A composition for treating thyroid nodules and a method of preparing the same

Abstract

The present application relates to a kind of composition for treating thyroid nodule and its preparation method, belong to biological medicine technical field, composition includes sea cucumber body wall fibrinolysis small peptide, laminaria small molecule active component, summer grass- cloud mume compound extract and safflower- chuanxiong compound extract;Sea cucumber body wall fibrinolysis small peptide is the product obtained by alkaline pectinase, bromelain and trypsin common enzymolysis after sea cucumber body wall powder, then by bacillus subtilis protease and earthworm kinase common enzymolysis. Laminaria small molecule active component is the product obtained by sea alginic acid lyase enzyme extraction, X-5 macroporous adsorption resin column purification, ultrafiltration after laminaria powder is enzymolysis. Summer grass- cloud mume compound extract is the product obtained by polyamide resin column purification, ultrafiltration after summer grass powder and cloud mume powder are extracted by ethanol aqueous solution;Safflower- chuanxiong compound extract is the product obtained by NKA-9 macroporous resin column purification, ultrafiltration after safflower powder and chuanxiong powder are extracted by ethanol aqueous solution.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a composition for treating thyroid nodules and a preparation method thereof. BACKGROUND

[0002] Thyroid nodules are a common endocrine disease in clinical practice, with a high prevalence rate. Most of them are benign, but some have the risk of malignant transformation or cause compression symptoms due to excessive volume, thus requiring significant treatment. At present, drug therapy is an important means of non-surgical intervention, covering various forms such as traditional Chinese medicine, Western medicine, oral administration and external application. If thyroid nodules are not treated in time, the nodules will gradually increase, and cysts, calcification and hemorrhage may occur, and in severe cases, compression symptoms may occur, affecting breathing, eating or malignant transformation.

[0003] The theoretical basis for the treatment of thyroid nodules with Western medicine is derived from the understanding of thyroid function and the causes of nodules in modern medicine. For nodules with hyperthyroidism, antithyroid drugs (such as methimazole and propylthiouracil) are commonly used oral drugs that control symptoms by inhibiting thyroid hormone synthesis, but have limited effect on reducing the volume of nodules. For iodine deficiency-related nodules, the "suppression therapy" of supplementing levothyroxine has been widely used. Based on the feedback regulation mechanism, the secretion of thyroid-stimulating hormone is reduced, which theoretically inhibits the growth of nodules. However, recent studies have shown that the therapeutic effect varies greatly among individuals, and long-term use may increase the risk of osteoporosis, and its clinical application has gradually been limited.

[0004] Traditional Chinese medicine believes that thyroid nodules are caused by long-term unbalanced diet or emotional internal injury leading to decreased spleen and stomach function, combined with internal liver stagnation, spleen dysfunction, internal production of phlegm and dampness, stagnation of qi and blood, and condensation of phlegm and dampness in front of the neck, thereby forming thyroid nodules. The most effective treatment for thyroid nodules, in addition to surgery, is traditional Chinese medicine therapy, which softens and dissolves nodules, gradually softening, decomposing and eliminating thyroid nodules.

[0005] Oral traditional Chinese medicine often uses compound preparations, such as kelp, seaweed, wu kelp, pinellia ternate, and spirea japonica, which can relieve stagnation. Clinical application has a history of hundreds of years, and modern pharmacological studies have confirmed that some of its components can regulate thyroid function and inhibit fibrous tissue proliferation, but if the compatibility is poor, long-term use may cause side effects such as kidney function. Topical drugs directly reach the lesion through local administration, reducing the side effects of systemic medication. Traditional Chinese medicine ointments and powders often contain cassia and musk, which can warm the meridians, unblock the collaterals, and dissolve and reduce swelling, and are suitable for nodules with hard texture and long duration. Modern external preparations are improved on the basis of traditional formulas, such as traditional Chinese medicine ultrasonic introduction patches, which combine physical penetration technology to enhance drug absorption efficiency. They are often used in clinical practice to improve the efficacy of oral drugs, but the effect is limited when used alone.

[0006] In general, the drug therapy for thyroid nodules still has limited efficacy, and safe and effective therapeutic drugs need to be developed continuously. SUMMARY

[0007] In view of the poor safety and side effects of existing western medicines and oral Chinese medicines, especially the slow treatment effect and long treatment course of Chinese medicine treatment, the present application provides a composition for treating thyroid nodules and a preparation method thereof. The sea cucumber body wall fibrinolytic small peptide is prepared by a special enzymatic method, the laminaria small molecule active component, the prunella vulgaris-bupleurum farrum complex extract, and the safflower-chuanxiong complex extract are prepared by a special extraction method, and each component is used in a specific proportion to realize the synergistic effect between the components and has significant anti-fibrosis and pro-apoptosis effects. In terms of anti-fibrosis, the composition can effectively inhibit the fibrosis process of human thyroid fibroblasts (HThyF) induced by TGF-β1. In terms of pro-apoptosis, the composition can promote the apoptosis of thyroid papillary carcinoma cells (TPC-1). The topical external application drug can reduce the side effects of oral drugs, and provides a safe and effective option for the treatment of thyroid nodules. The specific technical solutions are as follows:

[0008] A composition for treating thyroid nodules, the composition comprising the following raw materials in mass fractions: sea cucumber body wall fibrinolytic small peptide 3.0-5.0 parts, laminaria small molecule active component 2.0-3.5 parts, prunella vulgaris-bupleurum farrum complex extract 2.5-4.0 parts, and safflower-chuanxiong complex extract 2.0-3.5 parts. The sea cucumber body wall fibrinolytic small peptide is obtained by co-enzymolysis of sea cucumber body wall powder with alkaline pectinase, bromelain and trypsin, and then co-enzymolysis with bacillus subtilis protease and earthworm kinase to obtain a product below 1000 Da. The laminaria small molecule active component is obtained by enzymolysis extraction of laminaria powder with alginic acid lyase, purification with an X-5 macroporous adsorption resin column, and ultrafiltration to obtain a product below 1500 Da. The prunella vulgaris-bupleurum farrum complex extract is obtained by extracting prunella vulgaris powder and bupleurum farrum powder in a mass ratio of (3-4):(1-1.5) with an ethanol aqueous solution, purifying the extract with a polyamide resin column, and ultrafiltration to obtain a product below 2000 Da. The safflower-chuanxiong complex extract is obtained by extracting safflower powder and chuanxiong powder in a mass ratio of (2-3):(1-1.5) with an ethanol aqueous solution, purifying the extract with an NKA-9 macroporous resin column, and ultrafiltration to obtain a product below 1500 Da.

