Self-assembled nanomicellar drug for leukopenia and preparation method thereof

Abstract

This invention relates to the field of pharmaceutical technology, and more specifically, to a self-assembled nanomicelle drug for leukopenia and its preparation method. This invention utilizes self-assembled nanomicelle technology to significantly increase the content of key components such as astragaloside III, astragaloside A, tanshinone IIA, chlorogenic acid, and baicalin in the drug compared to traditional formulations, resulting in a significant increase in the content of active ingredients and effectively enhancing the drug's bioactivity. By integrating components such as tanshinone IIA, chlorogenic acid, and baicalin through self-assembly technology, non-covalent interactions are formed, fully leveraging the advantages of multi-target and multi-pathway synergistic therapy in traditional Chinese medicine. This invention combines traditional Chinese medicine theory with modern nanotechnology, providing a highly efficient and stable traditional Chinese medicine nanomedicine solution for leukopenia caused by radiotherapy and chemotherapy, thus overcoming the shortcomings of existing traditional Chinese medicine drugs for increasing white blood cell count.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically, to a self-assembled nanomicelle drug for leukopenia and its preparation method. Background Technology

[0002] Leukopenia is a syndrome caused by various factors, characterized by a persistently low total peripheral blood white blood cell count of less than 4.0 × 10⁻⁶. 9 Leukopenia induced by radiotherapy and chemotherapy is a common diagnostic criterion in clinical internal medicine. Based on its cause, it can be divided into two types: identifiable and secondary. Leukopenia secondary to radiotherapy and chemotherapy is particularly common. Approximately 90% of cancer patients experience adverse reactions such as leukopenia after radiotherapy and chemotherapy, leading to decreased immunity, significantly increased susceptibility, and in some cases, severe infections that can be life-threatening. It can also affect chemotherapy regimens, becoming a major obstacle to completing treatment. Therefore, elevating leukopenia caused by radiotherapy and chemotherapy is crucial.

[0003] Traditional Chinese medicine (TCM) understands leukopenia based on the patient's clinical symptoms, such as general weakness, dizziness, sallow complexion, poor appetite, shortness of breath, nausea and vomiting, fever, pale tongue, and weak pulse, classifying it under the categories of "asthenia" and "qi and blood deficiency." It believes the etiology of this disease stems from congenital insufficiency, weak constitution, acquired malnutrition damaging the five internal organs, and prolonged illness without proper care. Congenital insufficiency and acquired malnutrition can damage the five internal organs, especially the spleen and kidneys, leading to insufficient production of qi and blood. Prolonged illness further exacerbates the already weak constitution, causing the spleen to fail to produce blood, essence to fail to transform into blood, and dysfunction of the internal organs. Internal deficiency weakens the body's defenses, making it susceptible to external pathogens and recurrent fever.

[0004] The number of patients with malignant tumors is increasing year by year, and radiotherapy and chemotherapy are currently the main treatment methods for patients with malignant tumors. However, radiotherapy and chemotherapy drugs are strong and can easily generate "toxic fire". While attacking the cancer toxins in the body, the evil of heat toxins invades the body, depletes the yin and qi, resulting in deficiency of both qi and yin and damage to the functions of the internal organs, especially the spleen and kidneys, leading to deficiency of vital energy and deficiency of the source of qi and blood, resulting in an increase in the proportion of leukopenia.

[0005] Currently, drugs that increase white blood cell count include inosine, squalene, leukocyte-stimulating factor, and human granulocyte colony-stimulating factor, while there are few traditional Chinese medicine drugs that can increase white blood cell count. Based on this, this invention proposes a self-assembled nanomicelle drug for leukopenia and its preparation method. Summary of the Invention

[0006] To overcome the shortcomings of the prior art, this invention provides a self-assembled nanomicelle drug for leukopenia and its preparation method.

[0007] This invention is achieved through the following technical solution: In a first aspect, the present invention provides a self-assembled nanomicelle drug for leukopenia, wherein the drug comprises astragaloside III, astragaloside A, verrucoside isoflavone glucoside, gentianoside, tanshinone IIA, salvianolic acid B, cryptotanshinone, ferulic acid, ligustrol I, ligustrol A, curcuminol, curcumindione, resveratrol, diosgenin, chlorogenic acid, caffeic acid, baicalin, ellagic acid, apigenin-6-C-β-D-glucoside, bromelain, and ethyl caffeate; the self-assembling component comprises tanshinone IIA-chlorogenic acid-baicalin.

