Isothiocyanate bionic preparation and preparation method thereof
By designing biomimetic formulations of isothiocyanates, the stability and irritation issues of isothiocyanate compounds during storage and gastrointestinal release have been resolved, achieving long-term stable storage and slow release at room temperature.
Patent Information
- Application Number
- CN202511398832.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-28
- Publication Date
- 2025-12-12
AI Technical Summary
Isothiocyanate compounds have problems in clinical applications, such as poor stability, demanding storage conditions, low utilization rate, and strong gastrointestinal irritation.
The design employs a biomimetic formulation of isothiocyanate, which physically separates black myrosinase microparticles from a glucosinolate extract layer, forming a structure that ensures long-term stable storage under dry conditions at room temperature and slow release of isothiocyanate in the gastrointestinal tract.
This method enables long-term stable storage of isothiocyanates at room temperature and slow release in the gastrointestinal tract, avoiding strong irritation to the gastrointestinal tract and improving effective utilization.
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Figure CN121102148A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of pharmaceutical preparations, and particularly relates to a isothiocyanate biomimetic preparation and a preparation method thereof. BACKGROUND
[0002] Isothiocyanate compounds are an important class of natural active ingredients, which have the effects of anti-cancer, anti-inflammatory, anti-aging, etc. The compounds are mainly found in cruciferous plants, but do not exist in the form of isothiocyanate directly in the natural state. The precursor glucosinolate and the key enzyme myrosinase are physically separated in the plant cells, and only when the tissues are damaged, the two are in contact and then hydrolyzed to generate isothiocyanate. Although it has excellent biological activity, its clinical application faces severe challenges such as poor stability, harsh storage conditions, and low effective utilization rate. The characteristic group isothiocyanate group (-N=C=S) is easily attacked by nucleophilic attack and degraded, and needs to be strictly stored at low temperature and low water content. At the same time, isothiocyanate causes serious gastrointestinal irritation when taken orally, which makes it difficult to achieve an effective drug dose. SUMMARY
[0003] Therefore, the purpose of the present application is to provide an isothiocyanate biomimetic preparation and a preparation method thereof. The isothiocyanate biomimetic preparation provided by the present application physically separates myrosinase and glucosinolate, can be stored stably for a long time under dry room temperature conditions, can stably and slowly release an adequate dose of isothiocyanate when taken orally into the gastrointestinal tract, and effectively avoids the strong irritation of isothiocyanate to the gastrointestinal tract.
[0004] In order to achieve the above-mentioned purpose, the present application provides the following technical solutions: The present application provides an isothiocyanate biomimetic preparation, which comprises myrosinase microparticles and a glucosinolate extract layer coated on the outer layer of the myrosinase microparticles. The components of the myrosinase microparticles comprise: a myrosinase extract, a binder, a first filler, and a first lubricant. The components of the glucosinolate extract layer comprise: a glucosinolate extract, a second filler, a disintegrant, and a second lubricant.
[0005] Preferably, the extraction method of the glucosinolate extract is: first extracting a first cruciferous plant tissue to obtain a glucosinolate extract; the temperature of the first extraction is 60-100℃, and the time is 5-180 min.
[0006] Preferably, the extraction method of the myrosinase extract is: second extracting a second cruciferous plant tissue to obtain a myrosinase extract; the temperature of the second extraction is 4-40℃, and the time is 0.5-24 h.
[0007] Preferably, the binder is pregelatinized starch.
[0008] Preferably, the first filler and the second filler are microcrystalline cellulose.
[0009] Preferably, the first lubricant and the second lubricant are magnesium stearate.
[0010] Preferably, the mass ratio of the myrosinase extract, the binder, the first filler and the first lubricant is 1:0.1~1:0.2~2:0.01~0.05.
[0011] Preferably, the mass ratio of the glucosinolate extract, the second filler, the disintegrant and the second lubricant is 1:0.01~0.5:0.02~0.2:0.01~0.05.
[0012] Preferably, the mass ratio of the glucosinolate extract layer and the myrosinase microparticle is 1:0.05~2.
[0013] The present application also provides a preparation method of the isothiocyanate biomimetic preparation described in the above technical solution, comprising the following steps: mixing the myrosinase extract, the binder, the first filler and the first lubricant, granulating to obtain the myrosinase microparticle; mixing the myrosinase microparticle, the glucosinolate extract, the second filler, the disintegrant and the second lubricant, forming to obtain the isothiocyanate biomimetic preparation.
