffuse goiter is prepared through the following steps: S1. Add 10g of cat's claw grass, 15g of white flower tongue grass, 15g of verbena, 10g of pine needle grass, 10g of cardamom, 6g of calamus, 10g of Paris polyphylla, 10g of peach kernel, 30g of coix seed, 15g of patchouli, 10g of agastache rugosa, and 5g of licorice to 1700ml of cold water and soak for 2 hours. Add water to 1700ml, boil for 2 hours, reduce the liquid, and filter to obtain 150ml of the first decoction and the dregs. S2. Add 1000ml of warm water at 40℃ to the dregs, boil for 1 hour, reduce the liquid and filter to obtain 100ml of the second liquid.

[0046] S3. After mixing the first and second medicinal solutions, filter and boil to obtain the traditional Chinese medicine composition.

[0047] Comparative Example Comparative Example 1 A Western medicine for treating toxic diffuse goiter is methimazole tablets.

[0048] Comparative Example 2 A traditional Chinese medicine composition for treating toxic diffuse goiter, differing from Example 4 in that the weight of cat's claw herb is 30g.

[0049] Comparative Example 3 A traditional Chinese medicine composition for treating toxic diffuse goiter, differing from Example 4 in that the weight of Hedyotis diffusa is 5g and the weight of Verbena officinalis is 20g.

[0050] Comparative Example 4 A traditional Chinese medicine composition for treating toxic diffuse goiter, differing from Example 4 in that the weight of Hedyotis diffusa is 20g and the weight of Verbena officinalis is 4g.

[0051] Comparative Example 5 A traditional Chinese medicine composition for treating toxic diffuse goiter, differing from Example 4 in that the weight of coix seed is 10g.

[0052] Comparative Example 6 A traditional Chinese medicine composition for treating toxic diffuse goiter, differing from Example 4 in that the weight of coix seed is 60g.

[0053] Comparative Example 7 A traditional Chinese medicine composition for treating toxic diffuse goiter, differing from Example 4 in that the weight of patchouli is 5g and the weight of agastache is 15g.

[0054] Comparative Example 8 A traditional Chinese medicine composition for treating toxic diffuse goiter, differing from Example 4 in that the weight of patchouli is 15g and the weight of agastache is 5g.

[0055] The preparation methods of the traditional Chinese medicine compositions in Comparative Examples 2-8 are the same as those in Example 4.

[0056] Clinical trials

[0057] The following clinical trials were conducted on mice using the traditional Chinese medicine compositions provided in Examples 1-7 and Comparative Examples 2-8 of this invention, as well as methimazole tablets. Table 1 shows the performance test results.

[0058] Establishing the GD model group: At weeks 0, 3, and 6, mice were injected unilaterally into the vastus lateralis muscle with Ad-TSHR289 recombinant adenovirus expressing the TSHR-A subunit. The injection dosage was [missing data]. / time / mouse, diluted in 0.1ml PBS buffer, the control group was injected with the same amount of PBS buffer. The modeling status of mice was evaluated at week 10 of the experiment. Three mice were randomly selected from each group. Blood was collected through the retro-orbital venous plexus and clinical indicators such as triiodothyronine (T3), thyroxine (T4), free triiodothyronine (FT3), free thyroxine (FT4), and thyroid-stimulating hormone (TSH) were measured. At the same time, the activity status, mental status, weight, hair, excretion and tongue coating of the mice were observed.

[0059] The mice were randomly divided into groups of 10 each, as follows: ①Contro group (control group): Healthy mice were given 10 mL / kg / day of distilled water orally; ② Model group (GD model group): GD model was given 10 mL / kg / d of distilled water orally; ③ JZJD group (traditional Chinese medicine group): The traditional Chinese medicine composition of Example 4 was administered orally at 10 mL / kg / d to the GD model; ④ MMI group (Western medicine group): One methimazole tablet was crushed and added to 2 ml of physiological saline to prepare a 2 mL suspension. The GD model was orally administered 10 mL / kg / d of methimazole suspension. Each group had three groups of mice, and the intervention was carried out 3 times a week for 4 consecutive weeks.