[0008] Secondly, the present invention provides a method for preparing self-assembled nanomicelle drugs, comprising the following steps: S1: Take 500-800 parts by weight of black nightshade, 500-800 parts by weight of white nightshade, 500-800 parts by weight of wild strawberry, and 300-400 parts by weight of scutellaria barbata. Crush them into coarse powder and place them in an electromagnetic pyrolysis device. Add 13-15 times the total mass of black nightshade, white nightshade, wild strawberry, and scutellaria barbata ethanol each time. Extract 3 times, 10-30 minutes each time. Then filter to obtain filtrate. Combine the filtrates obtained after extraction and concentrate the combined filtrate under reduced pressure at 70℃ to a relative density of 1.12-1.18 (60℃) to obtain a concentrate. Extract the concentrate with 3-6 times the mass of n-butanol 3-5 times. Combine the extracts and dry the extract under reduced pressure at 70℃ to obtain the n-butanol fraction. Separate and purify the n-butanol fraction by high-speed countercurrent chromatography to obtain the purified n-butanol fraction. S2: Take 600-700 parts by weight of Astragalus membranaceus and 200-300 parts by weight of Salvia miltiorrhiza, grind them into coarse powder, add 11 times the total mass of Astragalus membranaceus and Salvia miltiorrhiza in 30% (volume fraction) ethanol, extract 3 times, filter, combine the filtrates, concentrate the combined filtrate to a relative density of 1.20-1.26 (60℃), add ethanol while stirring after concentration to make the alcohol content of the final concentrate reach 80%, then let it stand at 4℃ for 24h, filter, take the filtrate obtained after standing at 4℃ for 24h, wash the residue obtained after standing at 4℃ for 24h with 80% ethanol to obtain the washing liquid, combine the filtrate and washing liquid after standing at 4℃ for 24h and concentrate under reduced pressure to recover ethanol until there is no alcohol odor, dry the concentrate obtained after reduced pressure concentration to obtain the extract; S3: Take 200-300 parts by weight of Angelica sinensis and 200-300 parts by weight of Curcuma longa, grind them into fine powder, add water with a total weight of 20 times that of Angelica sinensis and Curcuma longa and soak for 3 hours, use a volatile oil extraction device to heat and boil gently for 6 hours, and collect the volatile oil; S4: Take the extract from step S2 and prepare a solution of 180-220 mg / mL with anhydrous ethanol; take the n-butanol purified fraction from step S1, the amount of which is 0.5-0.7 times the weight of the extract from step S2, and dissolve it in 5 times the weight of ultrapure water to obtain an aqueous solution of the n-butanol purified fraction; under stirring conditions, gradually add the ethanol solution of the extract from step S2 to the aqueous solution of the n-butanol purified fraction at a rate of 5 mL / min, and continue stirring for 2.5-3.5 h after the addition is complete, then sonicate under ice bath conditions for 10-20 min (power 180-220 W, frequency 35-45 kHz), and filter; add volatile oil to the filtrate, dissolve it completely, dilute it with water, mix it evenly, and filter to obtain the self-assembled nanomicelle drug.

[0009] Specifically: The extract from step S2 was prepared into a 200 mg / mL solution with anhydrous ethanol. The n-butanol purified fraction from step S1 was dissolved in 300 mL of ultrapure water. The ethanol solution of the extract was then prepared into a 200 mg / mL solution with anhydrous ethanol. The ethanol solution was gradually added dropwise to the n-butanol purified fraction at a rate of 5 mL / min. After the addition was complete, the mixture was stirred and sealed for 3 hours. Subsequently, the mixture was sonicated in an ice bath for 15 minutes (200 W, 40 kHz). The mixture was then filtered to obtain a filtrate. 20 mL of the volatile oil extracted in step S3 was added to the filtrate to ensure complete dissolution. The filtrate was then diluted with water to 1000 mL and mixed thoroughly. The mixture was then filtered through 0.45 μm and 0.22 μm microporous membranes, and the filtrate was collected to obtain the self-assembled nanomicelle drug.

[0010] For certain natural products with amphiphilic structures, a core-shell structure drug delivery system can be formed to load drugs into the core, which can be used as a drug delivery system for self-assembled nanomicelles.

[0011] The self-assembly principle of the amphiphilic molecules of this invention is as follows: Extract (hydrophobic core): The extract in step S2 (such as the ethanol solution of Astragalus membranaceus and Salvia miltiorrhiza) contains hydrophobic components (such as saponins and flavonoids), which form a hydrophobic environment after dissolving in anhydrous ethanol.

[0012] n-Butanol purification site (hydrophilic shell): The n-butanol purification site in step S1 (such as chlorogenic acid, baicalin, etc.) has hydrophilic groups, which form a hydrophilic environment after dissolving in water.

[0013] Self-assembly driven: When an ethanol solution is dropped into the aqueous phase, the ethanol is gradually diluted, and the hydrophobic components aggregate due to the decrease in solubility, forming a hydrophobic core; at the same time, the hydrophilic components wrap around the core surface to reduce the interfacial energy, forming a typical "core-shell" structure of micelles.

[0014] The auxiliary role of ultrasound treatment: Particle size uniformity: Ice bath ultrasound breaks down large particles through cavitation effect, reducing particle size distribution (PDI<0.25) and enhancing micelle stability.

[0015] Energy input: Ultrasound provides energy to promote molecular rearrangement, optimize hydrophobic / hydrophilic interactions, and make the micelle structure more compact.

[0016] Integration of volatile oils: Solubilization and stabilization: After the volatile oils (Angelica sinensis and Curcuma longa extracts) are added dropwise, their lipid-soluble components are embedded in the hydrophobic core of the micelles, which further stabilizes the structure and may enhance the drug loading.