[0014] The present application provides an isothiocyanate biomimetic preparation, comprising a myrosinase microparticle and a glucosinolate extract layer coated on the outer layer of the myrosinase microparticle; the components of the myrosinase microparticle comprise a myrosinase extract, a binder, a first filler and a first lubricant; the components of the glucosinolate extract layer comprise a glucosinolate extract, a second filler, a disintegrant and a second lubricant. The present application first prepares the extract rich in myrosinase into a myrosinase microparticle, and then coats a glucosinolate extract layer on the outer layer thereof, ensuring the physical separation of myrosinase and glucosinolate. Since the spatial separation state of the natural glucosinolate-myrosinase system is imitated, compared with unstable isothiocyanate, glucosinolate and myrosinase can be stored stably for a long time under room temperature and dry conditions, finally ensuring that the preparation can release sufficient dose of isothiocyanate when orally taken into the gastrointestinal tract. At the same time, the myrosinase in the microparticle can exhibit a more controllable catalytic rate than free myrosinase, so that isothiocyanate is stably and slowly generated in the gastrointestinal tract, effectively avoiding the strong irritability of isothiocyanate to the gastrointestinal tract. BRIEF DESCRIPTION OF DRAWINGS
[0015] Figure 1Figure showing the amount of sulforaphane generated from the sulforaphane biomimetic formulation prepared in Example 1 over a period of three months; Figure 2 Figure showing the amount of sulforaphane generated from the sulforaphane biomimetic formulation prepared in Example 2 over a period of three months; Figure 3 Figure showing the amount of sulforaphane generated from the sulforaphane biomimetic formulation prepared in Example 3 over a period of 6 hours; Figure 4 Figure showing the amount of sulforaphane generated from the sulforaphane biomimetic formulation prepared in Comparative Example 1 using low temperature extraction of glucosinolates over a period of 6 hours; Figure 5 Figure showing the amount of sulforaphane generated from the sulforaphane biomimetic formulation prepared in Comparative Example 2 using high temperature extraction of myrosinase over a period of 6 hours; Figure 6 Figure showing the amount of sulforaphane generated from the sulforaphane biomimetic formulation prepared in Comparative Example 3 without pre-preparation of myrosinase microparticles over a period of three months; Figure 7 Figure showing the amount of sulforaphane generated from the sulforaphane biomimetic formulation prepared in Comparative Example 4 by directly mixing glucosinolates and myrosinase extract over a period of 6 hours. DETAILED DESCRIPTION
[0016] The present application provides a isothiocyanate biomimetic formulation comprising myrosinase microparticles and a glucosinolate extract layer coated on the outer layer of the myrosinase microparticles; The components of the myrosinase microparticles comprise myrosinase extract, binder, first filler and first lubricant; The components of the glucosinolate extract layer comprise glucosinolate extract, second filler, disintegrant and second lubricant.
[0017] The source of the raw materials used in the present application is not particularly limited unless otherwise specified, and commercially available products known to those skilled in the art can be used.
[0018] As an embodiment, the extraction method of the glucosinolate extract comprises first extracting the first Brassicaceae plant tissue to obtain the glucosinolate extract.
[0019] As an embodiment, the glucosinolate extract comprises one or more of sinigrin, glucoraphanin, gluconasturtiin, myrosin, indole glucosinolate and benzyl glucosinolate, and in particular embodiments, sinigrin or glucoraphanin.
[0020] As an embodiment, the first Brassicaceae plant tissue is derived from one or more of broccoli, radish, arugula, and maca, and in particular embodiments, the first Brassicaceae plant tissue is derived from broccoli or radish; the first Brassicaceae plant tissue is a seed, and in particular embodiments, the first Brassicaceae plant tissue is a broccoli seed or a radish seed; the first extraction is performed at a temperature of 60-100°C, and in another embodiment, the first extraction is performed at a temperature of 90-100°C, and in particular embodiments, the first extraction is performed at a temperature of 90°C, and for a time period of 5-180 min, and in particular embodiments, the first extraction is performed for a time period of 30 min; the first extraction is performed under agitation; the agitation is performed at a rate of 100-1000 rpm, and in particular embodiments, the agitation is performed at a rate of 200 rpm; the first extraction is performed using water as the extraction solvent; and the ratio of the mass of the first Brassicaceae plant tissue to the volume of the extraction solvent used in the first extraction is 1 g to (5-50) mL, and in particular embodiments, the ratio of the mass of the first Brassicaceae plant tissue to the volume of the extraction solvent used in the first extraction is 1 g to 20 mL. Different plant sources can be selected in the art depending on the desired class of isothiocyanates.
