Composition for treating thyroid nodules and preparation method thereof

Abstract

The invention relates to a composition for treating thyroid nodules and a preparation method thereof, and belongs to the technical field of biological medicine, the composition comprises trepang body wall fibrinolytic small peptides, kelp small molecule active components, a selfheal-saussurea lappa composite extract and a safflower-ligusticum wallichii composite extract; the sea cucumber body wall fibrinolytic small peptide is a product obtained by performing common enzymolysis on sea cucumber body wall powder through alkaline pectinase, bromelain and trypsin and then performing common enzymolysis through subtilisin and lumbrukinase. The kelp micromolecular active component is a product obtained by performing enzymolysis extraction on kelp powder through alginate lyase, performing purification through an X-5 macroporous adsorption resin column and performing ultrafiltration. The selfheal-saussurea lappa composite extract is a product obtained by extracting selfheal powder and saussurea lappa powder with an ethanol water solution, purifying with a polyamide resin column and performing ultrafiltration; the safflower-Szechuan lovage rhizome composite extract is a product obtained by extracting safflower powder and Szechuan lovage rhizome powder with an ethanol aqueous solution, purifying with an NKA-9 macroporous resin column and performing ultrafiltration.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a composition for treating thyroid nodules and its preparation method. Background Technology

[0002] Thyroid nodules are a common endocrine disorder with a high prevalence. Most are benign, but some carry a risk of malignancy or cause compressive symptoms due to their large size, thus necessitating significant treatment. Currently, drug therapy is an important non-surgical intervention, encompassing various forms including traditional Chinese medicine, Western medicine, oral medication, and topical application. If left untreated, thyroid nodules can gradually enlarge, leading to cysts, calcification, and bleeding. In severe cases, they can cause compressive symptoms, affecting breathing and eating, or even become malignant.

[0003] The theoretical basis for Western medicine treatment of thyroid nodules stems from modern medicine's understanding of thyroid function and the causes of nodules. For nodules accompanied by hyperthyroidism, antithyroid drugs (such as methimazole and propylthiouracil) are commonly used orally, controlling symptoms by inhibiting thyroid hormone synthesis, but their effect on reducing nodule size is limited. For iodine deficiency-related nodules, "suppression therapy" of levothyroxine supplementation was once widely used. Based on the feedback regulation mechanism to reduce thyroid-stimulating hormone secretion, it is theoretically possible to inhibit nodule growth. However, recent studies have shown that its efficacy varies greatly among individuals, and long-term use may increase the risk of osteoporosis, thus its clinical application is gradually being limited.

[0004] Traditional Chinese medicine believes that thyroid nodules are caused by long-term unbalanced diet or emotional distress leading to decreased spleen and stomach function, coupled with liver qi stagnation, spleen dysfunction, phlegm and dampness accumulation, and qi and blood stagnation. The phlegm and dampness then condense in the neck, forming thyroid nodules. Currently, besides surgery, the most effective treatment for thyroid nodules is traditional Chinese medicine therapy to soften, break down, and eliminate the nodules.

[0005] Oral Chinese medicine often uses compound preparations, such as those containing kelp, seaweed, kelp, pinellia, and prunella vulgaris, which are known for their soothing properties. These have been used clinically for hundreds of years. Modern pharmacological studies have confirmed that some components can regulate thyroid function and inhibit fibrosis. However, if the combination is not properly balanced, long-term use may cause side effects such as kidney dysfunction. External application of medicine delivers medication directly to the affected area, reducing the side effects of systemic medication. Traditional Chinese medicine external applications often use ointments or powders containing cinnamon and musk, which are absorbed through the skin to warm the meridians, promote blood circulation, and reduce swelling. These are suitable for patients with hard nodules and long-term conditions. Modern external preparations are improvements on traditional formulas, such as ultrasonic transdermal patches, which combine physical penetration technology to enhance drug absorption efficiency. Clinically, these are often used in conjunction with oral medications to improve efficacy, but their effectiveness is limited when used alone.

[0006] Overall, drug treatment for thyroid nodules still has limitations in efficacy, and there is still a need to continuously develop safe and effective treatment drugs. Summary of the Invention

[0007] Addressing the issues of poor safety, side effects, and especially the slow efficacy and long treatment courses of traditional Chinese medicine (TCM) in existing Western and oral TCM treatments, this invention provides a composition for treating thyroid nodules and its preparation method. It utilizes a special enzymatic hydrolysis method to prepare fibrinolytic peptides from sea cucumber body walls, and a special extraction method to prepare small-molecule active components from kelp, a complex extract of Prunella vulgaris and Aucklandia lappa, and a complex extract of Carthamus tinctorius and Ligusticum chuanxiong. These components are used in specific proportions to achieve synergistic effects, exhibiting significant anti-fibrotic and pro-apoptotic effects. In terms of anti-fibrosis, it effectively inhibits the TGF-β1-induced fibrosis process in human thyroid fibroblasts (HThyF); in terms of pro-apoptosis, it promotes apoptosis in papillary thyroid carcinoma cells (TPC-1). Topical application reduces the systemic side effects of oral medications, providing a safe and efficient treatment option for thyroid nodules. The specific technical solution is as follows:

[0008] A composition for treating thyroid nodules, comprising the following raw materials in parts by weight: 3.0 to 5.0 parts of sea cucumber body wall fibrinolytic peptides, 2.0 to 3.5 parts of kelp small molecule active components, 2.5 to 4.0 parts of Prunella vulgaris-Saussurea costus complex extract, and 2.0 to 3.5 parts of Carthamus tinctorius-Ligusticum chuanxiong complex extract; wherein the sea cucumber body wall fibrinolytic peptides are products with a Da lower than 1000 obtained by enzymatic hydrolysis of sea cucumber body wall powder by alkaline pectinase, bromelain, and trypsin, followed by enzymatic hydrolysis by Bacillus subtilis protease and lumbrokinase; wherein the kelp small molecule active components are kelp powder hydrolyzed by alginate... The extract was obtained by enzymatic hydrolysis with lyase, purification by X-5 macroporous adsorption resin column, and ultrafiltration, yielding a product with a Da lower than 1500; the Prunella vulgaris-Saussurea costus complex extract was obtained by extracting Prunella vulgaris powder and Saussurea costus powder at a mass ratio of (3-4):(1-1.5) with ethanol-water solution, purifying the extract with polyamide resin column, and ultrafiltration, yielding a product with a Da lower than 2000; the Carthamus tinctorius-Ligusticum striatum complex extract was obtained by extracting Carthamus tinctorius powder and Ligusticum striatum powder at a mass ratio of (2-3):(1-1.5) with ethanol-water solution, purifying the extract with NKA-9 macroporous resin column, and ultrafiltration, yielding a product with a Da lower than 1500.

[0009] The preparation method of the sea cucumber body wall fibrinolytic peptide in the above composition includes: adding sea cucumber body wall powder to a phosphate buffer solution with a pH of 7.5-8.0, mixing well, adding alkaline pectinase, bromelain and trypsin, enzymatically hydrolyzing at 37℃-42℃ for 2h-2.5h, inactivating the enzymes, adding subtilisin and lumbrokinase, enzymatically hydrolyzing at 37℃-42℃ for 2h-2.5h, inactivating the enzymes, allowing to stand, centrifuging, taking the supernatant, ultrafiltration through an ultrafiltration membrane to obtain a component with less than 1000 Da, freeze-drying to obtain sea cucumber body wall fibrinolytic peptide.