[0017] Further, the high-speed countercurrent chromatography (HSCCC) separation and purification steps in step S1 are as follows: Prepare a solvent system of n-hexane-ethyl acetate-ethanol-water at a volume ratio of 3:7:1:9. After thorough mixing, allow the mixture to stand in a separatory funnel to separate into two phases. Once the two phases are in equilibrium, collect the upper and lower phases separately and place them in multiple brown reagent bottles. Degas the solution using ultrasonication for 20 minutes and set aside. Pump the stationary phase (the upper phase, which is one of the brown bottles used as the reserve upper phase) into the separation tube of the HSCCC. After the upper phase fills the separation tube, turn on the main unit and set the rotation speed to 9000 rpm. After the rotation speed stabilizes at 00 r / min, the mobile phase, i.e. the lower phase (one of the lower phases in the brown bottle is reserved), is pumped into the separation tube at a volumetric flow rate of 1.8 mL / min until the two phases are in equilibrium. Take the n-butanol fraction and dissolve it with an equal volume of the upper and lower phase mixture (the upper and lower phases reserved after ultrasonic degassing for 20 min, and the same volume is mixed). Inject it through the injection port. Set the wavelength of the ultraviolet detector to 320 nm and collect the eluent using an automatic collector. Concentrate and dry the eluent under reduced pressure at 70 °C to obtain the purified n-butanol fraction.

[0018] Furthermore, in step S1, take 678g of Solanum nigrum, 678g of Solanum lyratum, 678g of Duchesnea indica, and 339g of Scutellaria barbata; in step S2, take 678g of Astragalus membranaceus and 226g of Salvia miltiorrhiza; in step S3, take 226g of Angelica sinensis and 226g of Curcuma longa.

[0019] Furthermore, in step S1, the combined extract filtrate is concentrated under reduced pressure at 70°C to a relative density of 1.15 at 60°C, and in step S2, the combined filtrate is concentrated to a relative density of 1.23 at 60°C.

[0020] Thirdly, the present invention provides the application of self-assembled nanomicelle drugs in the treatment of leukopenia, wherein the leukopenia is caused by radiotherapy or chemotherapy.

[0021] Compared with the prior art, the advantages of the present invention are as follows: 1. Through self-assembled nanomicelle technology, the content of key components such as astragaloside III, astragaloside A, tanshinone IIA, chlorogenic acid, and baicalin in the drug is significantly higher than that in traditional preparations, resulting in a significant increase in the content of active ingredients and effectively improving the drug's bioactivity; 2. By integrating components such as tanshinone IIA, chlorogenic acid, and baicalin through self-assembly technology, non-covalent interactions are formed, fully leveraging the advantages of traditional Chinese medicine in multi-target and multi-pathway synergistic therapy; 2. This invention employs high-speed countercurrent chromatography (HSCCC) separation and purification technology and ultrasound-assisted self-assembly process, which improves the extraction efficiency of components and the uniformity of nanomicelles, solving the problems of complex components and low content in traditional Chinese medicine preparations. Animal experiments show that the self-assembled nanomicelle drug is superior to traditional preparations and positive control drugs in restoring the number of peripheral blood leukocytes and increasing the level of serum immune cytokines (such as IL-3, IL-4, and IFN-γ) in radiation-damaged mice, and the difference is statistically significant, thus enhancing the therapeutic effect. 3. The nanomicelles of this invention exhibit uniform particle size distribution (average particle size approximately 254 nm, PDI < 0.25), good dispersibility, and high stability. This nanostructure can improve drug solubility and bioavailability, enhance targeting, reduce side effects, and optimize the drug delivery system. This invention combines traditional Chinese medicine theory with modern nanotechnology to provide a highly efficient and stable traditional Chinese medicine nanomedicine solution for leukopenia caused by radiotherapy and chemotherapy, making up for the shortcomings of existing traditional Chinese medicine drugs for increasing white blood cell count. Attached Figure Description

[0022] The invention will now be further described with reference to the accompanying drawings.

[0023] Figure 1 Diagram of self-assembled nanomicelle drugs; Figure 2 Transmission electron microscope image of self-assembled nanomicelle drugs; Figure 3 Spectral characteristics of component self-assembled nanomicelles; Figure 4 Figure showing the effect of radiation-induced injury on the number of peripheral blood leukocytes in mice. Figure 5 Figure showing the effect of radiation-induced damage on serum immune cytokines in mice. Figure 6 The effect on organ indices. Detailed Implementation

[0024] Example 1: Traditional Drug Preparation Method Prescription: Astragalus membranaceus 678g, Angelica sinensis 226g, Solanum lyratum 678g, Solanum nigrum 6787g, Salvia miltiorrhiza 226g, Scutellaria barbata 678g, Duchesnea indica 339g, Curcuma longa 226g. Except for Salvia miltiorrhiza, the other seven herbs (including Astragalus membranaceus) are decocted twice with water, the first time for 2 hours and the second time for 1 hour. The decoctions are filtered, the filtrates are combined, concentrated under reduced pressure, and dried to obtain a dry extract powder. Salvia miltiorrhiza is extracted three times: first with ethanol under reflux for 1.5 hours, second with 50% ethanol under reflux for 1.5 hours, and third with water for 2 hours. The decoctions are filtered, the filtrates are combined with the ethanol extract, concentrated under reduced pressure, and dried to obtain a dry extract powder. The above dry extract powders are combined, pulverized, sieved, and mixed with an appropriate amount of microcrystalline cellulose. The mixture is then granulated, compressed into 1000 tablets, and coated with a film.