[0021] As an embodiment, prior to the first extraction, the first Brassicaceae plant tissue is comminuted to a particle size of 20-200 mesh, and in particular embodiments, the first Brassicaceae plant tissue is comminuted to a particle size of 60 mesh; and after the first extraction, the product of the first extraction is sequentially cooled, subjected to solid-liquid separation, and the resulting extract is sequentially subjected to ultrafiltration, nanofiltration, and drying to obtain a glucosinolate extract powder; the cooling is performed to room temperature, and in particular embodiments, the cooling is performed to a temperature of 20-25°C; the solid-liquid separation is performed by filtration; the ultrafiltration membrane used in the ultrafiltration has a molecular weight cut-off of 1-20 kDa, and in another embodiment, the ultrafiltration membrane has a molecular weight cut-off of 4-10 kDa, and in particular embodiments, the ultrafiltration membrane has a molecular weight cut-off of 4 kDa; the nanofiltration membrane used in the nanofiltration has a molecular weight cut-off of 100-400 Da, and in another embodiment, the nanofiltration membrane has a molecular weight cut-off of 200-300 Da, and in particular embodiments, the nanofiltration membrane has a molecular weight cut-off of 200 Da; the concentration of the solid content in the concentrated solution obtained by nanofiltration is 5-50 wt%, and in another embodiment, the concentration of the solid content in the concentrated solution obtained by nanofiltration is 30-35 wt%, and in particular embodiments, the concentration of the solid content in the concentrated solution obtained by nanofiltration is 30 wt%; the drying is performed at a temperature of 110-180°C, and in particular embodiments, the drying is performed at a temperature of 145°C; and the drying is performed by spray drying.
[0022] As an embodiment, the black mustard enzyme extract is obtained by performing a second extraction on a second Brassicaceae plant tissue.
[0023] As an embodiment, the second Brassica plant tissue is derived from one or more of broccoli, radish, rape and arugula, and in particular embodiments, the second Brassica plant tissue is derived from radish or rape; the second Brassica plant tissue is a seed, and in particular embodiments, the second Brassica plant tissue is a radish seed or a rape seed; the second extraction is performed at a temperature of 4-40°C, and in another embodiment, the second extraction is performed at a temperature of 16-20°C, and in particular embodiments, the second extraction is performed at a temperature of 20°C, and for a time period of 0.5-24 h, and in particular embodiments, the second extraction is performed for a time period of 4 h; the second extraction is performed under agitation; the agitation is performed at a rate of 200-2000 rpm, and in particular embodiments, the agitation is performed at a rate of 500 rpm; the extraction agent used in the second extraction is water; and the ratio of the mass of the second Brassica plant tissue to the volume of the extraction agent used in the second extraction is 1 g to (5-40) mL, and in particular embodiments, the ratio of the mass of the second Brassica plant tissue to the volume of the extraction agent used in the second extraction is 1 g to 10 mL.
[0024] As an embodiment, prior to the second extraction, the second Brassica plant tissue is comminuted to a particle size of 50-250 mesh, and in particular embodiments, the second Brassica plant tissue is comminuted to a particle size of 80 mesh; and after the second extraction, the product of the second extraction is subjected to solid-liquid separation, and the resulting extract is sequentially subjected to ultrafiltration and drying to obtain the myrosinase extract; the solid-liquid separation is filtration; the ultrafiltration membrane used in the ultrafiltration has a molecular weight cut-off of 1-100 kDa, and in particular embodiments, the ultrafiltration membrane has a molecular weight cut-off of 10 kDa; the concentration of the solid content in the concentrated solution obtained by the ultrafiltration is 1-30 wt%, and in particular embodiments, the concentration of the solid content in the concentrated solution obtained by the ultrafiltration is 25 wt%; the drying is performed at a temperature of 105-150°C, and in particular embodiments, the drying is performed at a temperature of 130°C; and the drying is spray drying.
[0025] As an embodiment, the binder is pregelatinized starch; the first filler is microcrystalline cellulose; the first lubricant is magnesium stearate; and the mass ratio of the myrosinase extract, the binder, the first filler and the first lubricant is 1:0.1-1:0.2-2:0.01-0.05, and in particular embodiments, the mass ratio of the myrosinase extract, the binder, the first filler and the first lubricant is 1:0.2:0.25:0.02; the myrosinase microparticles are spherical microparticles; the myrosinase microparticles have a particle weight of 1-20 mg, and in particular embodiments, the myrosinase microparticles have a particle weight of 5 mg, and a particle size of 1-5 mm, and in particular embodiments, the myrosinase microparticles have a particle size of 2.5 mm.