[0009] In the above composition, the preparation method of the sea cucumber body wall fibrinolytic small peptide comprises the following steps: sea cucumber body wall powder is added into a phosphate buffer solution with a pH of 7.5-8.0 and mixed uniformly, alkaline pectinase, bromelain and trypsin are added, and enzymolysis is performed at 37-42℃ for 2-2.5 hours, the enzymes are inactivated, bacillus subtilis protease and earthworm kinase are added, and enzymolysis is performed at 37-42℃ for 2-2.5 hours, the enzymes are inactivated, the mixture is allowed to stand, centrifuged, the supernatant is taken, ultrafiltration is performed with an ultrafiltration membrane, a component below 1000 Da is obtained, and freeze-drying is performed to obtain the sea cucumber body wall fibrinolytic small peptide.

[0010] The preparation method of the sea cucumber body wall fibrinolytic peptide comprises the following steps: sea cucumber body wall powder is obtained by freezing and drying sea cucumber body wall after removing the internal organs and then crushing the dried sea cucumber body wall into powder with a mesh size of 80-100; sea cucumber body wall powder is added into a phosphate buffer solution with a pH value of 7.5-8.0 at a material-liquid mass ratio of 1:8-10; the addition amount of alkaline pectinase is 0.5-1.0% of the mass of the sea cucumber body wall powder; the addition amount of bromelain is 0.5-1.0% of the mass of the sea cucumber body wall powder; the addition amount of trypsin is 0.3-0.5% of the mass of the sea cucumber body wall powder; the addition amount of subtilisin is 0.5-0.8% of the mass of the sea cucumber body wall powder; the addition amount of lumbrukinase is 0.3-0.5% of the mass of the sea cucumber body wall powder; enzyme inactivation is performed at 80-85℃ for 10-15 min; standing is performed at 4-6℃ for 1-2 h; and centrifugation is performed at 8000-10000g for 15-20 min.

[0011] In the composition, the preparation method of the laminaria small-molecule active component comprises the following steps: laminaria powder is added into deionized water, the pH value of the solution is adjusted to 5.5-6.0 by adding hydrochloric acid, and alginic acid lyase is added, and enzymolysis is performed at 30-35℃ for 1-1.5 h to obtain an enzymolysis solution; 60-70% of the volume of the enzymolysis solution is added into anhydrous ethanol, and standing, centrifugation, reduced-pressure concentration, and supernatant collection are performed; the supernatant is loaded onto an X-5 macroporous adsorption resin column, and the column is eluted with deionized water until the eluent is colorless, and then eluted with an ethanol aqueous solution; the eluent is collected, ultrafiltered through an ultrafiltration membrane, and components with a molecular weight of less than 1500 Da are collected; and the components are freeze-dried to obtain the laminaria small-molecule active component.

[0012] In the preparation method of the laminaria small-molecule active component, the laminaria powder is obtained by crushing dried laminaria after sun drying, and the powder has a mesh size of 80-100; the laminaria powder is added into deionized water at a material-liquid mass ratio of 1:15-20; the concentration of the hydrochloric acid aqueous solution is 0.5-1 mol / L; the addition amount of alginic acid lyase is 1.5-3% of the mass of the laminaria powder; enzyme inactivation is performed at 90-95℃ for 10-20 min; standing is performed at 4-6℃ for 1-2 h; centrifugation is performed at 8000-10000g for 10-15 min; reduced-pressure concentration is performed at 50-55℃ until the volume is reduced to 10-15%; and elution with an ethanol aqueous solution is performed by using 60-70% volume concentration ethanol aqueous solution to elute 2-3 column volumes.

[0013] The preparation method of the composite extract of Prunella vulgaris and Radix Aucklandiae in the composition comprises the following steps: mixing Prunella vulgaris powder and Radix Aucklandiae powder in a mass ratio of 3-4:1-1.5 to obtain a composite powder, adding the composite powder into an ethanol aqueous solution, heating and refluxing at 75-80 DEG C for 2-3 hours, concentrating under reduced pressure, centrifuging, taking supernatant, loading the supernatant onto a polyamide resin column, washing and removing impurities with deionized water, washing and removing impurities with an ethanol aqueous solution with a volume concentration of 40-50 %, eluting with an ethanol aqueous solution with a volume concentration of 60-70 %, collecting the eluate, ultrafiltering the eluate through an ultrafiltration membrane, taking components with a molecular weight of less than 2000 Da, concentrating under reduced pressure, and freeze-drying to obtain the composite extract of Prunella vulgaris and Radix Aucklandiae.

[0014] In the preparation method of the composite extract of Prunella vulgaris and Radix Aucklandiae, the Prunella vulgaris powder is a powder with a particle size of 60-80 mesh, the Radix Aucklandiae powder is a powder with a particle size of 60-80 mesh, the composite powder is added into an ethanol aqueous solution with a volume concentration of 70-75 % in a solid-liquid mass ratio of 1:10-12, the concentration under reduced pressure is concentration under reduced pressure at 50-55 DEG C, the centrifuging is centrifuging at 8000-10000 g for 10-15 min, the polyamide resin column is filled with polyamide resin with a mesh size of 80-120, the deionized water is used to wash and remove impurities for 1-2 column volumes, the ethanol aqueous solution with a volume concentration of 40-50 % is used to wash and remove impurities for 1.5-2 column volumes, the elution is carried out with an ethanol aqueous solution with a volume concentration of 60-70 % for 2-3 column volumes, and the concentration under reduced pressure is concentration under reduced pressure at 50-55 DEG C.