[0025] Example 2: Preparation method of each component of self-assembled nanomicelle drug S1: Take 500-800 parts by weight of black nightshade, 500-800 parts by weight of white nightshade, 500-800 parts by weight of wild strawberry, and 300-400 parts by weight of scutellaria barbata, crush them into coarse powder, place them in an electromagnetic pyrolysis device, add 13-15 times the total mass of black nightshade, white nightshade, wild strawberry, and scutellaria barbata ethanol each time, extract 3 times, 10-30 minutes each time, filter to obtain the extract filtrate, combine the extract filtrates, concentrate the combined extract filtrate under reduced pressure at 70℃ to a relative density of 1.12-1.18 at 60℃ (relative density is 1.15 in this example), to obtain the concentrate; extract the concentrate 3-5 times with 3-6 times the mass of n-butanol, to obtain the extract, combine the extracts, dry the combined extract under reduced pressure at 70℃ to obtain the n-butanol fraction; take the n-butanol fraction for high-speed countercurrent chromatography separation and purification to obtain the purified n-butanol fraction; S2: Take 600-700 parts by weight of Astragalus membranaceus and 200-300 parts by weight of Salvia miltiorrhiza, grind them into coarse powder, add 11 times the total mass of Astragalus membranaceus and Salvia miltiorrhiza in 30% (volume fraction) ethanol, extract 3 times, filter to obtain filtrate, combine the filtrates obtained in this step, concentrate the filtrate to a relative density of 1.20-1.26 at 60℃ (the relative density in this example is 1.23), add ethanol while stirring after concentration to make the alcohol content of the final concentrate reach 80%, then let it stand at 4℃ for 24 hours, filter, take the filtrate, let it stand at 4℃ for 24 hours, wash the residue obtained by filtration with 80% ethanol to obtain the washing liquid, combine the filtrate obtained by filtration after standing at 4℃ for 24 hours and the washing liquid, concentrate under reduced pressure to recover ethanol until there is no alcohol odor, dry the concentrate obtained after reduced pressure concentration to obtain the extract; wherein the reduced pressure concentration conditions are: temperature: 70℃, pressure: -0.09MPa; S3: Take 200-300 parts by weight of Angelica sinensis and 200-300 parts by weight of Curcuma longa, grind them into fine powder, add water with a total weight of 20 times that of Angelica sinensis and Curcuma longa and soak for 3 hours, use a volatile oil extraction device to heat to a gentle boil for 6 hours and collect the volatile oil. S4: Take the extract from step S2 and prepare a solution of 180-220 mg / mL with anhydrous ethanol; take the n-butanol purified fraction from step S1, the amount of which is 0.5-0.7 times the weight of the extract from step S2, and dissolve it in 5 times the weight of ultrapure water to obtain an aqueous solution of the n-butanol purified fraction; under stirring conditions, gradually add the ethanol solution of the extract from step S2 to the aqueous solution of the n-butanol purified fraction at a rate of 5 mL / min, and continue stirring for 2.5-3.5 h after the addition is complete, then sonicate under ice bath conditions for 10-20 min (power 180-220 W, frequency 35-45 kHz), and filter; add volatile oil to the filtrate, dissolve it completely, dilute it with water, mix it evenly, and filter it through 0.45 μm and 0.22 μm microporous membranes in sequence, collect the filtrate, and obtain the self-assembled nanomicelle drug.

[0026] Specifically: The extract from step S2 is prepared into a 200 mg / mL solution using anhydrous ethanol. The n-butanol purified fraction from step S1 is dissolved in 300 mL of ultrapure water. The ethanol solution of the extract is then prepared into a 200 mg / mL solution using anhydrous ethanol. This solution is gradually added dropwise to the n-butanol purified fraction at a rate of 5 mL / min. After the addition is complete, stirring is continued and the mixture is sealed and stirred for 3 hours. Subsequently, the mixture is sonicated under ice bath conditions for 15 minutes (200 W, 40 kHz). The mixture is then filtered to obtain a filtrate. 20 mL of the volatile oil extracted in step S3 is added to the filtrate to ensure complete dissolution. The filtrate is then diluted with water to 1000 mL, mixed thoroughly, and filtered to obtain a self-assembled nanomicelle drug.