[0026] As an embodiment, the second filler is microcrystalline cellulose; the disintegrant is low-substituted hydroxypropyl cellulose; the degree of substitution of the low-substituted hydroxypropyl cellulose is 5-10%; the second lubricant is magnesium stearate; the mass ratio of the thioglucoside extract, the second filler, the disintegrant and the second lubricant is 1:0.01-0.5:0.02-0.2:0.01-0.05, and in a specific embodiment, 1:0.1:0.15:0.02; the mass ratio of the thioglucoside extract layer and the myrosinase microparticle is 1:0.05-2, and in a specific embodiment, 1:0.35. The binder, the first filler, the first lubricant, the second filler, the disintegrant and the second lubricant used in the present application are all commercially available products.
[0027] The present application also provides a preparation method of the isothiocyanate biomimetic preparation described in the above technical solution, comprising the following steps: The myrosinase extract, the binder, the first filler and the first lubricant are mixed to perform granulation to obtain a myrosinase microparticle. The myrosinase microparticle, the thioglucoside extract, the second filler, the disintegrant and the second lubricant are mixed to perform molding to obtain an isothiocyanate biomimetic preparation.
[0028] The myrosinase extract, the binder, the first filler and the first lubricant are mixed to perform granulation to obtain a myrosinase microparticle.
[0029] As an embodiment, the granulation device is a granulator; the mixing and granulation process of the myrosinase extract, the binder, the first filler and the first lubricant is not specially limited, and a mixing and granulation process well known in the art can be used.
[0030] After the myrosinase microparticle and the thioglucoside extract are obtained, the myrosinase microparticle, the thioglucoside extract, the second filler, the disintegrant and the second lubricant are mixed to perform molding to obtain an isothiocyanate biomimetic preparation.
[0031] As an embodiment, the black mustard enzyme microparticles, the glucosinolate extract, the second filler, the disintegrant and the second lubricant are mixed as follows: the glucosinolate extract, the second filler, the disintegrant and the second lubricant are mixed first, and then the obtained mixture containing the glucosinolate extract is mixed with the black mustard enzyme microparticles; the molding is tabletting; the tabletting equipment is a tablet press; the tabletting pressure is 10-100 kN, and in particular embodiments, the pressure is 40 kN; the dwell time is 10-500 ms, and in particular embodiments, the dwell time is 50 ms; the round tablet obtained by tabletting has a tablet weight of 0.2-1.5 g, and in particular embodiments, the tablet weight is 0.5 g; the diameter is 0.5-1.5 cm, and in particular embodiments, the diameter is 1 cm; and the thickness is 0.2-1 cm, and in particular embodiments, the thickness is 0.5 cm.
[0032] The present application imitates the natural glucosinolate-myrosinase system, extracts glucosinolate and myrosinase from cruciferous plants by different processes, preferentially prepares myrosinase extract into microparticles, and then adds glucosinolate extract and appropriate excipients to prepare an isothiocyanate biomimetic preparation in which glucosinolate and myrosinase are physically separated. In the preparation, isothiocyanate is stored in the form of stable precursor glucosinolate, activated by myrosinase in the gastrointestinal tract, and slowly releases an adequate dose of isothiocyanate. The preparation greatly improves the effective storage time of isothiocyanate, and can be stored for a long time under dry room temperature conditions; at the same time, myrosinase exhibits controllable enzyme activity in the preparation, ensuring that isothiocyanate is slowly generated in the gastrointestinal tract, effectively avoiding the strong irritability of instant high-concentration isothiocyanate to the gastrointestinal tract. The isothiocyanate biomimetic preparation provided by the present application does not react with glucosinolate and myrosinase under dry and anhydrous conditions, and the contact on the preparation can be ignored. Only after disintegration in the digestive tract, the two will contact and react.
[0033] The technical solutions in the present application will be clearly and completely described below in combination with the embodiments in the present application, but they should not be understood as limitations to the protection scope of the present application.