[0010] In the above-mentioned method for preparing sea cucumber body wall fibrinolytic peptides, the sea cucumber body wall powder is obtained by freeze-drying the sea cucumber body wall after removing the viscera, and then pulverizing it into powder that passes through an 80-100 mesh sieve; the sea cucumber body wall powder is added to a phosphate buffer solution with a pH of 7.5-8.0 at a material-to-liquid mass ratio of 1:(8-10); the amount of alkaline pectinase added is 0.5%-1.0% of the mass of the sea cucumber body wall powder, the amount of bromelain added is 0.5%-1.0% of the mass of the sea cucumber body wall powder, and the amount of trypsin... The amount of white enzyme added is 0.3% to 0.5% of the mass of sea cucumber body wall powder, the amount of Bacillus subtilis protease added is 0.5% to 0.8% of the mass of sea cucumber body wall powder, and the amount of lumbrokinase added is 0.3% to 0.5% of the mass of sea cucumber body wall powder; the enzyme inactivation is carried out at 80℃ to 85℃ for 10 to 15 minutes; the standing is carried out at 4℃ to 6℃ for 1 to 2 hours; the centrifugation is carried out at 8000g to 10000g for 15 to 20 minutes.

[0011] The preparation method of the kelp small molecule active component in the above composition includes: adding kelp powder to deionized water, adjusting the pH to 5.5-6.0 with hydrochloric acid aqueous solution, adding alginate lyase, enzymatically hydrolyzing at 30℃-35℃ for 1-1.5h, inactivating the enzyme, and obtaining the enzymatic hydrolysate; adding 60%-70% of the volume of the enzymatic hydrolysate in anhydrous ethanol, allowing it to stand, centrifuging, concentrating under reduced pressure, taking the supernatant, loading it onto an X-5 macroporous adsorption resin column, rinsing with deionized water until the eluent is colorless, eluting with ethanol aqueous solution, collecting the eluent, ultrafiltration through an ultrafiltration membrane, taking the component below 1500 Da, freeze-drying, and obtaining the kelp small molecule active component.

[0012] In the above-mentioned method for preparing the small molecule active components of kelp, the kelp powder is obtained by pulverizing dried kelp and passing it through an 80-100 mesh sieve; the kelp powder is added to deionized water at a mass ratio of 1:(15-20); the concentration of hydrochloric acid aqueous solution is 0.5mol / L-1mol / L; the amount of alginate lyase added is 1.5%-3% of the mass of kelp powder; enzyme inactivation is performed at 90℃-95℃ for 10-20 minutes; standing is performed at 4℃-6℃ for 1-2 hours; centrifugation is performed at 8000g-10000g for 10-15 minutes; vacuum concentration is performed at 50℃-55℃ to 10%-15% of the volume; and elution is performed with 60%-70% volume concentration ethanol aqueous solution for 2-3 column volumes.

[0013] The preparation method of the above composition of the Prunella vulgaris-Saussurea costus complex extract includes: mixing Prunella vulgaris powder and Saussurea costus powder at a mass ratio of (3-4):(1-1.5) to obtain a complex powder; adding the complex powder to an ethanol aqueous solution; heating and refluxing at 75℃-80℃ for 2-3 hours; concentrating under reduced pressure to obtain a concentrated solution; centrifuging; taking the supernatant; loading the solution onto a polyamide resin column; rinsing with deionized water to remove impurities; rinsing with a 40%-50% volume concentration ethanol aqueous solution to remove impurities; eluting with a 60%-70% volume concentration ethanol aqueous solution; collecting the eluent; ultrafiltration through an ultrafiltration membrane; taking the fraction with a value below 2000 Da; concentrating under reduced pressure; and freeze-drying to obtain the Prunella vulgaris-Saussurea costus complex extract.

[0014] In the preparation method of the above-mentioned Prunella vulgaris-Saussurea costus compound extract, the Prunella vulgaris powder is Prunella vulgaris powder pulverized through a 60-80 mesh sieve; the Saussurea costus powder is Saussurea costus powder pulverized through a 60-80 mesh sieve; the compound powder is added to a 70%-75% volume concentration ethanol aqueous solution at a material-to-liquid mass ratio of 1:(10-12); the ethanol is removed by vacuum concentration at 50-55℃; the centrifugation is performed at 8000g-10000g for 10-15min; an 80-120 mesh polyamide resin is placed in a polyamide resin column; the column is rinsed with deionized water for 1-2 column volumes; the column is rinsed with a 40%-50% volume concentration ethanol aqueous solution for 1.5-2 column volumes; the column is eluted with a 60%-70% volume concentration ethanol aqueous solution for 2-3 column volumes; and the concentration is performed by vacuum concentration at 50-55℃.

[0015] The preparation method of the safflower-Ligusticum striatum compound extract in the above composition includes: mixing safflower powder and Ligusticum striatum powder at a mass ratio of (2-3):(1-1.5) to obtain a mixed powder; adding the mixed powder to an ethanol aqueous solution; heating and refluxing at 75℃-80℃ for 2-3 hours; concentrating under reduced pressure to obtain a concentrated extract; centrifuging; taking the supernatant; loading the extract onto an NKA-9 macroporous resin column; rinsing with deionized water to remove impurities; rinsing with a 20%-30% volume concentration ethanol aqueous solution to remove impurities; eluting with an aqueous solution containing 0.08%-0.12% ammonia and 50%-55% volume concentration ethanol; collecting the eluent; adjusting the pH to 6-7; ultrafiltration through an ultrafiltration membrane; taking the fraction below 1500 Da; concentrating under reduced pressure; and freeze-drying to obtain the safflower-Ligusticum striatum compound extract.

[0016] In the above preparation method of safflower-Ligusticum striatum compound extract, safflower powder is safflower powder pulverized through a 60-80 mesh sieve; Ligusticum striatum powder is Ligusticum striatum powder pulverized through a 60-80 mesh sieve; the mixed powder is added to a 70%-75% volume concentration ethanol aqueous solution at a material-to-liquid mass ratio of 1:(10-12); the ethanol is removed by vacuum concentration at 50-55℃; the mixture is centrifuged at 8000g-10000g for 10-15min; the mixture is rinsed with deionized water for 1-2 column volumes; the mixture is rinsed with a 20%-30% volume concentration ethanol aqueous solution for 1-2 column volumes; the mixture is eluted with an aqueous solution containing 0.08%-0.12% ammonia and 50%-55% volume concentration ethanol for 2-3 column volumes; the pH of the eluent is adjusted to 6-7 with an acetic acid aqueous solution; and the mixture is concentrated under vacuum at 50-55℃.

[0017] The preparation method of the above-mentioned composition for treating thyroid nodules includes the following steps:

[0018] The sea cucumber body wall fibrinolytic peptides, kelp small molecule active components, Prunella vulgaris-Saussurea costus complex extract and Carthamus tinctorius-Ligusticum striatum complex extract were mixed according to the mass fractions to obtain a composition; the composition was then used to formulate a dressing with a skin penetration enhancer and excipients available in pharmaceuticals.

[0019] The present invention provides a composition for treating thyroid nodules and a method for preparing the same, which has the following beneficial effects:

[0020] I. The pharmaceutical composition of this invention, through optimized raw material ratios and extraction processes, achieves synergistic effects among its components, exhibiting significant anti-fibrotic and pro-apoptotic effects. In terms of anti-fibrosis, it effectively inhibits the fibrotic process induced by TG F-β1 in human thyroid fibroblasts (HThyF); in terms of pro-apoptosis, it promotes apoptosis in papillary thyroid carcinoma cells (TPC-1). This multi-component synergistic therapeutic effect provides a safe and efficient option for the treatment of thyroid nodules.

[0021] II. Sea Cucumber Body Wall Fibrinolytic Peptides: Through specific enzymatic hydrolysis steps (using alkaline pectinase, bromelain, trypsin, subtilisin, and lumbrokinase in a stepwise manner, controlling parameters such as hydrolysis temperature, time, and pH), products with a value below 1000 Da are obtained. These peptides can effectively inhibit fibroblast activity and are a key component in anti-fibrosis. The absence of any of these enzymes or a change in the type of enzyme will lead to a decrease in fibrinolytic activity and a weakened anti-fibrotic effect.