[0027] More specifically: S1: Take 678g of black nightshade, 678g of white nightshade, 678g of wild strawberry, and 339g of Scutellaria barbata, grind them into coarse powder, place them in an electromagnetic pyrolysis device, add 13 times the amount of 60% ethanol each time, extract 3 times, 10 minutes each time, filter, combine the filtrates, concentrate under reduced pressure at 70℃ to a relative density of about 1.15 (60℃) to obtain a concentrated solution; extract the concentrated solution 3 times with 3 times the mass of the concentrated solution of n-butanol, combine the extracts, dry under reduced pressure at 70℃ to obtain the n-butanol fraction; prepare a solvent system of n-hexane-ethyl acetate-ethanol-water in a volume ratio of 3:7:1:9, mix thoroughly, let stand in a separatory funnel to separate the phases, collect the upper and lower phases separately after the two phases are in equilibrium, place them in multiple brown reagent bottles, and degas by sonication for 20 min. n, for later use; pump the stationary phase, i.e., the upper phase (one of the brown bottles as a spare upper phase), into the separation tube of the high-speed countercurrent chromatography (HSCCC). After the upper phase fills the separation tube, turn on the main unit and set the rotation speed to 900 r / min. After the rotation speed stabilizes, pump the mobile phase, i.e., the lower phase (one of the brown bottles as a spare lower phase), into the separation tube at a volumetric flow rate of 1.8 mL / min until the two phases are in equilibrium; take the n-butanol fraction and dissolve it with an equal volume of the upper and lower phase mixture (the upper and lower phases prepared for ultrasonic degassing after 20 min, and mix them in equal volumes) by ultrasonication, and inject it through the injection port; set the wavelength of the ultraviolet detector to 320 nm, collect the eluent using an automatic collector, concentrate and dry the eluent under reduced pressure at 70 °C to obtain the purified n-butanol fraction.

[0028] S2: Take 678g of Astragalus membranaceus and 226g of Salvia miltiorrhiza, grind them into coarse powder, add 11 times the total mass of Astragalus membranaceus and Salvia miltiorrhiza in 30% ethanol, extract three times, filter to obtain filtrate, combine the filtrates obtained in this step, concentrate the filtrate to a relative density of 1.23 (60℃), add ethanol while stirring after concentration to make the alcohol content of the final concentrate reach 80%, then let stand at 4℃ for 24h, filter, take the filtrate, let stand at 4℃ for 24h, wash the residue obtained by filtration with 80% ethanol to obtain the washing liquid, combine the filtrate obtained by filtration after standing at 4℃ for 24h with the washing liquid, concentrate under reduced pressure to recover ethanol until there is no alcohol odor, dry the concentrate obtained after reduced pressure concentration to obtain the extract; the reduced pressure concentration conditions are: temperature: 70℃, pressure: -0.09MPa.

[0029] S3: Take 226g of Angelica sinensis and 226g of Curcuma longa, grind them into fine powder, add water with a total weight of 20 times that of Angelica sinensis and Curcuma longa and soak for 3 hours. Use a volatile oil extraction device (a glass instrument commonly used in this example for extracting volatile oil from Chinese herbal medicine, which can be used to collect volatile oil) to heat to a gentle boil for 6 hours and collect the volatile oil.

[0030] Example 3: Preparation method of self-assembled nanomicelle drugs Take 100g of the extract from step S2 and prepare a 200mg / mL solution with anhydrous ethanol. Dissolve 60g of the n-butanol-purified fraction in 300mL of ultrapure water. Add the 200mg / mL solution of the extract from step S2 to the n-butanol-purified fraction at a rate of 5mL / min. Seal and stir for 3 hours. Then, sonicate under ice bath conditions for 15 minutes (200W, 40kHz). Filter the solution, add 20mL of volatile oil to the filtrate to ensure complete dissolution, dilute with water to 1000mL, mix thoroughly, and filter sequentially through 0.45μm and 0.22μm microporous membranes. Collect the filtrate to obtain the self-assembled nanomicelle drug.

[0031] Example 4: Determination of self-assembled nanomicelle drugs 1. Physicochemical Properties of Component Nanomicelles: The resulting nanomicelle solution is a transparent, pale red solution with good static stability. 10 μL of sample solution was placed on a carbon film copper grid. After standing for 1 min, excess liquid was removed from the grid edge. One drop of 3% phosphotungstic acid aqueous solution was added to the grid surface. After negative staining for 2 min, excess dye was absorbed with filter paper. After the liquid evaporated, the morphology was photographed using a transmission electron microscope, showing that the nanomicelles exhibited a spherical shape. 1 mL of the nanosolution was measured, and the particle size distribution was determined using a Litesizer 500 nanoparticle size analyzer, as shown in Table 1. The results showed that the average particle size was approximately 254 nm, and the polydispersity index (PDI) was less than 0.25, indicating a relatively uniform particle size distribution and good dispersibility. See [link to table]. Figures 1-2 The spectral characteristics of self-assembled nanomicelles are shown in the figure. Figure 3 .

[0032] Table 1 Measurement Results .