[0034] Example 1 (1) The broccoli seeds are ground to 60 mesh, and then 1 g of the powder is mixed with 20 mL of water at 90°C under stirring at 200 rpm for 30 min. After cooling to room temperature 20°C, the residue is removed by filtration. The obtained extract is filtered through a 4 kDa ultrafiltration membrane, and the permeate is concentrated to a solid content of 30 wt%. The concentrate is spray dried at 145°C to obtain a glucoraphanin (the precursor glucosinolate of isothiocyanate sulforaphane) extract powder, and the content of glucoraphanin in the powder is about 20 wt%. (2) The radish seeds were crushed to 80 mesh, and stirred at 500 rpm for 4 h at 20 °C according to the ratio of 1 g powder: 10 mL water. After stirring, the residue was removed by filtration, and concentrated to a solid content of 25 wt% by 10 kDa ultrafiltration membrane, and spray dried at 130 °C to obtain the myrosinase extract; (3) The myrosinase extract, pregelatinized starch, microcrystalline cellulose, and magnesium stearate were mixed uniformly according to the mass ratio of 1:0.2:0.25:0.02, and granulated into spherical microparticles with a particle size of 2.5 mm and a weight of 5 mg per particle in a granulator to obtain the myrosinase microparticles; (4) The sulforaphane extract, microcrystalline cellulose, low-substitution hydroxypropyl cellulose (the substitution degree of the commercially available product is marked as 5-10%), and magnesium stearate were mixed uniformly according to the mass ratio of 1:0.1:0.15:0.02, and injected into a tablet press according to the mass ratio of the mixture:myrosinase microparticles of 1:0.35, and pressed at a pressure of 40 kN for 50 ms to obtain circular tablets with a weight of 0.5 g, a diameter of 1 cm, and a thickness of 0.5 cm, which were the sulforaphane biomimetic preparation.
[0035] Example 2 (1) The radish seeds were crushed to 60 mesh, and stirred at 200 rpm for 30 min at 100 °C according to the ratio of 1 g powder:20 mL water. After cooling to room temperature of 20 °C, the residue was removed by filtration, and the permeate was obtained after 10 kDa ultrafiltration membrane. The permeate was concentrated to a solid content of 35% by 300 Da nanofiltration membrane, and spray dried at 145 °C to obtain the raphanin (pro-sulforaphane, thioglucoside) extract powder, and the content of raphanin in the powder was about 31 wt%; (2) The radish seeds were crushed to 80 mesh, and stirred at 500 rpm for 4 h at 20 °C according to the ratio of 1 g powder:10 mL water. After stirring, the residue was removed by filtration, and concentrated to a solid content of 25 wt% by 10 kDa ultrafiltration membrane, and spray dried at 130 °C to obtain the myrosinase extract; (3) The myrosinase extract, pregelatinized starch, microcrystalline cellulose, and magnesium stearate were mixed uniformly according to the mass ratio of 1:0.2:0.25:0.02, and granulated into spherical microparticles with a particle size of 2.5 mm and a weight of 5 mg per particle in a granulator to obtain the myrosinase microparticles; (4) The raphanin extract, microcrystalline cellulose, low-substitution hydroxypropyl cellulose (the substitution degree of the commercially available product is marked as 5-10%), and magnesium stearate were mixed uniformly according to the mass ratio of 1:0.1:0.15:0.02, and injected into a tablet press according to the mass ratio of the mixture:myrosinase microparticles of 1:0.35, and pressed at a pressure of 40 kN for 50 ms to obtain circular tablets with a weight of 0.5 g, a diameter of 1 cm, and a thickness of 0.5 cm, which were the sulforaphane biomimetic preparation.
[0036] Example 3 (1) Broccoli seeds were ground to 60 mesh, and 1 g of the powder was stirred and heated in 20 mL of water at 100°C for 30 min at 200 rpm. After cooling to room temperature (20°C), the residue was removed by filtration, and the permeate was obtained after passing through a 4 kDa ultrafiltration membrane. The permeate was concentrated to a solid content of 30 wt% by passing through a 200 Da nanofiltration membrane, and the resulting powder was spray-dried at 145°C to obtain a sulforaphane precursor (sulforaphane glucosinolate) extract powder. The content of sulforaphane glucosinolate in the powder was about 18 wt%; (2) Radish seeds were ground to 80 mesh, and 1 g of the powder was stirred in 15 mL of water at 16°C for 4 h at 500 rpm. After stirring, the residue was removed by filtration, and the solid content was concentrated to 20 wt% by passing through a 10 kDa ultrafiltration membrane. The resulting black mustard enzyme extract was spray-dried at 130°C. (3) The black mustard enzyme extract, pregelatinized starch, microcrystalline cellulose, and magnesium stearate were mixed in a mass ratio of 1:0.2:0.25:0.02, and spherical microparticles with a particle size of 5 mg and a diameter of 2.5 mm were prepared in a granulator. The resulting black mustard enzyme microparticles were obtained. (4) The sulforaphane glucosinolate extract, microcrystalline cellulose, low-substitution hydroxypropyl cellulose (commercial product with a substitution degree of 5-10%), and magnesium stearate were mixed in a mass ratio of 1:0.1:0.15:0.02. The mixture was injected into a tablet press at a mass ratio of 1:0.35 with the black mustard enzyme microparticles, and circular tablets with a mass of 0.5 g, a diameter of 1 cm, and a thickness of 0.5 cm were prepared under a pressure of 40 kN for 50 ms. The resulting sulforaphane biomimetic preparation was obtained.