[0022] Sea cucumber body wall fibrinolytic peptides possess fibrinolytic activity, targeting fibroblasts and inhibiting TGF-β1-induced fibroblast activity, thereby reducing fibrosis tissue proliferation and achieving an anti-fibrotic effect. TGF-β1 can induce bone marrow mesenchymal stem cells to differentiate into myofibroblasts in vivo, promoting fibrosis-related processes such as fibroblast proliferation and extracellular matrix synthesis. Fibrinolytic peptides inhibit TGF-β1-induced fibroblast activity by interfering with its signaling pathway. In experiments studying the effects of blocking peptides on fibroblast differentiation into myofibroblasts, the blocking peptides interfered with related signal transduction and inhibited the expression of pro-fibrosis genes.

[0023] III. Small molecule active components of kelp: After enzymatic hydrolysis with alginate lyase, combined with ethanol precipitation and purification using X-5 macroporous adsorption resin column (controlling parameters such as enzymatic hydrolysis pH, temperature, time, and eluent concentration), components with a molecular weight below 1500 Da are obtained, which can effectively enhance the anti-fibrotic effect.

[0024] IV. Prunella vulgaris-Saussurea costus compound extract: Mixed at a specific mass ratio, extracted with ethanol aqueous solution and purified by polyamide resin column (controlling extraction temperature, time, eluent concentration, etc.) to obtain components with less than 2000 Da, which can relieve depression and dissipate nodules, inhibit fibrous tissue proliferation and promote TPC-1 apoptosis.

[0025] V. Safflower-Ligusticum striatum compound extract: Mixed in a specific ratio, extracted with ethanol-water solution and purified by NKA-9 macroporous resin column (with controlled extraction and purification parameters), the fraction with less than 1500 Da was obtained. It has a synergistic effect in anti-inflammatory, anti-fibrotic and pro-apoptotic effects; it can also regulate qi and blood, promote blood circulation and drug penetration, and further promote the apoptosis process.

[0026] VI. Advantages of the extraction and purification method of the components in this invention compared with conventional extraction methods: Conventional extraction methods suffer from problems such as low or missing effective active ingredients, insufficient extraction, low purity of effective ingredients, and numerous impurities. This method, through multiple enzymatic hydrolysis techniques, specific resin purification, ultrafiltration, and other steps, can specifically extract and purify effective ingredients, remove impurities, and obtain low molecular weight active components. This improves the activity and purity of the components, enhances the therapeutic effect of the drug, facilitates skin penetration, reduces systemic side effects from oral administration, and minimizes side effects caused by impurities. Furthermore, precise control of parameters in each step ensures stable acquisition of effective ingredients, making the drug performance more reliable.

[0027] VII. The synergistic effect of the components in the drug formulation of this invention is significant: sea cucumber body wall fibrinolytic peptides mainly inhibit fibroblast activity, while kelp small molecule components enhance its anti-fibrotic effect; the Prunella vulgaris-Saussurea costus complex extract and the Carthamus tinctorius-Ligusticum chuanxiong complex extract, through rational matching, synergistically enhance the effect, softening and dispersing nodules, and promoting TPC-1 apoptosis. By optimizing the raw material ratio and extraction process, the four components work together to exert anti-fibrotic and apoptosis-promoting effects, thus improving the treatment effect of thyroid nodules. However, when the proportion of each component is unbalanced, the extraction process is defective, or the components are replaced, the synergistic effect is destroyed, and the effect will be significantly reduced. Detailed Implementation

[0028] The present invention will be further described below with reference to specific implementation examples, but the present invention is not limited to these embodiments.

[0029] Example 1

[0030] A composition for treating thyroid nodules, comprising the following raw materials in parts by weight: 4 parts sea cucumber body wall fibrinolytic peptides, 2.8 parts kelp small molecule active components, 3.2 parts Prunella vulgaris-Saussurea costus compound extract, and 3 parts Carthamus tinctorius-Ligusticum striatum compound extract.

[0031] The preparation method of sea cucumber body wall fibrinolytic peptides includes: freeze-drying the sea cucumber body wall after removing the viscera, pulverizing it into powder that passes through an 80-mesh sieve to obtain sea cucumber body wall powder; adding the sea cucumber body wall powder to a phosphate buffer solution at pH 7.8 at a mass ratio of 1:9, mixing well, adding 0.8% alkaline pectinase, 0.8% bromelain and 0.4% trypsin based on the mass of the sea cucumber body wall powder, enzymatically hydrolyzing at 40℃ for 2 hours, inactivating the enzyme at 82℃ for 12 minutes, adding 0.6% subtilisin and 0.4% lumbrokinase based on the mass of the sea cucumber body wall powder, enzymatically hydrolyzing at 40℃ for 2 hours, inactivating the enzyme at 82℃ for 12 minutes, letting it stand at 5℃ for 1.5 hours, centrifuging at 9000g for 18 minutes, taking the supernatant, ultrafiltration through an ultrafiltration membrane to obtain a component with a value below 1000 Da, freeze-drying to obtain sea cucumber body wall fibrinolytic peptides.

[0032] The preparation method of the small molecule active component of kelp includes: kelp is dried and then pulverized, passed through an 80-mesh sieve to obtain kelp powder; kelp powder is added to deionized water at a mass ratio of 1:18, the pH is adjusted to 5.8 with 0.8 mol / L hydrochloric acid aqueous solution, 2% (by mass) of alginate lyase is added, enzymatic hydrolysis is performed at 32℃ for 1 h, and enzyme inactivation is performed at 92℃ for 15 min to obtain the enzymatic hydrolysate; anhydrous ethanol (65% by volume of the enzymatic hydrolysate) is added, the mixture is allowed to stand at 5℃ for 1.5 h, centrifuged at 9000g for 12 min, the supernatant is collected, concentrated under reduced pressure at 52℃ to 12% of its volume, loaded onto an X-5 macroporous adsorption resin column (diameter-to-height ratio 1:10), washed with deionized water until the eluent is colorless, eluted with 65% (v / v) ethanol aqueous solution for 2.5 column volumes, the eluent is collected, ultrafiltered through an ultrafiltration membrane, the fraction with a value below 1500 Da is collected, and freeze-dried to obtain the small molecule active component of kelp.

[0033] The preparation method of the Prunella vulgaris-Saussurea costus compound extract includes: Prunella vulgaris is pulverized and passed through an 80-mesh sieve to obtain Prunella vulgaris powder; Saussurea costus is pulverized and passed through an 80-mesh sieve to obtain Saussurea costus powder; Prunella vulgaris powder and Saussurea costus powder are mixed at a mass ratio of 3.5:1.2 to obtain a compound powder; the compound powder is added to a 72% volume concentration ethanol aqueous solution at a material-to-liquid mass ratio of 1:11; the mixture is heated under reflux at 78℃ for 2.5 hours; the ethanol is removed by vacuum concentration at 52℃ to obtain a concentrated solution; and the solution is centrifuged at 9000g for 1 hour. After 2 minutes, the supernatant was collected and loaded onto a polyamide resin column (diameter-to-height ratio 1:10) with a particle size range of 80-120 mesh. The column was rinsed with deionized water for 1.5 column volumes to remove impurities, then rinsed with 45% ethanol aqueous solution for 1.5 column volumes to remove impurities, and finally eluted with 65% ethanol aqueous solution for 2.5 column volumes. The eluent was collected, ultrafiltered through an ultrafiltration membrane, and the fraction with a value below 2000 Da was collected. The fraction was concentrated under reduced pressure at 52℃ and freeze-dried to obtain the Prunella vulgaris-Saussurea costus complex extract.