[0033] 2. Spectral characteristics of component self-assembled nanomicelles Ultraviolet spectroscopy: After molecular interactions occur, the electronic configuration of conjugated groups is affected. The interaction patterns can be inferred from changes in the ultraviolet-visible spectra. Self-assembled nanomicelles were scanned in the 200–500 nm range using ultraviolet-visible absorption spectroscopy, and the spectra were compared with those of three free components. The results showed that the characteristic absorption peak of tanshinone IIA was at 270 nm, the characteristic absorption peaks of chlorogenic acid were at 327 and 360 nm, and the characteristic absorption peak of baicalin was at 335 nm. The self-assembled nanomicelles showed strong absorption peaks at 273 nm, 330 nm, and 362 nm, which were similar to those of free tanshinone IIA, chlorogenic acid, and baicalin. However, the absorption peaks of the self-assembled nanomicelles showed a slight blue shift from 270 to 273 nm for free tanshinone IIA, a slight blue shift from 327 to 330 nm and 360 to 362 nm for free chlorogenic acid, and a slight blue shift from 335 to 330 nm for free baicalin. This indicates that tanshinone IIA, chlorogenic acid, and baicalin have non-covalent interactions in the self-assembled nanomicelles.

[0034] Example 5: Component Detection of Two Preparation Methods (1) Detection of astragaloside III and astragaloside A Chromatographic conditions The column was filled with octadecylsilane-bonded silica gel (250 mm column length, 4.6 mm inner diameter, 5 μm particle size); acetonitrile was used as mobile phase A and 0.5% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the specifications in the table below; the column temperature was 40°C; and the column was detected by an evaporation photodetector.

[0035] .

[0036] Preparation of reference solutions: Accurately weigh appropriate amounts of astragaloside III and astragaloside A reference standards, add methanol to prepare a mixed reference solution containing 80 μg of each per mL.

[0037] Preparation of the test solution: Take about 0.5g of the preparations from Example 1 and Example 2, add 50mL of 60% methanol, extract by ultrasonication for 30 minutes, shake well, filter, and take the filtrate to obtain the test solution.

[0038] Determination method Accurately pipette 5 μL of the reference solution and 10 μL of the test solution into the liquid chromatograph and determine the result.

[0039] Table 1 Measurement Results .

[0040] The results showed that the contents of astragaloside III and astragaloside A in the self-assembled nanomicelle drug of the present invention were higher than those in the traditional formulation, thus the self-assembled nanomicelle drug of the present invention is superior.

[0041] (2)Component detection Chromatographic conditions: Octadecylsilyl silica gel was used as the filler (column length: 250 mm, inner diameter: 4.6 mm, particle size: 5 μm); acetonitrile was used as mobile phase A, and 0.02% formic acid solution was used as mobile phase B, and gradient elution was carried out according to the regulations in the following table; the column temperature was 35 °C; the detection wavelength was 280 nm.

[0042] .

[0043] Preparation of reference substance solution: Appropriate amounts of calycosin-7-O-β-D-glucoside, formononetin, tanshinone IIA, salvianolic acid B, cryptotanshinone, ferulic acid, senkyunolide I, senkyunolide A, curzerenone, curdione, resveratrol, diosgenin, chlorogenic acid, caffeic acid, scutellarin, ellagic acid, apigenin-6-C-β-D-glucoside, brevifolincarboxylic acid, ethyl caffeate reference substances were accurately weighed and dissolved in methanol to prepare a mixed reference solution containing 150 μg of each in 1 mL, and thus obtained.

[0044] Preparation of test solution: About 0.5 g of the preparations of Example 1 and Example 2 were taken, added with 50 mL of 60% methanol, ultrasonically extracted for 30 minutes, shaken well, filtered, and the continued filtrate was taken, and thus obtained. <00001​​​​​​​​​​​​​​​​​​​​​​​Modeling: Fifty rats were randomly divided into five groups: a blank control group, a model group, and an experimental group (traditional formulation group: obtained in Example 1; self-assembled nanomicelle drug group of this invention: obtained in Example 2; positive control group: Lecojun group: 20 mg / kg), with 10 rats in each group (n=10). The model group and experimental group underwent radiation modeling using a RAD SOURCE RS2000 X-ray biological irradiation system (ProBiological System), with a single whole-body irradiation session. The irradiation dose was 3.5 Gy, and the dose rate was 96.51 cGy / min. The filter was 0.3 mm copper, and the irradiation conditions were: energy 8 MeV, source-axis distance (SAD) 100 cm.

[0050] Dosage: During the experiment, the conventional formulation group and the self-assembled drug group of the present invention were each administered 10 mg / kg via intragastric gavage. −1 The mice were administered the drug once daily for 2 days before irradiation (the day of irradiation was recorded as day 0) and for 7 days after irradiation, for a total of 10 consecutive days. The model group and the control group were given an equal volume of solvent (0.5% sodium carboxymethyl cellulose) via intragastric gavage before and after irradiation. Two days after administration, except for the control group, all mice underwent a single whole-body irradiation with a 2.5 Gy X-ray dose. Peripheral blood leukocyte counts were measured in each group before irradiation and on days 3, 6, and 10 after irradiation.

[0051] 1.3 Peripheral blood leukocyte count Blood samples of 40 μL were collected from the distal end of each animal before irradiation and at the 3rd, 6th, and 10th tails after irradiation. The blood samples were added to 160 μL of diluent and white blood cell counts were performed using a blood analyzer.

[0052] 1.4 Measurement of serum immune cytokines On day 10, blood was collected from the tail tip of the mice, and blood was collected by enucleation of the eyeballs. The blood was centrifuged at 3000 r / min for 10 min, and the serum was separated. The levels of IL-3, IL-4 and IFN-γ in the mouse serum were measured using an ELISA kit.