[0060] like Figure 2 As shown, in the control group mice, the follicles in the thyroid tissue were flat, the cells were neatly arranged, the follicular lumen was smooth, and red, uniform colloid was visible. In the GD model group mice, the follicles in the thyroid tissue were of different sizes, the follicular lumen was not smooth, the colloid in the lumen was significantly reduced, and rupture occurred. In the drug-treated groups, the thyroid tissue of both groups of mice showed improved cell morphology compared to the GD model group, the cell morphology gradually returned to flat, and the colloid in the follicular lumen increased.

[0061] 1. Basic situation monitoring The feeding conditions for mice during the hyperthyroidism modeling stage are as follows: In the control group, mice showed minimal overall weight variation, smooth and glossy fur, typically yellow, rice-grain-sized feces, and yellow or clear, watery urine. Their tongues had a thin, even, bright red coating, and they responded normally to external stimuli, exhibiting good mental condition. In the GD model group, mice experienced a slight overall weight increase over time, with loose, disheveled fur, typically black or yellow, rice-grain-sized feces, yellow urine, a thick, greasy tongue coating, and an enlarged, slightly darker tongue. They exhibited excessive reactions to external stimuli, hyperactivity, and some mice displayed continuous circling. In the traditional Chinese medicine group, mice initially decreased in weight over time, then increased it to roughly the same as their initial weight. Their fur gradually increased in size and shape, their feces were typically granular, their urine was yellow, their tongue coating changed from white to yellow, and their tongues enlarged. They responded normally to external stimuli, maintained good mental condition, and exhibited no abnormal behavior. The mice in the Western medicine group generally increased in weight over time, had soft fur, usually had pellet-like stools, yellow or clear urine, thick white tongue coating, slightly enlarged tongue body, no excessive response to external stimuli, good mental state, and no abnormal behavior.

[0062] The activity status, mental state, weight, hair, excretion and tongue coating of mice in each group were recorded regularly. The basic information of mice in each group is shown in Table 1.

[0063] Table 1. Basic information of mice in each group As shown in Table 1, compared with the control group, the GD model group mice exhibited abnormal activity, hyperactivity, overreaction, and disheveled fur. The traditional Chinese medicine composition and methimazole tablets in Example 4 improved the clinical manifestations of the GD model, including activity and mental state.

[0064] 2. Histological examination After collecting thyroid tissue from each group of mice, the following steps were performed: 1. Dewaxing the slices: Place the slices in a 65℃ oven for 90 minutes; quickly immerse the baked slices in xylene I for 10 minutes, and then in xylene II solution for 10 minutes.

[0065] 2. Hydration: Gradient ethanol hydration, the slices are soaked in 100% ethanol I for 5 min, 100% ethanol II for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, 70% ethanol for 5 min, and then washed with distilled water for 10 min.

[0066] 3. HE staining: Stain the sections with hematoxylin for 8 min, wash with tap water for 10 min, differentiate with differentiation solution for 10 s, wash with tap water for 5 min twice, stain with eosin solution for 5 min, pour off excess staining solution and dehydrate quickly.

[0067] 4. Dehydration: Immerse the slices sequentially in 70% alcohol for 2-3 seconds, 80% alcohol for 2-3 seconds, 90% alcohol for 2-3 seconds, 100% alcohol I for 2-3 seconds, 100% alcohol II for 1 minute, and finally in xylene I and xylene II solutions for 10 minutes each. After dehydration and clearing, air dry in a fume hood.

[0068] 5. Mounting: Mount the sections with neutral resin for observation.

[0069] 6. Microscopic examination and photography: Observe the slide under a fluorescence microscope (Olympus) and acquire images.

[0070] The procedure involves fixation, embedding, sectioning, dewaxing, hydration, and staining. Finally, images are taken and analyzed using image software. Figure 2 .