[0034] The preparation method of the safflower-Ligusticum striatum compound extract includes: safflower is pulverized and passed through an 80-mesh sieve to obtain safflower powder; Ligusticum striatum is pulverized and passed through an 80-mesh sieve to obtain Ligusticum striatum powder; safflower powder and Ligusticum striatum powder are mixed at a mass ratio of 2.5:1.3 to obtain a mixed powder; the mixed powder is added to a 72% volume concentration ethanol aqueous solution at a material-liquid mass ratio of 1:11; the mixture is heated under reflux at 78℃ for 2.5 h; the ethanol is removed by vacuum concentration at 52℃ to obtain a concentrated extract; the extract is centrifuged at 9000g for 12 min; the supernatant is collected and loaded onto an NKA- A 9-pore resin column (diameter-to-height ratio 1:10) was used for 1.5 column volumes of rinsing with deionized water to remove impurities, followed by 1.5 column volumes of rinsing with a 25% (v / v) ethanol aqueous solution for further impurities. The eluent was then collected and adjusted to pH 6.5 with a 0.1% (v / v) acetic acid aqueous solution. The eluent was then subjected to ultrafiltration, and the fraction with a molecular weight below 1500 Da was collected. The fraction was concentrated under reduced pressure at 52°C and freeze-dried to obtain the safflower-Ligusticum striatum compound extract.

[0035] The preparation method of the above-mentioned composition for treating thyroid nodules includes the following steps: mixing sea cucumber body wall fibrinolytic peptides, kelp small molecule active components, Prunella vulgaris-Saussurea costus complex extract and safflower-Ligusticum striatum complex extract according to the mass fractions to obtain the composition.

[0036] Example 2

[0037] A composition for treating thyroid nodules, comprising the following raw materials in parts by weight: 3.0 parts of sea cucumber body wall fibrinolytic peptide, 3.5 parts of kelp small molecule active component, 2.5 parts of Prunella vulgaris-Saussurea costus compound extract and 3.5 parts of Carthamus tinctorius-Ligusticum striatum compound extract.

[0038] The preparation method of sea cucumber body wall fibrinolytic peptides includes: freeze-drying the sea cucumber body wall after removing the viscera, pulverizing it into powder that passes through an 80-mesh sieve to obtain sea cucumber body wall powder; adding the sea cucumber body wall powder to a phosphate buffer solution at a mass ratio of 1:10 (pH 7.5), mixing well, adding 1.0% alkaline pectinase, 0.5% bromelain and 0.5% trypsin by weight of sea cucumber body wall powder, enzymatically hydrolyzing at 37℃ for 2.5 h, inactivating the enzyme at 80℃ for 15 min, adding 0.5% subtilisin and 0.5% lumbrokinase by weight of sea cucumber body wall powder, enzymatically hydrolyzing at 37℃ for 2.5 h, inactivating the enzyme at 80℃ for 15 min, letting it stand at 4℃ for 2 h, centrifuging at 8000g for 20 min, taking the supernatant, ultrafiltration through an ultrafiltration membrane to obtain a component with a value below 1000 Da, freeze-drying to obtain sea cucumber body wall fibrinolytic peptides.

[0039] The preparation method of the small molecule active components of kelp includes: kelp is dried and then pulverized, passed through an 80-mesh sieve to obtain kelp powder; kelp powder is added to deionized water at a mass ratio of 1:20, the pH is adjusted to 6.0 with 0.5 mol / L hydrochloric acid aqueous solution, 1.5% alginate lyase by mass of kelp powder is added, enzymatic hydrolysis is performed at 35℃ for 1 h, and enzyme inactivation is performed at 95℃ for 10 min to obtain enzymatic hydrolysate; anhydrous ethanol is added at 70% of the volume of the enzymatic hydrolysate, the mixture is allowed to stand at 4℃ for 2 h, centrifuged at 8000g for 15 min, the supernatant is collected, concentrated under reduced pressure at 50℃ to 15% of the volume, loaded onto an X-5 macroporous adsorption resin column (diameter-to-height ratio 1:10), washed with deionized water until the eluent is colorless, eluted with 60% volume concentration ethanol aqueous solution for 3 column volumes, the eluent is collected, ultrafiltered through an ultrafiltration membrane, the fraction with a value below 1500 Da is collected, and freeze-dried to obtain the small molecule active components of kelp.

[0040] The preparation method of the Prunella vulgaris-Saussurea costus complex extract includes: Prunella vulgaris is pulverized and passed through a 60-mesh sieve to obtain Prunella vulgaris powder; Saussurea costus is pulverized and passed through an 80-mesh sieve to obtain Saussurea costus powder; Prunella vulgaris powder and Saussurea costus powder are mixed at a mass ratio of 3:1.5 to obtain a complex powder; the complex powder is added to a 75% volume concentration ethanol aqueous solution at a material-liquid mass ratio of 1:10; the mixture is heated and refluxed at 75℃ for 3 hours; the ethanol is removed by vacuum concentration at 50℃ to obtain a concentrated solution; the solution is centrifuged at 10000g for 10 minutes; the supernatant is collected and loaded onto a polyamide resin column (diameter-to-height ratio 1:10) with a particle size range of 80-120 mesh; the solution is washed with deionized water for 2 column volumes to remove impurities; washed with a 40% volume concentration ethanol aqueous solution for 2 column volumes to remove impurities; and eluted with a 60% volume concentration ethanol aqueous solution for 3 column volumes. The eluent is collected, ultrafiltered through an ultrafiltration membrane, and the fraction with a particle size below 2000 Da is collected; the solution is concentrated under vacuum at 50℃ and freeze-dried to obtain the Prunella vulgaris-Saussurea costus complex extract.

[0041] The preparation method of the safflower-Ligusticum striatum compound extract includes: safflower is pulverized through an 80-mesh sieve to obtain safflower powder; Ligusticum striatum is pulverized through a 60-mesh sieve to obtain Ligusticum striatum powder; safflower powder and Ligusticum striatum powder are mixed at a mass ratio of 3:1 to obtain a mixed powder; the mixed powder is added to a 70% volume concentration ethanol aqueous solution at a material-liquid mass ratio of 1:12; the mixture is heated under reflux at 80℃ for 2 hours; the ethanol is removed by vacuum concentration at 55℃ to obtain a concentrated extract; and the extract is centrifuged at 8000g for 15 minutes. n. Take the supernatant and load it onto an NKA-9 macroporous resin column (diameter-to-height ratio 1:10). Elute with deionized water for 1 column volume to remove impurities, then elute with a 30% (v / v) ethanol aqueous solution for 1 column volume to remove impurities. Elute with an aqueous solution containing 0.12% ammonia and 50% (v / v) ethanol for 3 column volumes. Collect the eluent and adjust the pH of the eluent to 6 with a 0.1% (v / v) acetic acid aqueous solution. Ultrafilter the eluent through an ultrafiltration membrane, collect the fraction below 1500 Da, concentrate under reduced pressure at 55℃, and freeze-dry to obtain the safflower-Ligusticum striatum compound extract.

[0042] The preparation method of the above-mentioned composition for treating thyroid nodules includes the following steps: mixing sea cucumber body wall fibrinolytic peptides, kelp small molecule active components, Prunella vulgaris-Saussurea costus complex extract and safflower-Ligusticum striatum complex extract according to the mass fractions to obtain the composition.

[0043] Example 3

[0044] A composition for treating thyroid nodules, comprising the following raw materials in parts by weight: 5.0 parts of sea cucumber body wall fibrinolytic peptide, 2.0 parts of kelp small molecule active component, 4.0 parts of Prunella vulgaris-Saussurea costus compound extract and 2.0 parts of Carthamus tinctorius-Ligusticum striatum compound extract.