[0037] Comparative Example 1 (compared with Example 3, the extraction temperature of the glucosinolate was as low as 50°C): (1) Broccoli seeds were ground to 60 mesh, and 1 g of the powder was stirred and heated in 20 mL of water at 50°C for 30 min at 200 rpm. After cooling to room temperature (20°C), the residue was removed by filtration, and the permeate was obtained after passing through a 4 kDa ultrafiltration membrane. The permeate was concentrated to a solid content of 30 wt% by passing through a 200 Da nanofiltration membrane, and the resulting powder was spray-dried at 145°C to obtain a sulforaphane precursor (sulforaphane glucosinolate) extract powder. The content of sulforaphane glucosinolate in the powder was about 4.7 wt%; (2) Radish seeds were ground to 80 mesh, and 1 g of the powder was stirred in 10 mL of water at 16°C for 4 h at 500 rpm. After stirring, the residue was removed by filtration, and the solid content was concentrated to 20 wt% by passing through a 10 kDa ultrafiltration membrane. The resulting black mustard enzyme extract was spray-dried at 130°C. (3) The black mustard enzyme extract, pregelatinized starch, microcrystalline cellulose and magnesium stearate were mixed in a mass ratio of 1:0.2:0.25:0.02, and then granulated in a granulator to form spherical particles with a particle size of 2.5 mm and a weight of 5 mg, namely black mustard enzyme particles; (4) The sulforaphane extract, microcrystalline cellulose, low-substitution hydroxypropyl cellulose (a commercially available product with a substitution degree of 5-10%) and magnesium stearate were mixed in a mass ratio of 1:0.1:0.15:0.02, and then injected into a tablet press in a mixture:black mustard enzyme particle mass ratio of 1:0.35, and pressed at a pressure of 40 kN for 50 ms to form circular tablets with a weight of 0.5 g, a diameter of 1 cm and a thickness of 0.5 cm, namely sulforaphane biomimetic preparations.
[0038] Comparative Example 2 (compared with Example 3, the black mustard enzyme extraction temperature is as high as 50°C): (1) The broccoli seeds were ground to 60 mesh, and then 1 g of the powder was mixed with 20 mL of water at 100°C under stirring at 200 rpm for 30 min. After cooling to room temperature of 20°C, the residue was removed by filtration, and the permeate was obtained after passing through a 4 kDa ultrafiltration membrane. The permeate was concentrated to a solid content of 30 wt% by passing through a 200 Da nanofiltration membrane, and then spray dried at 145°C to obtain a sulforaphane extract (a precursor of isothiocyanate sulforaphane, sulforaphane glucosinolate) powder. The content of sulforaphane glucosinolate in the powder was about 18%; (2) The radish seeds were ground to 80 mesh, and then 1 g of the powder was mixed with 15 mL of water at 16°C under stirring at 500 rpm for 4 h. After stirring, the residue was removed by filtration, and the concentrate was obtained after passing through a 10 kDa ultrafiltration membrane. The concentrate was spray dried at 130°C to obtain a black mustard enzyme extract; (3) The black mustard enzyme extract, pregelatinized starch, microcrystalline cellulose and magnesium stearate were mixed in a mass ratio of 1:0.2:0.25:0.02, and then granulated in a granulator to form spherical particles with a particle size of 2.5 mm and a weight of 5 mg, namely black mustard enzyme particles; (4) The sulforaphane extract, microcrystalline cellulose, low-substitution hydroxypropyl cellulose (a commercially available product with a substitution degree of 5-10%) and magnesium stearate were mixed in a mass ratio of 1:0.1:0.15:0.02, and then injected into a tablet press in a mixture:black mustard enzyme particle mass ratio of 1:0.35, and pressed at a pressure of 40 kN for 50 ms to form circular tablets with a weight of 0.5 g, a diameter of 1 cm and a thickness of 0.5 cm, namely sulforaphane biomimetic preparations.