[0015] The preparation method of the composite extract of Carthamus tinctorius and Rhizoma Chuanxiong in the composition comprises the following steps: mixing Carthamus tinctorius powder and Rhizoma Chuanxiong powder in a mass ratio of 2-3:1-1.5 to obtain a mixed powder, adding the mixed powder into an ethanol aqueous solution, heating and refluxing at 75-80 DEG C for 2-3 hours, concentrating under reduced pressure to obtain a concentrated extract, centrifuging, taking supernatant, loading the supernatant onto a NKA-9 macroporous resin column, washing and removing impurities with deionized water, washing and removing impurities with an ethanol aqueous solution with a volume concentration of 20-30 %, eluting with an aqueous solution containing ammonia with a mass concentration of 0.08-0.12 % and ethanol with a volume concentration of 50-55 %, adjusting the pH to 6-7, ultrafiltering the eluate through an ultrafiltration membrane, taking components with a molecular weight of less than 1500 Da, concentrating under reduced pressure, and freeze-drying to obtain the composite extract of Carthamus tinctorius and Rhizoma Chuanxiong.

[0016] In the preparation method of the safflower-tetrapanax papyriferus composite extract, the safflower powder is a powder obtained by crushing safflower through a 60-80 mesh sieve; the tetrapanax papyriferus powder is a powder obtained by crushing tetrapanax papyriferus through a 60-80 mesh sieve; the mixed powder is added into 70-75% volume concentration ethanol aqueous solution at a material-liquid mass ratio of 1:10-12; the ethanol is removed by concentration under reduced pressure at 50-55 DEG C; the eluate is centrifuged at 8000-10000 g for 10-15 min; the deionized water is eluted for 1-2 column volumes; the 20-30% volume concentration ethanol aqueous solution is eluted for 1-2 column volumes; the aqueous solution containing ammonia with a mass concentration of 0.08-0.12% and containing ethanol with a volume concentration of 50-55% is eluted for 2-3 column volumes; the eluate is adjusted to pH 6-7 with acetic acid aqueous solution; and the eluate is concentrated under reduced pressure at 50-55 DEG C.

[0017] The preparation method of the composition for treating thyroid nodules comprises the following steps:

[0018] The sea cucumber body wall fibrinolytic small peptides, laminaria japonica small molecule active components, prunella vulgaris-tetrapanax papyriferus composite extract and safflower-tetrapanax papyriferus composite extract are mixed according to mass parts to obtain the composition; and the composition is formulated with a skin penetration enhancer and an excipient which are available in the pharmaceutical field.

[0019] The composition for treating thyroid nodules and the preparation method thereof have the following beneficial effects:

[0020] Firstly, the pharmaceutical composition realizes the synergistic effect among the components by optimizing the raw material ratio and the extraction process, has significant anti-fibrosis and pro-apoptosis effects, can effectively inhibit the fibrosis process of human thyroid fibroblasts (HThyF) induced by TG F-β1, and can promote the apoptosis of thyroid papillary carcinoma cells (TPC-1), and can play a therapeutic role through multi-component synergy, thereby providing a safe and efficient choice for the treatment of thyroid nodules.

[0021] Secondly, the sea cucumber body wall fibrinolytic small peptides are obtained through specific enzymolysis steps (step-by-step enzymolysis using alkaline pectinase, bromelain, trypsin, subtilisin and earthworm kinase, and control of enzymolysis temperature, time and pH), and the product below 1000 Da can effectively inhibit the activity of fibroblasts and is a key component for anti-fibrosis. If a certain enzyme is missing or the enzyme type is replaced, the fibrinolytic activity will decrease and the anti-fibrosis effect will weaken.

[0022] The sea cucumber body wall fibrinolytic small peptide has fibrinolytic activity, can act on fibroblasts, inhibits the activity of fibroblasts induced by TGF-β1, thereby reducing the proliferation of fibrous tissue, and achieving the effect of anti-fibrosis. TGF-β1 can induce bone marrow mesenchymal stem cells to differentiate into myofibroblasts in vivo, promote fibroblast proliferation, extracellular matrix synthesis and other fibrosis-related processes. The fibrinolytic small peptide inhibits the activity of fibroblasts induced by TGF-β1 by interfering with the signal transduction pathway of TGF-β1. In the experiment of blocking peptide on the influence of fibroblasts on myofibroblasts differentiation, the blocking peptide can interfere with the related signal transduction and inhibit the expression of fibrosis-promoting genes.

[0023] Three, laminaria small molecule active component: after enzymolysis by alginic acid lyase, combined with ethanol precipitation, X-5 macroporous adsorption resin column purification and other steps (controlling parameters such as enzymolysis pH, temperature, time, and eluent concentration), components below 1500 Da are obtained, which can effectively enhance the anti-fibrosis effect.

[0024] Four, Prunella vulgaris-Cosmos composite extract: mixed according to a specific mass ratio, extracted with ethanol aqueous solution, and purified by polyamide resin column (controlling extraction temperature, time, and eluent concentration), components below 2000 Da are obtained, which can soothe depression and dispel knots, inhibit fibrous tissue proliferation and promote TPC-1 apoptosis.

[0025] Five, safflower-chuanxiong composite extract: mixed according to a specific ratio, extracted with ethanol aqueous solution, and purified by NKA-9 macroporous resin column (controlling extraction and purification parameters), components below 1500 Da are obtained, which have synergistic effects of anti-inflammatory, anti-fibrosis and pro-apoptosis; can also regulate qi and blood, promote blood circulation and drug penetration, and further promote the apoptosis process.

[0026] Six, the advantages of the extraction and purification method of each component of the present application compared with the conventional extraction method: the conventional extraction method has the problems of less or lack of effective active ingredients, insufficient extraction, low purity of effective ingredients, and many impurities. And the method can extract and purify effective ingredients by various enzymolysis techniques, specific resin purification, ultrafiltration and other steps, remove impurities, obtain low molecular weight active components, improve the activity and purity of the components, enhance the therapeutic effect of the drug, easily penetrate the skin, reduce the systemic side effects of oral administration, and at the same time reduce the side effects caused by impurities; and the accurate control of each step parameter ensures the stable acquisition of effective components, making the drug performance more reliable.

[0027] Seven, the synergistic effect of each component in the pharmaceutical formula of the application is remarkable: the sea cucumber body wall fibrinolytic small peptide mainly inhibits the activity of fibroblasts, the laminaria small molecule component can enhance the anti-fibrosis effect; the prunella vulgaris-bupleurum falcatum complex extract and the safflower-chuanxiong complex extract synergistically enhance each other, soften and remove the hard mass, and promote TPC-1 apoptosis. Through optimization of the raw material ratio and extraction process, the four components cooperate with each other to play the roles of anti-fibrosis and promoting apoptosis, thereby improving the effect of treating thyroid nodules. When the proportion of each component is unbalanced, the extraction process has defects or the components are replaced, the synergistic effect is destroyed, and the effect will be significantly reduced. DETAILED DESCRIPTION

[0028] The application will be further described below in combination with specific implementation examples, but the application is not limited to these examples.