[0045] The preparation method of sea cucumber body wall fibrinolytic peptides includes: freeze-drying the sea cucumber body wall after removing the viscera, pulverizing it into powder that passes through a 100-mesh sieve to obtain sea cucumber body wall powder; adding the sea cucumber body wall powder to a phosphate buffer solution at a mass ratio of 1:8 (pH 8.0), mixing well, adding 0.5% alkaline pectinase, 1.0% bromelain and 0.3% trypsin based on the mass of the sea cucumber body wall powder, enzymatically hydrolyzing at 42℃ for 2 hours, inactivating the enzyme at 85℃ for 10 minutes, adding 0.8% subtilisin and 0.3% lumbrokinase based on the mass of the sea cucumber body wall powder, enzymatically hydrolyzing at 42℃ for 2 hours, inactivating the enzyme at 85℃ for 10 minutes, letting it stand at 6℃ for 1 hour, centrifuging at 10000g for 15 minutes, taking the supernatant, ultrafiltration through an ultrafiltration membrane to obtain a component with a value below 1000 Da, freeze-drying to obtain sea cucumber body wall fibrinolytic peptides.

[0046] The preparation method of the small molecule active components of kelp includes: kelp is dried and then pulverized, passed through a 100-mesh sieve to obtain kelp powder; kelp powder is added to deionized water at a mass ratio of 1:15, pH is adjusted to 5.5 with 1 mol / L hydrochloric acid aqueous solution, 3% alginate lyase by mass of kelp powder is added, enzymatic hydrolysis is performed at 30℃ for 1.5 h, and enzyme inactivation is performed at 90℃ for 20 min to obtain enzymatic hydrolysate; anhydrous ethanol is added at 60% of the volume of enzymatic hydrolysate, the mixture is allowed to stand at 6℃ for 1 h, centrifuged at 10000g for 10 min, the supernatant is collected, concentrated under reduced pressure at 55℃ to 10% of the volume, loaded onto an X-5 macroporous adsorption resin column (diameter-to-height ratio 1:10), washed with deionized water until the eluent is colorless, eluted with 70% volume concentration ethanol aqueous solution for 2 column volumes, the eluent is collected, ultrafiltered through an ultrafiltration membrane, and the fraction with a value below 1500 Da is obtained, freeze-dried to obtain the small molecule active components of kelp.

[0047] The preparation method of the Prunella vulgaris-Saussurea costus complex extract includes: Prunella vulgaris is pulverized and passed through an 80-mesh sieve to obtain Prunella vulgaris powder; Saussurea costus is pulverized and passed through a 60-mesh sieve to obtain Saussurea costus powder; Prunella vulgaris powder and Saussurea costus powder are mixed at a mass ratio of 4:1 to obtain a complex powder; the complex powder is added to a 70% volume concentration ethanol aqueous solution at a material-liquid mass ratio of 1:12; the mixture is heated and refluxed at 80℃ for 2 hours; the ethanol is removed by vacuum concentration at 55℃ to obtain a concentrated solution; the solution is centrifuged at 8000g for 15 minutes; the supernatant is collected and loaded onto a polyamide resin column (diameter-to-height ratio 1:10) with a particle size range of 80-120 mesh; the solution is washed with deionized water for 1 column volume to remove impurities; washed with 50% volume concentration ethanol aqueous solution for 1.5 column volumes to remove impurities; and eluted with 70% volume concentration ethanol aqueous solution for 2 column volumes. The eluent is collected, ultrafiltered through an ultrafiltration membrane, and the fraction with a particle size below 2000 Da is collected; the solution is concentrated under vacuum at 55℃ and freeze-dried to obtain the Prunella vulgaris-Saussurea costus complex extract.

[0048] The preparation method of the safflower-Ligusticum striatum compound extract includes: safflower is pulverized through a 60-mesh sieve to obtain safflower powder; Ligusticum striatum is pulverized through an 80-mesh sieve to obtain Ligusticum striatum powder; safflower powder and Ligusticum striatum powder are mixed at a mass ratio of 2:1.5 to obtain a mixed powder; the mixed powder is added to a 75% volume concentration ethanol aqueous solution at a material-liquid mass ratio of 1:10; the mixture is heated under reflux at 75℃ for 3 hours; the ethanol is removed by vacuum concentration at 50℃ to obtain a concentrated extract; the extract is centrifuged at 10000g for 10 minutes; the supernatant is collected and loaded onto NK cells. The extract was prepared by eluting an A-9 macroporous resin column (diameter-to-height ratio 1:10) for two column volumes with deionized water, followed by two column volumes with a 20% (v / v) ethanol aqueous solution. The extract was then eluted for two column volumes with an aqueous solution containing 0.08% ammonia and 55% (v / v) ethanol. The eluent was collected and adjusted to pH 7 with a 0.1% (v / v) acetic acid aqueous solution. The extract was then subjected to ultrafiltration, and the fraction below 1500 Da was collected, concentrated under reduced pressure at 50°C, and freeze-dried to obtain the safflower-Ligusticum striatum complex extract.

[0049] The preparation method of the above-mentioned composition for treating thyroid nodules includes the following steps: mixing sea cucumber body wall fibrinolytic peptides, kelp small molecule active components, Prunella vulgaris-Saussurea costus complex extract and safflower-Ligusticum striatum complex extract according to the mass fractions to obtain the composition.

[0050] The compositions of the above embodiments are formulated with pharmaceutically available skin penetration enhancers and excipients to create dressings for the treatment of thyroid nodules.

[0051] The raw materials used in the above embodiments are as follows: Sea cucumber (Liaoning sea cucumber); Kelp (kelp) from Anhui Kangweifu Pharmaceutical Co., Ltd.; Prunella vulgaris from Bozhou Shengdetang Pharmaceutical Co., Ltd.; Costus root from Chengdu Deyin Runzhu Biotechnology Co., Ltd.; Safflower from Bozhou Renhong Pharmaceutical Co., Ltd. (Xinjiang premium grade safflower); Ligusticum chuanxiong from Badong Zhenyu Traditional Chinese Medicine Planting Co., Ltd.; Alkaline pectinase from Zhengzhou Weifeng Biotechnology Co., Ltd. (enzyme activity 100,000 U / g); Bromelain from Xi'an Shouhe Biotechnology Co., Ltd. (enzyme activity 100,000 U / g); Trypsin from Shandong Pingju Biotechnology Co., Ltd. (enzyme activity 10,000 U / g); Bacillus subtilis protease from Wuhan Xinxin Jiali Biotechnology Co., Ltd. (enzyme activity 400,000 U / g); Lumbrokinase from Shaanxi Yuanshengte Biotechnology Co., Ltd. (enzyme activity 20,000 IU / mg). The alginate lyase was sourced from Hebei Runbu Biotechnology Co., Ltd., while the X-5 macroporous adsorption resin column, 80-120 mesh polyamide resin column, and NKA-9 macroporous resin column were sourced from Shanghai Yuanye Biotechnology Co., Ltd.

[0052] Comparative Example 1

[0053] The difference from Example 1 is that the sea cucumber body wall fibrinolytic peptide is changed to 1 part and the kelp small molecule active component is changed to 5.8 parts.

[0054] Comparative Example 2

[0055] The difference from Example 1 is that the sea cucumber body wall fibrinolytic peptides in the composition are changed to 6 parts, and the kelp small molecule active components are changed to 0.8 parts.

[0056] Comparative Example 3

[0057] The difference from Example 1 is that the amount of Prunella vulgaris-Saussurea costus compound extract in the composition is changed to 1.2 parts, and the amount of Carthamus tinctorius-Ligusticum chuanxiong compound extract is 5 parts.