[0071] according to Figure 2 As shown, compared with the control group, the GD model group showed thyroid follicular hyperplasia, with columnar epithelial cells and significant proliferation. The follicles were larger, multilayered, with rough edges and irregular inward convexity. The follicular lumen was not fully filled and there was obvious inflammatory cell infiltration. Compared with the GD model group, the thyroid follicular hyperplasia and epithelial cell proliferation were reduced in the traditional Chinese medicine group and the Western medicine group. The follicular edges were smooth, the inward convexity was significantly reduced, and the follicular lumen was fully filled.

[0072] 3. Serum testing The levels of T3, T4, FT3, FT4, and TSH in serum were detected using ELISA. The ELISA kit was brought to room temperature. Standard solutions were prepared according to the manufacturer's instructions. Blank wells, standard wells, and sample wells were set up. No sample or enzyme-labeled reagent was added to the blank wells. 50 μL of sample or standard was added to each of the remaining wells, followed by 100 μL of enzyme-labeled reagent. The plates were incubated at 37°C for 1 hour. After washing the plates five times, chromogenic reagent was added, and the plates were incubated at 37°C for 15 minutes. The reaction was terminated by adding stop solution. The absorbance of each well was measured at 450 nm. Specific concentrations are shown in Tables 2-6.

[0073] Table 2 Comparison of T3 results in mice of different groups ( ) Table 3 Comparison of T4 results in mice of different groups ( ) Table 4 Comparison of FT3 results in mice of different groups ( ) Table 5 Comparison of FT4 results in mice of different groups ( ) Table 6 Comparison of TSH results in mice of different groups ( ) (* indicates a significant difference compared to the control group, P < 0.05; # indicates a significant difference compared to the GD model group, P < 0.05).

[0074] according to Figure 3 It was found that, compared with the control group, the GD model group had increased levels of T3, T4, FT3, and FT4, and decreased levels of TSH (P < 0.05). Compared with the GD model group, the traditional Chinese medicine group had decreased levels of T3, T4, FT3, and FT4, and increased levels of TSH (P < 0.05); the Western medicine group showed no significant difference in FT4 levels compared with the GD model group, while the remaining levels were consistent with the JZJD trend and showed significant differences (P < 0.05).

[0075] 4. Western blot protein quantification analysis Thyroid tissue was placed in RIPA lysis buffer to extract total protein. After grinding, the tissue was centrifuged at 12,000 rpm and 4°C for 10 min, and the supernatant was collected. The total protein of phosphorylated phosphatidylinositol 3-kinase (P13K), protein kinase B (AKT), mechanistic target of rapamycin (mTOR), and tuberous sclerosis complex 2 (TSC2) was quantified using a BCA protein quantification kit. After denaturation, the protein samples were subjected to sodium dodecylbenzenesulfonate gel electrophoresis (SDS-PAGE) for 0.5 h, followed by membrane transfer at 300 mA constant current for 1.5 h. After blocking the PVDF membrane (Millipore) with skim milk powder, it was incubated with primary antibody overnight at 4°C. The next day, the PVDF membrane was incubated with secondary antibody at room temperature for 1 hour. The PVDF membrane was then moistened with chemiluminescence solution and developed in a fully automated chemiluminescence imaging analysis system (SCG-W2000, Servicebio). Figure 4 As shown. The reagents were sourced from Wuhan Sanying Biotechnology Co., Ltd., Wuhan Huamei Bioengineering Co., Ltd., Aibisin Biotechnology, Jiangsu and Wuhan Sewell Biotechnology Co., Ltd.