[0029] Example 1

[0030] A composition for treating thyroid nodules, the composition comprising the following raw materials in mass parts: sea cucumber body wall fibrinolytic small peptide 4 parts, laminaria small molecule active component 2.8 parts, prunella vulgaris-bupleurum falcatum complex extract 3.2 parts, and safflower-chuanxiong complex extract 3 parts.

[0031] The preparation method of the sea cucumber body wall fibrinolytic small peptide comprises the following steps: after removing the internal organs of the sea cucumber body wall, the sea cucumber body wall is freeze-dried and crushed into a powder passing through an 80-mesh sieve to obtain sea cucumber body wall powder; the sea cucumber body wall powder is added into a pH 7.8 phosphate buffer according to a solid-liquid mass ratio of 1:9, mixed uniformly, 0.8% alkaline pectinase, 0.8% bromelain and 0.4% trypsin are added according to the mass of the sea cucumber body wall powder, 40℃ enzymolysis is performed for 2h, 82℃ enzyme inactivation is performed for 12min, 0.6% bacillus subtilis protease and 0.4% earthworm kinase are added according to the mass of the sea cucumber body wall powder, 40℃ enzymolysis is performed for 2h, 82℃ enzyme inactivation is performed for 12min, 5℃ standing is performed for 1.5h, 9000g centrifugation is performed for 18min, the supernatant is taken, ultrafiltration is performed through an ultrafiltration membrane, components below 1000Da are obtained, freeze-drying is performed to obtain the sea cucumber body wall fibrinolytic small peptide.

[0032] The preparation method of the Laminaria japonica small molecule active component comprises the following steps: drying and pulverizing Laminaria japonica to obtain Laminaria japonica powder; adding deionized water into the Laminaria japonica powder according to a material-liquid mass ratio of 1:18, adjusting pH to 5.8 by adding 0.8 mol / L hydrochloric acid aqueous solution, adding 2% of the mass of the Laminaria japonica powder of alginic acid lyase, and performing enzymatic hydrolysis at 32 ℃ for 1 h, and then performing enzyme inactivation at 92 ℃ for 15 min to obtain an enzymatic hydrolysis liquid; adding 65% of the volume of the enzymatic hydrolysis liquid of anhydrous ethanol, and then standing at 5 ℃ for 1.5 h; centrifuging at 9000 g for 12 min, and then taking supernatant; concentrating the supernatant at 52 ℃ under reduced pressure to 12% of the volume; loading the supernatant into an X-5 macroporous adsorption resin column (diameter-height ratio 1:10), and then washing with deionized water until the effluent liquid is colorless; eluting 2.5 column volumes of the supernatant with 65% volume concentration ethanol aqueous solution; collecting the eluate; performing ultrafiltration on the eluate through an ultrafiltration membrane; taking components with a molecular weight of less than 1500 Da; and performing freeze-drying to obtain the Laminaria japonica small molecule active component.

[0033] The preparation method of the Prunella vulgaris and Vladimiria souliei composite extract comprises the following steps: pulverizing Prunella vulgaris to obtain Prunella vulgaris powder; pulverizing Vladimiria souliei to obtain Vladimiria souliei powder; mixing the Prunella vulgaris powder and the Vladimiria souliei powder according to a mass ratio of 3.5:1.2 to obtain composite powder; adding 72% volume concentration ethanol aqueous solution into the composite powder according to a material-liquid mass ratio of 1:11, and then performing heating reflux extraction at 78 ℃ for 2.5 h; removing ethanol at 52 ℃ under reduced pressure to obtain a concentrated liquid; centrifuging at 9000 g for 12 min, and then taking supernatant; loading the supernatant into a polyamide resin column (diameter-height ratio 1:10) with a particle size range of 80 mesh-120 mesh; washing 1.5 column volumes of the supernatant with deionized water to remove impurities; washing 1.5 column volumes of the supernatant with 45% volume concentration ethanol aqueous solution to remove impurities; eluting 2.5 column volumes of the supernatant with 65% volume concentration ethanol aqueous solution; collecting the eluate; performing ultrafiltration on the eluate through an ultrafiltration membrane; taking components with a molecular weight of less than 2000 Da; concentrating at 52 ℃ under reduced pressure; and performing freeze-drying to obtain the Prunella vulgaris and Vladimiria souliei composite extract.

[0034] The preparation method of the Carthamus tinctorius and Ligusticum chuanxiong composite extract comprises the following steps: pulverizing Carthamus tinctorius to obtain Carthamus tinctorius powder; pulverizing Ligusticum chuanxiong to obtain Ligusticum chuanxiong powder; mixing the Carthamus tinctorius powder and the Ligusticum chuanxiong powder according to a mass ratio of 2.5:1.3 to obtain mixed powder; adding 72% volume concentration ethanol aqueous solution into the mixed powder according to a material-liquid mass ratio of 1:11, and then performing heating reflux extraction at 78 ℃ for 2.5 h; removing ethanol at 52 ℃ under reduced pressure to obtain a concentrated extract liquid; centrifuging at 9000 g for 12 min, and then taking supernatant; loading the supernatant into an NKA-9 macroporous resin column (diameter-height ratio 1:10); washing 1.5 column volumes of the supernatant with deionized water to remove impurities; washing 1.5 column volumes of the supernatant with 25% volume concentration ethanol aqueous solution to remove impurities; eluting 2.5 column volumes of the supernatant with an aqueous solution containing 0.1% mass concentration of ammonia and 52% volume concentration of ethanol; collecting the eluate; adjusting the pH of the eluate to 6.5 by adding 0.1% volume concentration acetic acid aqueous solution; performing ultrafiltration on the eluate through an ultrafiltration membrane; taking components with a molecular weight of less than 1500 Da; concentrating at 52 ℃ under reduced pressure; and performing freeze-drying to obtain the Carthamus tinctorius and Ligusticum chuanxiong composite extract.