[0053] 1.5 organ index The spleen, liver, and thymus of each group of mice were removed, and the fatty tissue adhering to the surface of the organs was carefully removed while keeping the organs as intact as possible. The liquid on the surface of the organs was then absorbed with absorbent paper, and the weight was measured on an analytical balance. The organ indices were calculated separately.

[0054] Organ Index = Organ Mass / Body Mass 2. Results 2.1 Effects on the number of peripheral blood leukocytes in radiation-damaged mice Peripheral blood leukocytes originate from bone marrow hematopoietic stem cells, and their increase or decrease can indirectly reflect the functional status of bone marrow hematopoietic tissue. Therefore, the number of leukocytes in peripheral blood is a biomarker for assessing radiation damage. As shown in the table, 3 days after irradiation, compared with the control group, the peripheral blood leukocyte levels in the model group, traditional preparation group, self-assembled nanomicelle drug group, and positive control group were significantly decreased (p < 0.05). 6–10 days after irradiation, compared with the model group, the peripheral blood leukocyte levels in the traditional preparation group, self-assembled nanomicelle drug group, and positive control group were significantly increased (p < 0.05). Compared with the traditional preparation group, the peripheral blood leukocyte level in the self-assembled nanomicelle drug group was significantly increased (p < 0.05). Compared with the positive control group, the peripheral blood leukocyte level in the self-assembled nanomicelle drug group was significantly increased (p < 0.05). The effect of radiation damage on the number of peripheral blood leukocytes in mice is shown in the figure below. Figure 4 .

[0055] Table 3 Effects on peripheral blood leukocyte count in radiation-injured mice (n=10) .

[0056] Note: * Compared with the control group, p < 0.05; # =P < 0.05 compared with the model group; ^P < 0.05 compared with the traditional preparation group; -P < 0.05 compared with the positive control group. 2.2 Results of serum immune cytokine assay IFN-γ is a cytokine secreted by activated T lymphocytes and plays an important regulatory role in the immune response. Compared with the control group, the levels of IFN-γ, IL-4, and IL-3 in the model group were significantly decreased (P < 0.05); compared with the model group, the levels of IFN-γ, IL-4, and IL-3 in the traditional formulation group, the self-assembled nanomicelle drug group, and the positive control group were significantly increased (P < 0.05); compared with the traditional formulation group, the levels of IFN-γ, IL-4, and IL-3 in the self-assembled nanomicelle drug group were significantly increased (P < 0.05); compared with the positive control group, the levels of IFN-γ, IL-4, and IL-3 in the self-assembled nanomicelle drug group were significantly increased (P < 0.05). The effect of radiation-induced damage on serum immune cytokines in mice is shown in the figure. Figure 5 .

[0057] Table 4. Effects of radiation-damaged mice on serum immune cytokines (n=10) .

[0058] Note: * Compared with the control group, p < 0.05; #=P < 0.05 compared with the model group; ^P < 0.05 compared with the traditional preparation group; -P < 0.05 compared with the positive control group. 2.3 Organ Index The liver, spleen, and thymus are crucial immune organs in the human body and also the most common sites of extramedullary hematopoiesis. Organ indices can reflect the body's immune and extramedullary hematopoietic status to some extent. However, as shown in the table below, the situation has improved somewhat, and no significant changes have been observed. (See the diagram showing the impact of organ indices.) Figure 6 .

[0059] Table 5. Effects on organ indices (n=10) .

Claims

1. A self-assembled nanomicelle drug for leukopenia, characterized in that, The drug contains astragaloside III, astragaloside A, verbascoside, gentianoside, tanshinone IIA, salvianolic acid B, cryptotanshinone, ferulic acid, ligustrol I, ligustrol A, curcuminol, curcumindione, resveratrol, diosgenin, chlorogenic acid, caffeic acid, baicalin, ellagic acid, apigenin-6-C-β-D-glucoside, bromelain, and ethyl caffeate; the self-assembling components include tanshinone IIA-chlorogenic acid-baicalin.