[0076] The results of Caspase3, Beclin-1, P-PI3K, P-AKT, P-mTOR, and P-TSC2 in each group of mice are shown in Tables 2-7. The protein expression of P-PI3K, P-AKT, Caspase3, Beclin1, P-mTOR, and P-TSC2 in mouse thyroid tissue was detected by Western blotting. The results showed that compared with the control group, the expression of P-PI3K and P-AKT, key factors of the anti-apoptotic signaling pathway PI3K / AKT, was significantly increased in the GD model group; the expression of P-mTOR and P-TSC2, key factors of the mTOR signaling pathway, was significantly increased; and the expression of Caspase3, a key molecule of apoptosis, and Beclin-1, a key gene of autophagy, was significantly increased. Compared with the GD model group, the expression of key anti-apoptotic signaling pathway PI3K / AKT factors P-PI3K and P-AKT decreased in the traditional Chinese medicine group and the Western medicine group; the expression of key mTOR signaling pathway factors P-mTOR and P-TSC2 decreased; and the expression of key apoptosis molecule Caspase3 and key autophagy gene Beclin-1 decreased significantly (p<0.05), as detailed in Tables 7-12.

[0077] Table 7 Comparison of Caspase 3 results in mice of different groups ( ) Table 8 Comparison of Beclin-1 results in mice of different groups ( ) Table 9 Comparison of P-TSC2 results in mice of different groups ( ) Table 10 Comparison of P-AKT results in mice of different groups ( ) Table 11 Comparison of P-PI3K results in mice of different groups ( ) Table 12 Comparison of P-mTOR results in mice of different groups ( ) In the Western blot results (* indicates a significant difference compared to the control group, P < 0.05; # indicates a significant difference compared to the GD model group, P < 0.05).

[0078] according to Figure 5 As shown, compared with the control group, the expression of P-P13K, P-AKT, P-mTOR, and P-TSC2 proteins was increased in the GD model group (P < 0.05). Compared with the GD model group, the expression of P-P13K, P-mTOR, and P-TSC2 proteins was decreased in both the traditional Chinese medicine group and the Western medicine group (P < 0.05). Although there was no difference in P-AKT protein expression between the groups, it showed a decreasing trend.

[0079] 5. PCR gene relative quantification analysis Total RNA of beclin-1, microtubule-associated protein 1 light chain 3 beta Isoform I (LC3BI), B-cell lymphoma 2 (BCL-2), BCL2-associated X protein (BAX), and caspase 3 was extracted from thyroid tissue using Trizon reagent. mRNA was extracted using an RNA ultrapure extraction kit, and the concentration and purity of the mRNA were determined using a UV-Vis spectrophotometer. cDNA was synthesized using an RNA reverse transcription kit, and quantitative real-time PCR was performed using a fluorescence PCR instrument. The reaction steps were as follows: pre-denaturation 95℃, 10 min; denaturation 95℃, 10 s; annealing 58℃, 30 s; extension 72℃, 30 s; 40 cycles. GAPDH was used as an internal control, and the relative expression level of the gene was determined according to... Calculation by method, see Figure 6 .

[0080] according to Figure 6 Compared with the control group, the expression of Beclin-1 and LC3BI genes was increased in the GD model group (P < 0.05). Compared with the GD model group, the expression of Beclin-1 and LC3BI genes was decreased in both the traditional Chinese medicine group and the Western medicine group (P < 0.05).

[0081] Each experiment was repeated three times. * indicates a significant difference compared to the control group (P < 0.05); # indicates a significant difference compared to the GD model group (P < 0.05). All results are expressed as mean ± standard deviation. The results indicate that SPSS 19.0 was used for statistical analysis, and ANOVA was used for one-way ANOVA among multiple groups. A p-value < 0.05 was considered statistically significant. Graphpad 9.0 software was used for plotting.

[0082] Autophagy and apoptosis are two important types of programmed cell death, dynamically coordinated; imbalance promotes the progression of Graves' disease (GD). During disease, autophagy maintains homeostasis by clearing damaged organelles, while apoptosis mediates the clearance of abnormal cells. Beclin-1 is a recognized regulator of autophagy, modulating endocytosis and LC3-related phagocytosis. LC3B is an LC3 subtype involved in phagocytosis; the conversion of LC3B I to LC3B II during autophagy is crucial for phagocytic vesicle expansion and autophagosome formation. Bcl-2 is considered a key regulator of apoptosis, exhibiting different functions under different conditions. Generally, it primarily functions as an anti-apoptotic agent, but Bcl-2 can also participate in the cleavage of some pro-apoptotic proteins, including Bax, or progressively activate Caspase-3 to induce cell membrane permeability and cell death.