[0035] The preparation method of the composition for treating thyroid nodules comprises the following steps: mixing sea cucumber body wall fibrinolytic small peptides, laminaria small molecule active components, prunella vulgaris-bupleurum falcatum complex extracts and safflower-chuanxiong complex extracts by mass fraction to obtain the composition.

[0036] Example 2

[0037] A composition for treating thyroid nodules, the composition comprising the following mass fractions of raw materials: sea cucumber body wall fibrinolytic small peptides 3.0 parts, laminaria small molecule active components 3.5 parts, prunella vulgaris-bupleurum falcatum complex extracts 2.5 parts and safflower-chuanxiong complex extracts 3.5 parts.

[0038] The preparation method of the sea cucumber body wall fibrinolytic small peptides comprises: removing the internal organs of the sea cucumber body wall, freeze-drying, and crushing into a powder that passes through an 80-mesh sieve to obtain sea cucumber body wall powder; adding the sea cucumber body wall powder to a pH 7.5 phosphate buffer at a solid-liquid mass ratio of 1:10, mixing well, adding 1.0% alkaline pectinase, 0.5% bromelain and 0.5% trypsin based on the mass of the sea cucumber body wall powder, and enzymatically digesting at 37°C for 2.5 h, then inactivating the enzymes at 80°C for 15 min, adding 0.5% bacillus subtilis protease and 0.5% earthworm kinase based on the mass of the sea cucumber body wall powder, and enzymatically digesting at 37°C for 2.5 h, then inactivating the enzymes at 80°C for 15 min, standing at 4°C for 2 h, centrifuging at 8000g for 20 min, taking the supernatant, and ultrafiltering through an ultrafiltration membrane to obtain components below 1000 Da, and freeze-drying to obtain the sea cucumber body wall fibrinolytic small peptides.

[0039] The preparation method of the laminaria small molecule active components comprises: drying and crushing the laminaria, passing through an 80-mesh sieve to obtain laminaria powder; adding the laminaria powder to deionized water at a solid-liquid mass ratio of 1:20, adjusting the pH to 6.0 with 0.5 mol / L hydrochloric acid aqueous solution, adding 1.5% alginate lyase based on the mass of the laminaria powder, and enzymatically digesting at 35°C for 1 h, then inactivating the enzymes at 95°C for 10 min to obtain an enzymatic digestion liquid; adding 70% anhydrous ethanol by volume of the enzymatic digestion liquid, standing at 4°C for 2 h, centrifuging at 8000g for 15 min, taking the supernatant, concentrating to 15% of the volume at 50°C under reduced pressure, loading onto an X-5 macroporous adsorption resin column (diameter-height ratio 1:10), washing with deionized water until the effluent is colorless, eluting with 60% by volume concentration of an ethanol aqueous solution for 3 column volumes, collecting the eluate, ultrafiltering through an ultrafiltration membrane, taking components below 1500 Da, and freeze-drying to obtain the laminaria small molecule active components.

[0040] The preparation method of the Prunella vulgaris-Aucklandiae Radix composite extract includes the following steps: Prunella vulgaris is crushed and passed through a 60-mesh sieve to obtain Prunella vulgaris powder; Aucklandiae Radix is crushed and passed through an 80-mesh sieve to obtain Aucklandiae Radix powder; the Prunella vulgaris powder and the Aucklandiae Radix powder are mixed according to a mass ratio of 3:1.5 to obtain a composite powder; the composite powder is added into 75% volume concentration ethanol aqueous solution according to a solid-liquid mass ratio of 1:10, heated and refluxed at 75 DEG C for 3 h, concentrated and dehydrated at 50 DEG C under reduced pressure to remove ethanol, to obtain a concentrated solution; the concentrated solution is centrifuged at 10,000 g for 10 min, and the supernatant is collected; the supernatant is loaded onto a polyamide resin column (diameter-height ratio 1:10) with a particle size range of 80-120 meshes; the column is washed with deionized water for 2 column volumes to remove impurities; the column is washed with 40% volume concentration ethanol aqueous solution for 2 column volumes to remove impurities; the column is eluted with 60% volume concentration ethanol aqueous solution for 3 column volumes; the eluate is collected, ultrafiltered through an ultrafiltration membrane, and components with a molecular weight of less than 2,000 Da are collected; the components are concentrated under reduced pressure at 50 DEG C, and freeze-dried to obtain the Prunella vulgaris-Aucklandiae Radix composite extract.

[0041] The preparation method of the Prunella vulgaris-Aucklandiae Radix composite extract includes the following steps: Prunella vulgaris is crushed and passed through a 60-mesh sieve to obtain Prunella vulgaris powder; Aucklandiae Radix is crushed and passed through an 80-mesh sieve to obtain Aucklandiae Radix powder; the Prunella vulgaris powder and the Aucklandiae Radix powder are mixed according to a mass ratio of 3:1.5 to obtain a composite powder; the composite powder is added into 75% volume concentration ethanol aqueous solution according to a solid-liquid mass ratio of 1:10, heated and refluxed at 75 DEG C for 3 h, concentrated and dehydrated at 50 DEG C under reduced pressure to remove ethanol, to obtain a concentrated solution; the concentrated solution is centrifuged at 10,000 g for 10 min, and the supernatant is collected; the supernatant is loaded onto a polyamide resin column (diameter-height ratio 1:10) with a particle size range of 80-120 meshes; the column is washed with deionized water for 2 column volumes to remove impurities; the column is washed with 40% volume concentration ethanol aqueous solution for 2 column volumes to remove impurities; the column is eluted with 60% volume concentration ethanol aqueous solution for 3 column volumes; the eluate is collected, ultrafiltered through an ultrafiltration membrane, and components with a molecular weight of less than 2,000 Da are collected; the components are concentrated under reduced pressure at 50 DEG C, and freeze-dried to obtain the Prunella vulgaris-Aucklandiae Radix composite extract.

[0042] The preparation method of the composition for treating thyroid nodules includes the following steps: sea cucumber body wall fibrinolytic small peptides, laminaria small molecule active components, Prunella vulgaris-Aucklandiae Radix composite extract and safflower-Chuanxiong composite extract are mixed according to mass parts to obtain the composition.

[0043] Example 3

[0044] The composition for treating thyroid nodules includes the following mass parts of raw materials: sea cucumber body wall fibrinolytic small peptides 5.0 parts, laminaria small molecule active components 2.0 parts, Prunella vulgaris-Aucklandiae Radix composite extract 4.0 parts and safflower-Chuanxiong composite extract 2.0 parts.