[0058] Comparative Example 4

[0059] The difference from Example 1 is that the amount of Prunella vulgaris-Saussurea costus compound extract in the composition is changed to 5.2 parts, and the amount of Carthamus tinctorius-Ligusticum chuanxiong compound extract is 1 part.

[0060] Comparative Example 5

[0061] The difference from Example 1 is that the sea cucumber body wall fibrinolytic peptide is changed to 1 part, the kelp small molecule active component is changed to 5.8 parts, the Prunella vulgaris-Saussurea costus complex extract is changed to 1.2 parts, and the safflower-Ligusticum chuanxiong complex extract is changed to 5 parts.

[0062] Comparative Example 6

[0063] The difference from Example 1 is that trypsin is not added in the preparation method of sea cucumber body wall fibrinolytic peptides.

[0064] Comparative Example 7

[0065] The difference from Example 1 is that no bromelain is added in the preparation method of sea cucumber body wall fibrinolytic peptides.

[0066] Comparative Example 8

[0067] The difference from Example 1 is that no Bacillus subtilis protease is added in the preparation method of sea cucumber body wall fibrinolytic peptides.

[0068] Comparative Example 9

[0069] The difference from Example 1 is that in the preparation method of sea cucumber body wall fibrinolytic peptides, Bacillus subtilis protease is replaced with Serratia marcescens enzyme (enzyme activity 300,000 U / g).

[0070] Comparative Example 10

[0071] The difference from Example 1 is that no lumbrokinase is added in the preparation method of sea cucumber body wall fibrinolytic peptides.

[0072] Comparative Example 11

[0073] The difference from Example 1 is that in the preparation method of the small molecule active component of kelp, the X-5 macroporous adsorption resin column is replaced with a polyamide resin column with a particle size range of 80-120 mesh.

[0074] Comparative Example 12

[0075] The difference from Example 1 is that in the preparation method of the Prunella vulgaris-Saussurea costus complex extract, the polyamide resin column is replaced with an NKA-9 macroporous resin column.

[0076] Comparative Example 13

[0077] The difference from Example 1 is that in the preparation method of the Prunella vulgaris-Saussurea costus compound extract, the mass ratio of Prunella vulgaris powder to Saussurea costus powder is changed to 1.2:3.5.

[0078] Comparative Example 14

[0079] The difference from Example 1 is that in the preparation method of safflower-Ligusticum striatum compound extract, the NKA-9 macroporous resin column is replaced with a polyamide resin column with a particle size range of 80-120 mesh.

[0080] Comparative Example 15

[0081] The difference from Example 1 is that in the preparation method of safflower-Ligusticum chuanxiong compound extract, the mass ratio of safflower powder to Ligusticum chuanxiong powder is changed to 1.3:2.5.

[0082] Comparative Example 16

[0083] The difference from Example 1 is as follows: the fibrinolytic peptides of sea cucumber body wall were replaced with commercially available sea cucumber peptides (short sea cucumber peptides from Shaanxi Langde Biotechnology Co., Ltd., with small molecule active protein peptides ≤500 Daltons), the small molecule active components of kelp were replaced with kelp powder, and the herbs Prunella vulgaris and Saussurea costus were replaced with powder, as were Carthamus tinctorius and Ligusticum chuanxiong. In the preparation and testing, the sample was dispersed in sterile DMSO to prepare a 10 mg / mL stock solution, soaked for 24 h, centrifuged at 10000 g for 10 min, and the supernatant was collected and stored at -20℃. Samples of different concentrations were subsequently prepared.

[0084] I. Detection of anti-fibrotic effect (TGF-β1 inhibition rate)

[0085] 1. Establishment of cell model:

[0086] Cell line: HThyF (human thyroid fibroblasts).

[0087] Culture conditions: DMEM medium containing 10% fetal bovine serum, incubated at 37°C in a 5% CO2 incubator.

[0088] Experimental preparation: Passage to the 3rd generation, using 5×10 4Inoculate 1 cell / well into a 24-well plate, culture for 24 hours until adhesion, then replace with medium containing 10 ng / mL TGF-β1, and continue culture for 48 hours to establish a fibrosis model.

[0089] 2. Experimental Groups:

[0090] Normal control group: normal cells + TGF-β1-free culture medium.

[0091] Model group: TGF-β1 induction + drug-free culture medium.

[0092] Drug group: TGF-β1 induction + culture medium containing the composition.

[0093] Solvent control group: TGF-β1 induction + medium containing 0.1% DMSO.

[0094] Each group has 6 duplicate holes.

[0095] 3. Sample preparation:

[0096] The composition was dissolved in sterile DMSO to prepare a 10 mg / mL stock solution, which was stored at -20°C. Before use, it was diluted with complete culture medium to working concentrations of 20 μg / mL, 50 μg / mL, and 100 μg / mL (final DMSO concentration ≤ 0.1%).

[0097] 4. Drug administration:

[0098] After the model was established, the drug group was replaced with culture medium containing the working concentration of the composition. The solvent control group was in medium containing 0.1% DMSO. The normal control group was replaced with fresh drug-free culture medium. The model group was replaced with fresh drug-free culture medium. The supernatant was collected after incubation at 37℃ for 24 hours.

[0099] 5. Collagen I content detection (ELISA):

[0100] Centrifuge the supernatant at 10000×g for 10 min at 4℃, and collect the supernatant. Follow the instructions in the Human COL1A1 ELISA Kit: Add 100 μL each of standard (0-320 ng / mL) and sample to a 96-well plate, and incubate at 37℃ for 90 min. After washing, add 100 μL of biotinylated antibody and incubate at 37℃ for 60 min. After washing, add 100 μL of HRP conjugate and incubate at 37℃ for 30 min. After washing, add 100 μL of TMB substrate and incubate at 37℃ in the dark for 15 min, then add 50 μL of stop solution. Measure the absorbance at 450 nm (reference wavelength 570 nm) using a microplate reader and calculate the inhibition rate.

[0101] II. Detection of Apoptosis-Promoting Effect (Flow Cytometry):

[0102] 1. Cell model:

[0103] Cell line: Thyroid papillary carcinoma cells (TPC-1).

[0104] Culture conditions: RPMI-1640 medium containing 10% fetal bovine serum, 37℃, 5% CO2.

[0105] 2. Experimental Groups:

[0106] Negative control group: TPC-1 cells + drug-free culture medium.

[0107] Drug group: TPC-1 cells + culture medium containing the composition.

[0108] Solvent control group: TPC-1 cells + medium containing 0.1% DMSO.

[0109] Each group has 3 duplicate holes.

[0110] 3. Sample preparation:

[0111] Same as anti-fibrosis experiment (mother liquor 10 mg / mL, working concentrations 20 μg / mL, 50 μg / mL, 100 μg / mL, final DMSO concentration ≤0.1%).

[0112] 4. Drug administration:

[0113] With 2×10 5 Cells were seeded per well in 6-well plates and cultured for 24 hours until adherence. The drug-treated group was then replaced with medium containing the working concentration of the drug composition. The solvent control group was replaced with medium containing 0.1% DMSO. The negative control group was replaced with fresh, drug-free medium. Cells were harvested after another 48 hours of culture.

[0114] 5. Apoptosis detection (Annexin V-FITC / PI double staining):

[0115] Cells were digested with trypsin (without EDTA), centrifuged at 300×g for 5 min, and washed twice with PBS. The cell concentration was adjusted to 1×10⁻⁶. 6 Apoptosis rate was calculated by adding 100 μL of suspension to 5 μL of Annexin V-FITC and 5 μL of LPI (20 μg / mL), incubating at room temperature in the dark for 15 min, and then adding 400 μL of Binding Buffer. The apoptosis rate was calculated using FlowJo 10.0 within 1 h and analyzed using BD FACSCanto II.

