Pharmaceutical composition for preventing and treating intracranial aneurysms based on inhibiting ROS and preparation method thereof

Abstract

The invention discloses a pharmaceutical composition for preventing and treating intracranial aneurysms based on inhibiting ROS and a preparation method in the technical field of preventing and treating intracranial aneurysms. The pharmaceutical composition for preventing and treating intracranial aneurysms based on inhibiting ROS comprises the following components in parts by weight: 54-66 parts of Spirulina platensis powder, 120-150 parts of Hydrangea sphaerocarpa powder, 4.2-5.1 parts of 3,3-diselenodipronic acid and 7-9 parts of phellodendron. The invention proposes to prepare a ROS-responsive polysaccharide drug-loaded micelle with a diselenide bond, and to improve the drug response rate by the response of Se-Se to ROS, extract polysaccharide as a hydrophilic unit, and prepare ROS-responsive drug-adjuvant integrated nano-micelles as a hydrophobic unit, and coat the drug, thereby achieving the technical effect of reducing the use of ineffective components and reducing the toxicity of high-concentration drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of preventing and treating intracranial aneurysms, and specifically refers to a pharmaceutical composition for preventing and treating intracranial aneurysms based on inhibiting ROS and a preparation method thereof. Background Art

[0002] Intracranial aneurysm refers to an abnormal bulge formed by the gradual expansion of the intracranial artery due to local damage to the vascular wall caused by congenital abnormalities or acquired injuries, under the influence of hemodynamic load and other factors; intracranial aneurysm rupture generally has no precursors, has high disability and mortality rates, and has a poor prognosis; about 3% of the world's population suffers from intracranial aneurysms, and subarachnoid hemorrhage caused by ruptured intracranial aneurysms accounts for about 75% of the total cases; ROS is a series of oxygen-containing substances produced during cell metabolism, including superoxide anions, hydroxyl free radicals, and hydrogen peroxide. Under normal physiological conditions, cells maintain ROS balance through the antioxidant system. During oxidative stress, ROS destroys biological macromolecules and causes cell damage. Injury and then carcinogenic effect; based on inhibiting ROS to prevent and treat tumors is a new idea for anti-cancer. Compared with the microenvironment of normal tissues and cells, the tumor microenvironment has significant differences, such as glutathione-related metabolic enzymes and reactive oxygen species are highly expressed in different subcellular structures; selenium, as a homologous element of sulfur, is an important component of several enzymes such as glutathione peroxidase, plays an important role in protecting cells from oxidative damage, has certain redox reactivity and anti-tumor activity, and compared with S, Se has a larger atomic radius and weaker electronegativity. The bond energy of C-Se is lower than CS and is more sensitive to ROS. At the same time, nano-drugs can enter the capillaries and enter the lesions through the blood circulation to achieve targeted drug delivery.

[0003] The existing technologies currently have the following main problems: low drug response rate and toxicity of high-concentration drugs. Summary of the invention

[0004] In view of the above situation, in order to overcome the defects of the prior art, the present invention provides a pharmaceutical composition for preventing and treating intracranial aneurysms based on inhibiting ROS and a preparation method. In order to solve the problems of low drug response rate and high-concentration drug toxicity, the present invention proposes to prepare ROS-responsive polysaccharide drug-loaded micelles with diselenide bonds, and achieve the improvement of drug response rate through the response of Se-Se to ROS. ROS-responsive drug-excipient integrated nanomicelles are prepared with polysaccharides as hydrophilic units and polypeptides as hydrophobic units, and the drugs are coated, thereby achieving the technical effect of reducing the use of ineffective ingredients and reducing the toxicity of high-concentration drugs.

[0005] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present invention is as follows: The present invention proposes a pharmaceutical composition for preventing and treating intracranial aneurysms based on inhibiting ROS, and the pharmaceutical composition for preventing and treating intracranial aneurysms based on inhibiting ROS comprises the following components in parts by weight: 54-66 parts of Spirulina platensis powder, 120-150 parts of Hydrangea macrophylla powder, 4.2-5.1 parts of 3,3-diselenodipionic acid and 7-9 parts of phellodendron chinense.