[0045] The preparation method of the sea cucumber body wall fibrinolytic small peptide comprises the following steps: removing the internal organs of the sea cucumber body wall, freezing and drying the sea cucumber body wall, and crushing the sea cucumber body wall into powder with a particle size of 100 mesh to obtain sea cucumber body wall powder; adding the sea cucumber body wall powder into a phosphate buffer solution with a pH value of 8.0 at a material-liquid mass ratio of 1:8, mixing uniformly, adding 0.5% alkaline pectinase, 1.0% bromelain and 0.3% trypsin, and carrying out enzymolysis at 42°C for 2 hours; adding 0.8% bacillus subtilis protease and 0.3% earthworm kinase, and carrying out enzymolysis at 42°C for 2 hours; standing at 6°C for 1 hour; centrifuging at 10 000 g for 15 minutes; taking the supernatant; and carrying out ultrafiltration through an ultrafiltration membrane to obtain a component with a molecular weight of less than 1000 Da, and freezing and drying to obtain the sea cucumber body wall fibrinolytic small peptide.

[0046] The preparation method of the laminaria small molecule active component comprises the following steps: drying and crushing laminaria to obtain laminaria powder; adding the laminaria powder into deionized water at a material-liquid mass ratio of 1:15, adjusting the pH value to 5.5 by adding a 1 mol / L hydrochloric acid aqueous solution, adding 3% laminaria powder mass of alginate lyase, and carrying out enzymolysis at 30°C for 1.5 hours; adding anhydrous ethanol with a volume of 60% of the enzymolysis liquid, standing at 6°C for 1 hour, centrifuging at 10 000 g for 10 minutes, taking the supernatant, and concentrating to 10% of the volume at 55°C under reduced pressure; loading the concentrated solution into an X-5 macroporous adsorption resin column (diameter-height ratio 1:10), washing with deionized water until the effluent is colorless, eluting with 70% volume concentration ethanol aqueous solution for 2 column volumes, collecting the eluate, carrying out ultrafiltration through an ultrafiltration membrane, taking a component with a molecular weight of less than 1500 Da, and freezing and drying to obtain the laminaria small molecule active component.

[0047] The preparation method of the prunella vulgaris and radix notopterygii composite extract comprises the following steps: crushing prunella vulgaris to obtain prunella vulgaris powder with a particle size of 80 mesh; crushing radix notopterygii to obtain radix notopterygii powder with a particle size of 60 mesh; mixing the prunella vulgaris powder and the radix notopterygii powder at a mass ratio of 4:1 to obtain composite powder; adding 70% volume concentration ethanol aqueous solution to the composite powder at a material-liquid mass ratio of 1:12, heating and refluxing at 80°C for 2 hours, removing ethanol by concentrating at 55°C under reduced pressure, and obtaining a concentrated solution; centrifuging at 8000 g for 15 minutes, taking the supernatant, and loading the supernatant into a polyamide resin column (diameter-height ratio 1:10) with a particle size range of 80 mesh to 120 mesh; washing with deionized water for 1 column volume to remove impurities, washing with 50% volume concentration ethanol aqueous solution for 1.5 column volumes to remove impurities, eluting with 70% volume concentration ethanol aqueous solution for 2 column volumes, collecting the eluate, carrying out ultrafiltration through an ultrafiltration membrane, taking a component with a molecular weight of less than 2000 Da, concentrating at 55°C under reduced pressure, and freezing and drying to obtain the prunella vulgaris and radix notopterygii composite extract.

[0048] The preparation method of the safflower-cowherb composite extract includes: crushing safflower to pass through a 60-mesh sieve to obtain safflower powder; crushing cowherb to pass through an 80-mesh sieve to obtain cowherb powder; mixing the safflower powder and the cowherb powder at a mass ratio of 2:1.5 to obtain mixed powder; adding 75% by volume concentration ethanol aqueous solution to the mixed powder at a material-liquid mass ratio of 1:10, heating and refluxing at 75°C for 3 hours, removing ethanol at 50°C under reduced pressure to obtain concentrated extract liquid, centrifuging at 10,000 g for 10 min, taking supernatant, and loading the supernatant onto an NKA-9 macroporous resin column (diameter-height ratio 1:10); washing impurities with deionized water for 2 column volumes, washing impurities with 20% by volume concentration ethanol aqueous solution for 2 column volumes, eluting with an aqueous solution containing 0.08% by mass concentration of ammonia and 55% by volume concentration of ethanol for 2 column volumes, collecting the eluate, and adjusting the pH of the eluate to 7 with 0.1% by volume concentration acetic acid aqueous solution; ultrafiltrating through an ultrafiltration membrane, taking components below 1,500 Da, concentrating under reduced pressure at 50°C, and freeze-drying to obtain the safflower-cowherb composite extract.

[0049] The preparation method of the composition for treating thyroid nodules includes the following steps: mixing sea cucumber body wall fibrinolytic peptides, laminaria small molecule active components, prunella vulgaris-nardostachys jatamansi composite extract, and safflower-cowherb composite extract according to mass parts to obtain the composition.

[0050] The composition of each of the above embodiments is formulated with a skin penetration enhancer and an excipient that is pharmaceutically acceptable, and is used as an external application agent for treating thyroid nodules.

[0051] The raw material sources in each of the above embodiments are as follows: sea cucumber is selected from Liaoning sea cucumber. Laminaria is from Anhui Kangweifuk Pharmaceutical Co., Ltd. Prunella vulgaris is from Bozhou Shengdetang Pharmaceutical Co., Ltd. Nardostachys jatamansi is from Chengdu Deyin Runzhu Biological Technology Co., Ltd. Safflower is from Bozhou Renhong Pharmaceutical Co., Ltd., Xinjiang special grade safflower. Cowherb is from Badong County Zhenyu Chinese Herbal Medicine Planting Co., Ltd. Alkaline pectinase is from Zhengzhou Weifeng Biological Technology Co., Ltd., with an enzyme activity of 100,000 U / g. Bromelain is from Xi'an Shouhe Biological Technology Co., Ltd., with an enzyme activity of 100,000 U / g. Trypsin is from Shandong Pingju Biological Technology Co., Ltd., with an enzyme activity of 10,000 U / g. Subtilisin is from Wuhan Xinxinjiali Biological Technology Co., Ltd., with an enzyme activity of 400,000 U / g. Avisin is from Shaanxi Yuanshengte Biological Technology Co., Ltd., with an enzyme activity of 20,000 iu / mg. Alginic acid lyase is from Hebei Runbu Biological Technology Co., Ltd. X-5 macroporous adsorption resin column, 80-mesh-120-mesh polyamide resin column, and NKA-9 macroporous resin column are all from Shanghai Yuanye Biological Technology Co., Ltd.