2. The method for preparing self-assembled nanomicelle drugs according to claim 1, characterized in that, Includes the following steps: S1: Take black nightshade, white nightshade, wild strawberry, and scutellaria barbata, crush them into coarse powder, place them in an electromagnetic pyrolysis device, and then extract them multiple times with ethanol. Filter to obtain the extract filtrate. Combine the extract filtrates obtained from each filtration and concentrate the combined extract filtrate under reduced pressure at 70℃ to a relative density of 1.12-1.18 at 60℃ to obtain a concentrated solution. Extract the concentrated solution 3-5 times with n-butanol at a volume of 3-6 times the volume of the concentrated solution. Combine the extracts and dry the combined extract under reduced pressure at 70℃ to obtain the n-butanol fraction. Separate and purify the n-butanol fraction using high-speed countercurrent chromatography to obtain the purified n-butanol fraction. S2: Take Astragalus membranaceus and Salvia miltiorrhiza, crush them into coarse powder, add 30% ethanol to extract multiple times, filter, and obtain filtrate. Combine the filtrates obtained after each filtration, and concentrate the combined filtrate to a relative density of 1.20-1.26 at 60℃. After concentration, add ethanol while stirring to make the alcohol content of the final concentrate reach 80%. Then let it stand and filter, take the filtrate, wash the residue after standing and filtering with 80% ethanol to obtain the washing liquid, combine the filtrate after standing and filtering and the washing liquid, concentrate the filtrate and washing liquid under reduced pressure to recover ethanol, and obtain the concentrate after concentration. Dry the concentrate to obtain the extract. S3: Take Angelica sinensis and Curcuma longa, grind them into fine powder, soak them in water, use a volatile oil extraction device, heat to a gentle boil, and collect the volatile oil; S4: Take the extract from step S2 and prepare a solution of 180-220 mg / mL with anhydrous ethanol; take the n-butanol purified fraction from step S1, the amount of which is 0.5-0.7 times the weight of the extract from step S2, and dissolve it in ultrapure water to obtain an aqueous solution of the n-butanol purified fraction; take the extract from step S2, i.e., the solution prepared with anhydrous ethanol at 180-220 mg / mL, and add it dropwise to the aqueous solution of the n-butanol purified fraction, seal and stir, and then sonicate under ice bath conditions. After sonication, filter to obtain the filtrate, add volatile oil to the filtrate, and after the volatile oil is fully dissolved, dilute with water, mix evenly, and then filter to obtain the self-assembled nanomicelle drug.

3. The method for preparing self-assembled nanomicelle drugs according to claim 2, characterized in that, The high-speed countercurrent chromatography (HSCCC) separation and purification steps in step S1 are as follows: The solvent system (n-hexane-ethyl acetate-ethanol-water) is allowed to stand and separate into layers. After the two phases reach equilibrium, the upper and lower phases are collected separately and placed in multiple brown reagent bottles. The mixture is then degassed by sonication and set aside. Next, one bottle of stationary phase (upper phase) is pumped into the HSCCC separation tube. Once the tube is full, the main unit is turned on, and the rotation speed is set to 900 r / min. After the rotation speed stabilizes, one bottle of mobile phase (lower phase) is pumped into the separation tube at a volumetric flow rate of 1.8 mL / min until the two phases reach equilibrium. The n-butanol fraction is taken, and equal volumes of the degassed upper and lower phases are mixed to obtain a mixed solution. This solution is then dissolved by sonication and injected through the injection port. The UV detector wavelength is set to 320 nm, and the eluent is collected using an automatic collector. The eluent is then concentrated and dried under reduced pressure at 70 °C to obtain the purified n-butanol fraction.

4. The method for preparing self-assembled nanomicelle drugs according to claim 3, characterized in that, The solvent system is n-hexane-ethyl acetate-ethanol-water, with a volume ratio of n-hexane-ethyl acetate-ethanol-water of 3:7:1:

9.

5. The method for preparing self-assembled nanomicelle drugs according to claim 2, characterized in that, In step S1, there are 500-800 parts by weight of black nightshade, 500-800 parts by weight of white nightshade, 500-800 parts by weight of wild strawberry, and 300-400 parts by weight of scutellaria barbata; in step S2, there are 600-700 parts by weight of astragalus and 200-300 parts by weight of salvia miltiorrhiza; in step S3, there are 200-300 parts by weight of angelica sinensis and 200-300 parts by weight of turmeric.

6. The method for preparing self-assembled nanomicelle drugs according to claim 5, characterized in that, In step S1, take 678g of Solanum nigrum, 678g of Solanum lyratum, 678g of Duchesnea indica, and 339g of Scutellaria barbata; in step S2, take 678g of Astragalus membranaceus and 226g of Salvia miltiorrhiza; in step S3, take 226g of Angelica sinensis and 226g of Curcuma longa.

7. The method for preparing self-assembled nanomicelle drugs according to claim 2, characterized in that, In step S1, add 13-15 times the total mass of black nightshade, white nightshade, wild strawberry, and scutellaria barbata with 60-70% ethanol each time, and extract 3 times, 10-30 minutes each time; in step S2, add 11 times the total mass of astragalus and salvia miltiorrhiza with 30% ethanol, and extract 3 times; in step S3, add 20 times the total mass of angelica and turmeric with water and soak for 3 hours, then heat to a gentle boil for 6 hours using a volatile oil extraction device.

8. The method for preparing self-assembled nanomicelle drugs according to claim 2, characterized in that, In step S4, the ultrasonic treatment conditions are: ultrasonic treatment for 15 min, ultrasonic conditions are 200 W and 40 kHz; the drop rate of the extract in step S2 is 5 mL / min.

9. The method for preparing self-assembled nanomicelle drugs according to claim 2, characterized in that, In step S1, the combined extract filtrate is concentrated under reduced pressure at 70°C to a relative density of 1.15 at 60°C. In step S2, the combined filtrate is concentrated to a relative density of 1.23 at 60°C.

10. The application of the self-assembled nanomicelle drug according to claim 1 in the treatment of leukopenia, wherein the leukopenia is leukopenia caused by radiotherapy or chemotherapy.

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