[0039] Comparative Example 3 (compared with Example 1, no pre-preparation of black mustard enzyme particles to change the stability): (1) Broccoli seeds were crushed to 60 mesh, and 1 g of the powder was stirred and heated in 20 mL of water at 90°C for 30 min at 200 rpm. After cooling to room temperature (20°C), the residue was removed by filtration, and the permeate was obtained after passing through a 4 kDa ultrafiltration membrane. The permeate was concentrated to a solid content of 30 wt% by passing through a 200 Da nanofiltration membrane and spray dried at 145°C to obtain a powder of glucoraphanin (a precursor of sulforaphane isothiocyanate), with a content of about 20 wt% glucoraphanin in the powder; (2) Radish seeds were crushed to 80 mesh, and 1 g of the powder was stirred in 10 mL of water at 20°C for 4 h at 500 rpm. After stirring, the residue was removed by filtration, and the concentrate was obtained after passing through a 10 kDa ultrafiltration membrane to a solid content of 25 wt%. The concentrate was spray dried at 130°C to obtain a myrosinase extract; (3) The glucoraphanin extract, microcrystalline cellulose, low-substituted hydroxypropyl cellulose (a commercially available product with a substitution degree of 5-10%), and magnesium stearate were mixed in a mass ratio of 1:0.1:0.15:0.02, and the mixture was injected into a tablet press in a mass ratio of 1:0.35 to the myrosinase extract. The mixture was pressed at a pressure of 40 kN for 50 ms to form circular tablets with a weight of 0.5 g, a diameter of 1 cm, and a thickness of 0.5 cm. The sulforaphane preparation was obtained.
[0040] Comparative Example 4 (comparison with Example 1, release effect of sulforaphane when no preparation is formed): (1) Broccoli seeds were crushed to 60 mesh, and 1 g of the powder was stirred and heated in 20 mL of water at 90°C for 30 min at 200 rpm. After cooling to room temperature (20°C), the residue was removed by filtration, and the permeate was obtained after passing through a 4 kDa ultrafiltration membrane. The permeate was concentrated to a solid content of 30 wt% by passing through a 200 Da nanofiltration membrane and spray dried at 145°C to obtain a powder of glucoraphanin (a precursor of sulforaphane isothiocyanate), with a content of about 20 wt% glucoraphanin in the powder; (2) Radish seeds were crushed to 80 mesh, and 1 g of the powder was stirred in 10 mL of water at 20°C for 4 h at 500 rpm. After stirring, the residue was removed by filtration, and the concentrate was obtained after passing through a 10 kDa ultrafiltration membrane to a solid content of 25 wt%. The concentrate was spray dried at 130°C to obtain a myrosinase extract; (3) No excipients were added, and the glucoraphanin extract and the myrosinase extract were mixed in a mass ratio of 2.25:1 (the content ratio of the glucoraphanin extract to the myrosinase extract in the biomimetic preparation prepared in Example 1) to obtain a sulforaphane preparation.
[0041] Performance test (1) The sulforaphane biomimetic preparation prepared in Example 1 was prepared according to the simulated intestinal fluid in the Chinese Pharmacopoeia 2020 edition, to simulate the release effect of sulforaphane in the intestinal tract of sulforaphane biomimetic preparation, and the amount of sulforaphane generated in the sulforaphane biomimetic preparation within three months was as shown in Table 1. From Table 1, it can be seen that the amount of sulforaphane generated was 20.6 mg / tablet, the conversion rate of sinigrin was 94.3%, and the amount of sulforaphane generated within three months did not decrease significantly. Figure 1 Figure 1
[0042] (2) The erucin biomimetic preparation prepared in Example 2 was prepared according to the simulated intestinal fluid in the Chinese Pharmacopoeia 2020 edition, to simulate the release effect of erucin in the intestinal tract of erucin biomimetic preparation, and the amount of erucin generated in the erucin biomimetic preparation within three months was as shown in Table 2. From Table 2, it can be seen that the amount of erucin generated was 32.8 mg / tablet, the conversion rate of erucin was 98.1%, and the amount of erucin generated within three months did not decrease significantly. Figure 2 Figure 2
[0043] (3) The sulforaphane biomimetic preparation prepared in Example 3 was prepared according to the simulated intestinal fluid in the Chinese Pharmacopoeia 2020 edition, to simulate the release effect of sulforaphane in the intestinal tract of sulforaphane biomimetic preparation, and the amount of sulforaphane generated within 6h was as shown in Table 3. From Table 3, it can be seen that the amount of sulforaphane generated slowly increased. Figure 3 Figure 3
[0044] (4) The sulforaphane biomimetic preparation prepared in Comparative Example 1 was prepared according to the simulated intestinal fluid in the Chinese Pharmacopoeia 2020 edition, to simulate the release effect of sulforaphane in the intestinal tract of sulforaphane biomimetic preparation, and the amount of sulforaphane generated within 6h was as shown in Table 4. From Table 4, it can be seen that the amount of sulforaphane generated in the sulforaphane biomimetic preparation prepared by low-temperature extraction of glucosinolate was significantly lower than that of Example 3, and the increasing trend was slow. Figure 4 Figure 4