[0052] Comparative Example 1

[0053] The difference from Example 1 is that in the composition, sea cucumber body wall fibrinolytic peptides are changed to 1 part, and laminaria small molecule active components are changed to 5.8 parts.

[0054] Comparative Example 2

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

[0056] Comparative Example 3

[0057] The difference from Example 1 is that in the composition, the prunella vulgaris and artemisia japonica complex extract is changed to 1.2 parts, and the safflower and ligusticum wallichii complex extract is 5 parts.

[0058] Comparative Example 4

[0059] The difference from Example 1 is that in the composition, the prunella vulgaris and artemisia japonica complex extract is changed to 5.2 parts, and the safflower and ligusticum wallichii complex extract is 1 part.

[0060] Comparative Example 5

[0061] The difference from Example 1 is that in the composition, the sea cucumber body wall fibrinolytic small peptide is changed to 1 part, the laminaria small molecule active component is changed to 5.8 parts, the prunella vulgaris and artemisia japonica complex extract is changed to 1.2 parts, and the safflower and ligusticum wallichii complex extract is 5 parts.

[0062] Comparative Example 6

[0063] The difference from Example 1 is that in the preparation method of the sea cucumber body wall fibrinolytic small peptide, no trypsin is added.

[0064] Comparative Example 7

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

[0066] Comparative Example 8

[0067] The difference from Example 1 is that in the preparation method of the sea cucumber body wall fibrinolytic small peptide, no subtilisin is added.

[0068] Comparative Example 9

[0069] The difference from Example 1 is that in the preparation method of the sea cucumber body wall fibrinolytic small peptide, the subtilisin is replaced by serratia peptidase (enzyme activity 300,000 U / g) in the preparation method of the sea cucumber body wall fibrinolytic small peptide.

[0070] Comparative Example 10

[0071] The difference from Example 1 is that in the preparation method of the sea cucumber body wall fibrinolytic small peptide, no lumbrukinase is added.

[0072] Comparative Example 11

[0073] The difference from Example 1 is that, in the preparation method of the Laminariaia small molecule active component, the X-5 macroporous adsorption resin column is replaced by 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-Aucklandia lappa composite extract, the polyamide resin column is replaced by 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-Aucklandia lappa composite extract, the mass ratio of Prunella vulgaris powder to Aucklandia lappa 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 the Carthamus tinctorius-Ligusticum chuanxiong composite extract, the NKA-9 macroporous resin column is replaced by 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 the Carthamus tinctorius-Ligusticum chuanxiong composite extract, the mass ratio of Carthamus tinctorius powder to Ligusticum chuanxiong powder is changed to 1.3:2.5.

[0082] Comparative Example 16

[0083] The difference from Example 1 is that, in the preparation method of the Carthamus tinctorius-Ligusticum chuanxiong composite extract, the NKA-9 macroporous resin column is replaced by a polyamide resin column with a particle size range of 80-120 mesh.

[0084] I. Anti-fibrosis effect detection (TGF-β1 inhibition rate)

[0085] 1. Cell model establishment:

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

[0087] Culture conditions: DMEM medium containing 10% fetal bovine serum, 37°C, 5% CO2 constant temperature incubator.

[0088] Experimental preparation: passaged to the 3rd generation, 5x10 4Cells were seeded at 1 x 104 / well in 24-well plates and cultured for 24 h. Then, the medium was replaced with a medium containing 10 ng / mL TGF-β1 to establish a fibrosis model.

[0089] 2. Experimental grouping:

[0090] Normal control group: normal cells + medium without TGF-β1.

[0091] Model group: TGF-β1 induction + medium without drug.

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

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

[0094] Each group had 6 replicate wells.

[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 medium to working concentrations of 20 μg / mL, 50 μg / mL, and 100 μg / mL (final DMSO concentration ≤0.1%).

[0097] 4. Drug treatment:

[0098] After the model was established, the drug group was replaced with a medium containing the working concentration of the composition. The solvent control group contained a medium with 0.1% DMSO. The normal control group was replaced with fresh medium without drug. The model group was replaced with fresh medium without drug. After 24 h of incubation at 37°C, the supernatant was collected.

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

[0100] The supernatant was centrifuged at 10,000 x g for 10 min at 4°C, and the supernatant was taken. According to the kit (Human COL1A1 ELISA Kit) operation: add 100 μL of standard (0-320 ng / mL) and sample to a 96-well plate, and incubate at 37°C for 90 min. After washing the plate, add 100 μL of biotinylated antibody, and incubate at 37°C for 60 min. After washing the plate, add 100 μL of HRP conjugate, and incubate at 37°C for 30 min. After washing the plate, add 100 μL of TMB substrate, and incubate at 37°C for 15 min in the dark. Add 50 μL of stop solution. Detect the absorbance at 450 nm (reference wavelength 570 nm) using a microplate reader, and calculate the inhibition rate.

[0101] II. Detection of cell apoptosis promotion (flow cytometry):

[0102] 1. Cell model:

[0103] Cell line: thyroid papillary carcinoma cell (TPC-1).

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

[0105] 2. Experimental grouping:

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

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

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

[0109] Each group has 3 replicate wells.

[0110] 3. Sample preparation:

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

[0112] 4. Drug treatment:

[0113] Inoculate 2×10 5 cells / well in a 6-well plate, culture for 24 h, then replace the drug group with medium containing the working concentration of the composition. The solvent control group is replaced with medium containing 0.1% DMSO. The negative control group is replaced with fresh drug-free medium. Continue to culture for 48 h and collect the cells.