[0116] Table 1 Test Results

[0117]

[0118] Note: All experiments were repeated 3 times independently. Data are expressed as mean ± standard deviation. Analysis of variance was performed using SPSS 22.0 and all results were p < 0.05.

[0119] III. Skin test on the inner forearm (patch test): The compositions of Examples 1 to 3 above were diluted with water to 10 mg / mL. Five people were in each group for the skin sensitization test. The diluted drug was applied to the marked area on the inner forearm, and light pressure was applied to ensure close contact between the drug and the skin. The application time was 48 hours. No allergic symptoms were observed during the application period and within 96 hours after the application.

[0120] The above results show that, in the compositions of Examples 1 to 3, the synergistic effect of the components is achieved by optimizing the raw material ratio and extraction process: the fibrinolytic peptides of sea cucumber body wall mainly inhibit the activity of fibroblasts, and the small molecule components of kelp can enhance the anti-fibrotic effect; the compound extracts of Prunella vulgaris and Aucklandia lappa and the compound extracts of Carthamus tinctorius and Ligusticum chuanxiong are synergistically enhanced through reasonable matching; they soften and disperse nodules and promote TPC-1 apoptosis.

[0121] Comparative Example 1 (1 part sea cucumber peptide, 5.8 parts kelp): The proportion of fibrinolytic peptides in the body wall of sea cucumber was too low, which weakened its inhibitory effect on TGF-β1-induced fibrosis; the excessive amount of kelp component may interfere with thyroid metabolism due to impurities, which may reduce the synergistic effect and lead to a decrease in inhibition rate and apoptosis rate.

[0122] Comparative Example 2 (6 parts sea cucumber peptide, 0.8 parts kelp): Excessive sea cucumber peptide leads to component imbalance, with excessive fibrinolytic activity exceeding the cell's absorption and metabolism capacity. The excess may also trigger excessive inhibition leading to inflammatory response. Insufficient kelp weakens the effect of softening and dispersing nodules, and cannot synergistically inhibit fibrosis and promote apoptosis. Therefore, the effect is inferior to that of Comparative Example 1.

[0123] Comparative Examples 3 and 4 (imbalance in the ratio of Prunella vulgaris-Saussurea costus and Carthamus tinctorius-Ligusticum chuanxiong): Excessive amounts of a certain component introduce impurities and cause side reactions; excessive amounts of a certain component exceed metabolic needs and result in component waste; insufficient amounts of a certain component lead to insufficient efficacy and weakened synergistic effects.

[0124] Comparative Example 5 (all four components were unbalanced): The proportions of all four components deviated from the optimal range, and the synergistic effects of sea cucumber peptide, kelp, prunella vulgaris-saussurea costus, and safflower-chuanxiong were completely destroyed. The inhibition rate and apoptosis rate were the lowest at each concentration, confirming the key role of the raw material ratio.

[0125] Comparative Example 6 (Sea Cucumber Peptide Preparation without Trypsin): The absence of trypsin leads to incomplete hydrolysis of sea cucumber body wall proteins, increased macromolecular peptide residues, and decreased fibrinolytic activity, thereby reducing the inhibitory effect on TGF-β1, but it is still better than the imbalanced ratio group.

[0126] Comparative Example 7 (Sea Cucumber Peptide Preparation without Bromelain): Bromelain can specifically degrade collagen, and the inhibitory effect of sea cucumber peptide on fibrosis is weakened after its absence.

[0127] Comparative Example 8 (Preparation of sea cucumber peptides without Bacillus subtilis protease): Bacillus subtilis protease is a key enzyme for degrading fibrin, and its absence reduces the core anti-fibrotic activity of sea cucumber peptides.

[0128] Comparative Example 9 (subtilisin replaced with serratiopeptidase): Although serratiopeptidase has thrombolytic activity, its specific hydrolytic ability on sea cucumber body wall proteins is weaker than that of subtilisin, resulting in a decrease in sea cucumber peptide activity.

[0129] Comparative Example 10 (Sea cucumber peptide preparation without lumbrokinase): Lumbrokinase enhances the activity of the fibrinolytic system, and without it, sea cucumber peptide cannot effectively inhibit fibroblast proliferation.

[0130] Comparative Example 11 (Kelp with polyamide resin instead of X-5 macroporous resin): X-5 macroporous resin has a higher adsorption and purification efficiency for fucoidan (the core anti-fibrotic component) in kelp. Polyamide resin is more likely to adsorb impurities with lower polarity, resulting in a decrease in the purity of the small molecule active components of kelp and a weakening effect.

[0131] Comparative Example 12 (Prunella vulgaris-Saussurea costus with NKA-9 replacing polyamide resin): Polyamide resin showed strong adsorption specificity for flavonoids in Prunella vulgaris (inhibiting fibrosis) and costus lactones in Saussurea costus (promoting apoptosis), while NKA-9 resin did not retain enough lipid-soluble components, resulting in a decrease in the activity of the extract.

[0132] Comparative Example 13 (Prunella vulgaris: Aucklandia lappa = 1.2:3.5): The proportion of Prunella vulgaris (containing prunellain, the core component for dispersing nodules) was too low, while the proportion of Aucklandia lappa (an auxiliary component for regulating qi) was too high. As a result, it could not effectively inhibit TGF-β1-induced fibrosis, and thus the effect was significantly reduced.

[0133] Comparative Example 14 (Safflower-Ligusticum striatum with polyamide replacing NKA-9 resin): NKA-9 macroporous resin has a higher retention rate of safflower yellow pigment (promoting apoptosis) in safflower and tetramethylpyrazine (enhancing penetration) in Ligusticum striatum. Polyamide resin has an excessively strong adsorption of phenolic acids, which leads to a decrease in the activity of the extract.

[0134] Comparative Example 15 (Carthamus tinctorius: Ligusticum chuanxiong = 1.3:2.5): The proportion of carthamus tinctorius was insufficient and the amount of Ligusticum chuanxiong was excessive, which weakened the synergistic effect of promoting apoptosis.

[0135] Comparative Example 16 (Powder Impregnation): The content and purity of effective active substances decreased significantly, resulting in poor efficacy.

[0136] Conclusion: Examples 1 to 3 achieved synergistic effects of the four components through optimal raw material ratio and process optimization, with significant anti-fibrotic and apoptosis-promoting effects; the comparative examples showed significantly reduced effects due to imbalanced ratios, process defects, or component substitution, resulting in insufficient active ingredients or disruption of synergistic effects.

Claims

1. A composition for treating thyroid nodules, characterized in that, The composition comprises the following raw materials in parts by weight: 3.0 to 5.0 parts of sea cucumber body wall fibrinolytic peptides, 2.0 to 3.5 parts of kelp small molecule active components, 2.5 to 4.0 parts of Prunella vulgaris-Saussurea costus complex extract, and 2.0 to 3.5 parts of Carthamus tinctorius-Ligusticum chuanxiong complex extract; The sea cucumber body wall fibrinolytic peptides are products with a value of less than 1000 Da obtained by enzymatic hydrolysis of sea cucumber body wall powder by alkaline pectinase, bromelain and trypsin, followed by enzymatic hydrolysis by subtilisin and lumbrokinase. The small molecule active component of kelp is a product with a value of less than 1500 Da obtained by enzymatic hydrolysis of kelp powder with alginate lyase, purification by X-5 macroporous adsorption resin column, and ultrafiltration. The Prunella vulgaris-Saussurea costus compound extract is obtained by extracting Prunella vulgaris powder and Saussurea costus powder at a mass ratio of (3-4):(1-1.5) with ethanol aqueous solution, purifying the extract with polyamide resin column, and ultrafiltration to obtain a product with a value of less than 2000 Da. The safflower-Ligusticum striatum compound extract is obtained by extracting safflower powder and Ligusticum striatum powder at a mass ratio of (2-3):(1-1.5) with ethanol aqueous solution, purifying the extract with NKA-9 macroporous resin column, and ultrafiltration to obtain a product with a value of less than 1500 Da.