[0045] (5) The sulforaphane biomimetic preparation prepared in Comparative Example 2 was prepared according to the simulated intestinal fluid in the Chinese Pharmacopoeia 2020 edition, to simulate the release effect of sulforaphane in the intestinal tract of sulforaphane biomimetic preparation, and the amount of sulforaphane generated within 6h was as shown in Table 5. From Table 5, it can be seen that the amount of sulforaphane generated in the sulforaphane biomimetic preparation prepared by high-temperature extraction of myrosinase was significantly lower than that of Example 3, and the increasing trend was slow. Figure 5 Figure 5
[0046] (6) The sulforaphane preparation prepared in Comparative Example 3 was prepared according to the simulated intestinal fluid in the Chinese Pharmacopoeia 2020 edition to simulate the release effect of sulforaphane in the intestinal tract of the sulforaphane bionic preparation, and the sulforaphane production amount of the sulforaphane preparation prepared by the myrosinase microparticles without pre-preparation within three months was as shown in Table 6. Figure 6 As can be seen from Table 6, the sulforaphane production amount of the sulforaphane preparation prepared in Comparative Example 3 was 18.4 mg / tablet, the conversion rate of sinigrin was 85.3%, and the sulforaphane production amount slowly decreased within three months. Figure 6
[0047] (7) The sulforaphane preparation prepared in Comparative Example 4 was prepared according to the simulated intestinal fluid in the Chinese Pharmacopoeia 2020 edition to simulate the release effect of sulforaphane in the intestinal tract of the sulforaphane preparation, and the change of the sulforaphane production amount within 6h was evaluated, and the sulforaphane production amount of the direct mixture of glucosinolate and myrosinase extract within 6h was as shown in Table 7. Figure 7 As can be seen from Table 7, the sulforaphane production amount rapidly reached a peak within 10 min. Figure 7
[0048] Although the above examples make a detailed description of the present application, it is only a part of the embodiments of the present application but not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which all belong to the protection scope of the present application.
Claims
1. A biomimetic isothiocyanate formulation, characterized in that, Includes black myrosinase microparticles and a glucosinolate extract layer coating the outer layer of the black myrosinase microparticles; The components of the black myrosinase microparticles include: black myrosinase extract, binder, first filler and first lubricant; The components of the glucosinolate extract layer include: glucosinolate extract, a second filler, a disintegrant, and a second lubricant.
2. The isothiocyanate biomimetic formulation according to claim 1, characterized in that, The extraction method of the glucosinolate extract is as follows: the tissue of a first cruciferous plant is subjected to a first extraction to obtain the glucosinolate extract; the temperature of the first extraction is 60~100℃ and the time is 5~180min.
3. The isothiocyanate biomimetic formulation according to claim 1, characterized in that, The extraction method of the black myristica enzyme extract is as follows: a second extraction is performed on the tissue of a second cruciferous plant to obtain the black myristica enzyme extract; the temperature of the second extraction is 4~40℃ and the time is 0.5~24h.
4. The isothiocyanate biomimetic formulation according to claim 1, characterized in that, The adhesive is pregelatinized starch.
5. The isothiocyanate biomimetic formulation according to claim 1, characterized in that, The first filler and the second filler are microcrystalline cellulose.
6. The isothiocyanate biomimetic formulation according to claim 1, characterized in that, The first and second lubricants are magnesium stearate.
7. The isothiocyanate biomimetic formulation according to claim 1, characterized in that, The mass ratio of the black mustard enzyme extract, binder, first filler and first lubricant is 1:0.1~1:0.2~2:0.01~0.
05.
8. The isothiocyanate biomimetic formulation according to claim 1, characterized in that, The mass ratio of the glucosinolate extract, the second filler, the disintegrant, and the second lubricant is 1:0.01~0.5:0.02~0.2:0.01~0.
05.
9. The isothiocyanate biomimetic formulation according to claim 1, characterized in that, The mass ratio of the glucosinolate extract layer to the black myrosinase microparticles is 1:0.05~2.
10. The method for preparing the isothiocyanate biomimetic formulation according to any one of claims 1 to 9, characterized in that, Includes the following steps: Black myristica enzyme extract, binder, first filler and first lubricant are mixed and granulated to obtain black myristica enzyme microparticles; The black myristase microparticles, glucosinolate extract, second filler, disintegrant, and second lubricant are mixed and molded to obtain a biomimetic isothiocyanate formulation.