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

[0115] Trypsin (without EDTA) digest the cells, centrifuge at 300×g for 5 min, wash with PBS twice. Adjust the cell concentration to 1×10 6 cells / mL, take 100 μL of the suspension, add 5 μL Annexin V-FITC and 5 μL PI (20 μg / mL), incubate in the dark at room temperature for 15 min, add 400 μL Binding Buffer and mix well. Within 1 h, perform machine operation (BD FACSCanto II), analyze with FlowJo 10.0, and calculate the apoptosis rate.

[0116] Table 1 detection results

[0117]

[0118] Note: All experiments were repeated 3 times, and the data were expressed as mean ± standard deviation. SPSS 22.0 was used for variance analysis, and all *p<0.05.

[0119] III. Human forearm skin experiment (patch test): The compositions of the above-mentioned examples 1 to 3 were diluted with water to 10 mg / mL, and a skin sensitization experiment was performed on 5 people in each group. The drug was attached to the marked area on the inner side of the forearm, and the drug was in close contact with the skin by light pressure. The attachment time was 48 hours, and there were no allergic symptoms during the attachment period and within 96 hours after the attachment.

[0120] The above results show that, in the compositions of examples 1 to 3, by optimizing the raw material ratio and extraction process, the synergistic effect of the components is achieved: the sea cucumber body wall fibrinolytic small peptide mainly inhibits the activity of fibroblasts, the laminaria small molecule component can enhance the anti-fibrosis effect; the prunella vulgaris-bupleurum compound extract and the safflower-chuanxiong compound extract can synergistically enhance the effect by reasonable matching; softening and resolving, promoting TPC-1 apoptosis.

[0121] Comparative example 1 (sea cucumber peptide 1 part, laminaria 5.8 parts): the proportion of sea cucumber body wall fibrinolytic small peptide is too low, and its inhibitory effect on TGF-β1 induced fibrosis is weakened; the excessive laminaria component may interfere with thyroid metabolism due to impurity content, thereby reducing the synergistic effect, resulting in a decrease in inhibition rate and apoptosis rate.

[0122] Comparative example 2 (sea cucumber peptide 6 parts, laminaria 0.8 parts): excessive sea cucumber peptide leads to imbalance of components, and its fibrinolytic activity is excessive and redundant, which exceeds the metabolic capacity of cells, and the excess amount also may cause excessive inhibition leading to inflammatory reaction; insufficient laminaria weakens the softening and resolving effect, and cannot synergistically inhibit fibrosis and promote apoptosis, so the effect is worse than that of comparative example 1.

[0123] Comparative examples 3 and 4 (prunella vulgaris-bupleurum, safflower-chuanxiong ratio imbalance): excessive introduction of impurities by a certain component causes side reactions, excessive amount of a certain component exceeds the metabolic needs, resulting in waste of components, and insufficient amount of a certain component, which weakens the synergistic effect.

[0124] Comparative example 5 (four components are imbalanced): the ratio of the four components deviates from the optimal range, and the synergistic effect of sea cucumber peptide, laminaria, prunella vulgaris-bupleurum and safflower-chuanxiong is completely destroyed, and the inhibition rate and apoptosis rate are the lowest at each concentration, which confirms the key role of raw material ratio.

[0125] Comparative example 6 (preparation of sea cucumber peptide without trypsin): the absence of trypsin leads to incomplete hydrolysis of sea cucumber body wall proteins, and the increase of residual large molecule peptides, which reduces the fibrinolytic activity and further reduces the inhibitory effect on TGF-β1, but is still better than the imbalance group.

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

[0127] Comparative Example 8 (Preparation of sea cucumber peptide without subtilisin): Subtilisin is a key enzyme for degrading fibrous proteins, and the anti-fibrosis core activity of sea cucumber peptide is reduced after the absence of subtilisin.

[0128] Comparative Example 9 (Subtilisin replaced with serratia peptidase): Serratia peptidase has thrombolytic activity, but its specific hydrolysis ability for sea cucumber body wall proteins is weaker than that of subtilisin, resulting in reduced activity of sea cucumber peptide.

[0129] Comparative Example 10 (Preparation of sea cucumber peptide without lumbrukinase): Lumbrukinase enhances the activity of the fibrinolytic system, and sea cucumber peptide cannot effectively inhibit the proliferation of fibroblasts after the absence of lumbrukinase.

[0130] Comparative Example 11 (Kelp replaced with polyamide resin instead of X-5 macroporous resin): X-5 macroporous resin has higher adsorption and purification efficiency for fucoidan (anti-fibrosis core component) in kelp, and polyamide resin is more prone to adsorb impurities with lower polarity, resulting in reduced purity of active components in kelp and weakened effect.

[0131] Comparative Example 12 (Summer-Cloudy grass replaced with NKA-9 instead of polyamide resin): Polyamide resin has strong adsorption specificity for flavonoids (inhibiting fibrosis) in summer grass and costunolide (promoting apoptosis) in cloudwood, and NKA-9 resin is insufficient for retaining fat-soluble components, resulting in reduced activity of the extract.

[0132] Comparative Example 13 (Summer grass: Cloudy grass = 1.2:3.5): The proportion of summer grass (containing prunus mume glycoside, core dissolving component) is too low, and the proportion of cloudwood (auxiliary component for regulating qi) is excessive, which cannot effectively inhibit TGF-β1-induced fibrosis, resulting in a significant decrease in effect.

[0133] Comparative Example 14 (Red flower-Cowherb replaced with polyamide instead of NKA-9 resin): NKA-9 macroporous resin has higher retention rate for safflower yellow pigment (promoting apoptosis) in red flower and cnidanzine (enhancing permeability) in cowherb, and polyamide resin has too strong adsorption for phenolic acids, resulting in reduced activity of the extract.

[0134] Comparative Example 15 (Red flower: Cowherb = 1.3:2.5): The proportion of red flower is insufficient, and the proportion of cowherb is excessive, resulting in weakened synergistic effect of promoting apoptosis.

[0135] Comparative Example 16 (Powder immersion): The content and purity of effective active substances are greatly reduced, and the effect is very poor.

[0136] Conclusion: Examples 1 to 3 achieve synergistic effect of four components by optimal proportioning of raw materials and process optimization, and have significant anti-fibrosis and pro-apoptosis effects; each comparative example has significant decrease in effect due to insufficient active ingredients or destruction of synergistic effect caused by unbalanced proportioning, process defects or component replacement.

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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