[0083] Autophagy and apoptosis are interconnected through the Beclin-1 and Bcl-2 complex. Beclin-1 expression promotes Bcl-2 expression, while Bcl-2 expression inhibits Beclin-1 function. Therefore, the herbal composition of this application inhibited apoptosis in thyroid cells of the GD mouse model, and the increase in Bcl-2 expression further inhibited autophagy. Thus, the herbal composition of this application and the decrease in thyroid hormone levels are related to thyroid cell survival resulting from autophagy and apoptosis homeostasis.

[0084] The PI3K / AKT / mTOR signaling pathway is closely related to autophagy and apoptosis. Beclin-1 can further affect PI3K activity. AKT is a key downstream target of PI3K, and its signal activation plays a crucial role in promoting cell growth, proliferation, and inhibiting apoptosis. mTOR can form two complexes, mTORC1 and mTORC2. mTORC2 is both a downstream effector of AKT and can activate AKT signaling by phosphorylating AKT, jointly promoting cell survival, growth, and proliferation. This study demonstrates that the traditional Chinese medicine composition of this application inhibited the expression of the PI3K / AKT / mTOR signaling pathway in a GD mouse model, and the multiple effects of traditional Chinese medicine on thyroid cells may be related to this. In conclusion, the traditional Chinese medicine composition of this application can improve the activity and mental state of a GD mouse model and inhibit the release of thyroid hormones into the bloodstream during the disease process.

[0085] Finally, it should be noted that the above descriptions are merely optional examples of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A traditional Chinese medicine composition for treating toxic diffuse goiter, characterized in that, According to parts by weight, it includes the following ingredients: 10-25 parts of Hedyotis diffusa, 10-25 parts of Verbena officinalis, 8-20 parts of Pinellia ternata, 8-15 parts of Amomum tsao-ko, 3-10 parts of Acorus tatarinowii, 8-20 parts of Paris polyphylla, 8-15 parts of Prunus persica, 8-15 parts of Pogostemon cablin, 8-15 parts of Eupatorium fortunei, and 3-8 parts of Glycyrrhiza uralensis.

2. The traditional Chinese medicine composition for treating toxic diffuse goiter according to claim 1, characterized in that, The weight ratio of Hedyotis diffusa and Verbena officinalis is 1:

1.

3. The traditional Chinese medicine composition for treating toxic diffuse goiter according to claim 1, characterized in that, The traditional Chinese medicine composition also includes cat's claw grass, and the mass ratio of cat's claw grass to white flower tongue grass and verbena is 1:

3.

4. The traditional Chinese medicine composition for treating toxic diffuse goiter according to claim 1, characterized in that, It also includes 24-40 portions of Job's tears; The mass ratio of the Job's tears to Hedyotis diffusa and Verbena officinalis is 1:

1.

5. The traditional Chinese medicine composition for treating toxic diffuse goiter according to claim 1, characterized in that, The mass ratio of the coix seed to the licorice is 6:

1.

6. The traditional Chinese medicine composition for treating toxic diffuse goiter according to claim 1, characterized in that, The mass ratio of patchouli to eupatorium is (1-1.5):

1.

7. A traditional Chinese medicine preparation for treating toxic diffuse goiter, characterized in that, Includes the traditional Chinese medicine composition as described in any one of claims 1-6.

8. A method for preparing a traditional Chinese medicine composition for treating toxic diffuse goiter, used to prepare the traditional Chinese medicine composition as described in any one of claims 1-6, characterized in that, Includes the following steps: S1. Weigh all the raw materials according to the stated weight proportions, add cold water and soak for 2-3 hours, boil for 2-3 hours and then filter to obtain the first medicinal liquid and the dregs. S2. Add warm water to the dregs, boil for 1-2 hours, and then filter to obtain the second medicinal liquid; S3. After mixing the first and second medicinal solutions, filter and boil to obtain the traditional Chinese medicine composition.

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