2. The composition for treating thyroid nodules according to claim 1, characterized in that, The preparation method of the sea cucumber body wall fibrinolytic peptides includes: adding sea cucumber body wall powder to a phosphate buffer solution with a pH of 7.5-8.0, mixing well, adding alkaline pectinase, bromelain and trypsin, enzymatically hydrolyzing at 37℃-42℃ for 2h-2.5h, inactivating the enzymes, adding subtilisin and lumbrokinase, enzymatically hydrolyzing at 37℃-42℃ for 2h-2.5h, inactivating the enzymes, allowing to stand, centrifuging, taking the supernatant, ultrafiltration through an ultrafiltration membrane to obtain a component with less than 1000 Da, freeze-drying to obtain sea cucumber body wall fibrinolytic peptides.

3. The composition for treating thyroid nodules according to claim 2, characterized in that, Sea cucumber body wall powder is obtained by freeze-drying the body wall of sea cucumbers after removing the internal organs, and then pulverizing it into powder that passes through an 80-100 mesh sieve. The sea cucumber body wall powder is added to a phosphate buffer solution with a pH of 7.5-8.0 at a material-to-liquid mass ratio of 1:(8-10). The amounts of alkaline pectinase, bromelain, trypsin, subtilisin, and lumbrokinase added are 0.5%-1.0% of the sea cucumber body wall powder mass, respectively. The enzymes are inactivated at 80-85℃ for 10-15 minutes. The mixture is then allowed to stand at 4-6℃ for 1-2 hours. Finally, it is centrifuged at 8000-10000g for 15-20 minutes.

4. The composition for treating thyroid nodules according to claim 1, characterized in that, The preparation method of the kelp small molecule active component includes: adding kelp powder to deionized water, adjusting the pH to 5.5-6.0 with hydrochloric acid aqueous solution, adding alginate lyase, enzymatically hydrolyzing at 30℃-35℃ for 1-1.5h, inactivating the enzyme, and obtaining the enzymatic hydrolysate; adding 60%-70% of the volume of the enzymatic hydrolysate in anhydrous ethanol, allowing it to stand, centrifuging, taking the supernatant, concentrating under reduced pressure, loading the sample onto an X-5 macroporous adsorption resin column, rinsing with deionized water until the eluent is colorless, eluting with ethanol aqueous solution, collecting the eluent, ultrafiltration through an ultrafiltration membrane, taking the component below 1500 Da, freeze-drying, and obtaining the kelp small molecule active component.

5. The composition for treating thyroid nodules according to claim 4, characterized in that, The kelp powder is obtained by pulverizing dried kelp and passing it through an 80-100 mesh sieve. The kelp powder is added to deionized water at a material-to-liquid mass ratio of 1:(15-20). The hydrochloric acid aqueous solution concentration is 0.5 mol / L to 1 mol / L. The amount of alginate lyase added is 1.5% to 3% of the kelp powder mass. Enzyme inactivation is performed at 90℃ to 95℃ for 10-20 minutes. The mixture is allowed to stand at 4℃ to 6℃ for 1-2 hours. Centrifugation is performed at 8000g to 10000g for 10-15 minutes. Vacuum concentration is achieved at 50℃ to 55℃ to 10% to 15% of the volume. Eluting is performed with a 60% to 70% volume concentration of ethanol aqueous solution for 2-3 column volumes.

6. The composition for treating thyroid nodules according to claim 1, characterized in that, The preparation method of the Prunella vulgaris-Saussurea costus complex extract includes: mixing Prunella vulgaris powder and Saussurea costus powder at a mass ratio of (3-4):(1-1.5) to obtain a complex powder; adding the complex powder to an ethanol aqueous solution; heating and refluxing at 75℃-80℃ for 2-3 hours; concentrating under reduced pressure to obtain a concentrated solution; centrifuging; taking the supernatant; loading the solution onto a polyamide resin column; rinsing with deionized water to remove impurities; rinsing with a 40%-50% volume concentration ethanol aqueous solution to remove impurities; eluting with a 60%-70% volume concentration ethanol aqueous solution; collecting the eluent; ultrafiltration through an ultrafiltration membrane; taking the fraction with a value below 2000 Da; concentrating under reduced pressure; and freeze-drying to obtain the Prunella vulgaris-Saussurea costus complex extract.

7. The composition for treating thyroid nodules according to claim 6, characterized in that, The powder of Prunella vulgaris is the powder of Prunella vulgaris pulverized through a 60-80 mesh sieve; the powder of Aucklandia lappa is the powder of Aucklandia lappa pulverized through a 60-80 mesh sieve; the compound powder is added to a 70%-75% volume concentration ethanol aqueous solution at a material-to-liquid mass ratio of 1:(10-12); the ethanol is removed by vacuum concentration at 50-55℃; the ethanol is removed by centrifugation at 8000g-10000g for 10-15min; the ethanol is placed in a polyamide resin column with 80-120 mesh polyamide resin; the column is rinsed with deionized water for 1-2 column volumes; the column is rinsed with a 40%-50% volume concentration ethanol aqueous solution for 1.5-2 column volumes; the column is eluted with a 60%-70% volume concentration ethanol aqueous solution for 2-3 column volumes; the ethanol is then concentrated under vacuum at 50-55℃.

8. The composition for treating thyroid nodules according to claim 1, characterized in that, The preparation method of the safflower-Ligusticum striatum compound extract includes: mixing safflower powder and Ligusticum striatum powder at a mass ratio of (2-3):(1-1.5) to obtain a mixed powder; adding the mixed powder to an ethanol aqueous solution; heating and refluxing at 75℃-80℃ for 2-3 hours; concentrating under reduced pressure to obtain a concentrated extract; centrifuging; taking the supernatant; loading the extract onto an NKA-9 macroporous resin column; rinsing with deionized water to remove impurities; rinsing with a 20%-30% volume concentration ethanol aqueous solution to remove impurities; eluting with an aqueous solution containing 0.08%-0.12% ammonia and 50%-55% volume concentration ethanol; collecting the eluent; adjusting the pH to 6-7; ultrafiltration through an ultrafiltration membrane; taking the fraction below 1500 Da; concentrating under reduced pressure; and freeze-drying to obtain the safflower-Ligusticum striatum compound extract.

9. The composition for treating thyroid nodules according to claim 8, characterized in that, Safflower powder is safflower powder pulverized through a 60-80 mesh sieve; Ligusticum chuanxiong powder is Ligusticum chuanxiong powder pulverized through a 60-80 mesh sieve; the mixed powder is added to a 70%-75% volume concentration ethanol aqueous solution at a material-to-liquid mass ratio of 1:(10-12); the mixture is concentrated under reduced pressure at 50-55℃ to remove ethanol; centrifuged at 8000g-10000g for 10-15min; rinsed with deionized water for 1-2 column volumes; rinsed with a 20%-30% volume concentration ethanol aqueous solution for 1-2 column volumes; eluted with an aqueous solution containing 0.08%-0.12% ammonia and 50%-55% volume concentration ethanol for 2-3 column volumes; the eluent is adjusted to pH 6-7 with an acetic acid aqueous solution; and concentrated under reduced pressure at 50-55℃.

10. A method for preparing the composition for treating thyroid nodules according to claim 1, characterized in that, Includes the following steps: The sea cucumber body wall fibrinolytic peptides, kelp small molecule active components, Prunella vulgaris-Saussurea costus complex extract and Carthamus tinctorius-Ligusticum striatum complex extract were mixed according to the mass fractions to obtain a composition; the composition was then used to formulate a dressing with a skin penetration enhancer and excipients available in pharmaceuticals.

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