[0006] Preferably, the method for preparing the pharmaceutical composition for preventing and treating intracranial aneurysms based on inhibiting ROS specifically comprises the following steps:

[0007] S1. Add Spirulina platensis powder to 120 mL ultrapure water, dissolve under magnetic stirring at 60 rpm in a 37°C water bath for 30 min, then freeze at -20°C for 150-200 min, thaw in a 30°C water bath, freeze and thaw repeatedly for 3 times, homogenize for 2 min, then sonicate for 6-8 s at an interval of 15-20 s at an ultrasonic power of 300 W in an ice bath, for a total of 150-180 times to obtain a protein mixture;

[0008] S2, centrifuge the mixed solution obtained in S1, take the supernatant, freeze-dry, and obtain spirulina protein powder, prepare the spirulina protein powder into a protein solution with a mass fraction of 2%, add trypsin, the enzyme addition amount is 2.8-3.5% of the mass fraction of the spirulina protein powder, adjust the pH to 8, bathe in 42°C water for 8-10h, inactivate in 100°C oil bath for 10min, centrifuge at 6000rpm for 15min, take the supernatant, and freeze-dry to obtain spirulina peptide;

[0009] S3, adding 3,3-diselenodipronic acid to 15 mL of dimethyl sulfoxide, then adding N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide under nitrogen protection, stirring at room temperature for 60 min to obtain a mixed solution, adding the spirulina peptide obtained in S2 to the mixed solution, stirring at room temperature for 24 h, filtering, dialyzing with deionized water for 3 d, and freeze-drying to obtain the modified spirulina peptide;

[0010] S4, adding hydrangea powder to 200 mL of composite enzyme solution, wherein the mass ratio of cellulase to papain is 1:0.5-0.6, enzymolysis at 37-42°C for 50-60 min, and ultrasonication at 80W for 20 min to obtain a polysaccharide mixture;

[0011] S5, adding 3 times the volume of anhydrous ethanol solution to the polysaccharide mixture obtained in S4, centrifuging, filtering, washing 3 times with anhydrous ethanol, freeze-drying to obtain crude sugar, preparing the crude sugar into a crude sugar solution with a mass fraction of 5%, and removing the protein by Sevage method to obtain hydrangea polysaccharide;

[0012] S6, adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, 0.01-0.02 g of p-dimethylaminopyridine, the hydrangea polysaccharide obtained in S5, and the modified spirulina peptide obtained in S2 to 50 mL of dimethyl sulfoxide, stirring at room temperature for 48 h, dialyzing with deionized water for 3 d to remove impurities, and freeze-drying to obtain ROS-responsive drug-excipient integrated nanomicelles;

[0013] S7. Add the ROS-responsive drug-excipient integrated nanomicelles obtained in S6 to 1000 mL of phosphate buffered saline solution with a pH of 7.4, add phellodendron hydroxide and triethylamine, perform ultrasound at 100W for 30 min in a 30°C water bath, dialyze with deionized water until phellodendron hydroxide is undetectable, and freeze-dry to obtain a pharmaceutical composition for preventing and treating intracranial aneurysms based on inhibiting ROS.

[0014] Preferably, the ROS response principle of the pharmaceutical composition for preventing and treating intracranial aneurysms based on inhibiting ROS is:

[0015] ;

[0016] Preferably, in S1, the amount of the Spirulina platensis powder added to the ultrapure water is 0.45-0.55 g / mL;

[0017] Preferably, in S2, the mixed solution is centrifuged at a speed of 8000-10000 rpm for 25-35 min;

[0018] Preferably, in S3, the amount of 3,3-diselenodiprolic acid added to dimethyl sulfoxide is 0.028-0.034 g / mL; the amount of N,N'-dicyclohexylcarbodiimide added to dimethyl sulfoxide is 0.018-0.022 g / mL; the amount of N-hydroxysuccinimide added to dimethyl sulfoxide is 0.01-0.013 g / mL;

[0019] Preferably, in S4, the amount of the hydrangea powder added to the complex enzyme solution with a concentration of 2-2.5% is 0.06-0.075 g / mL;

[0020] Preferably, in S5, the centrifugal speed is 3000-3500 rpm, and the time is 5-10 min;

[0021] Preferably, in S6, the amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride added to dimethyl sulfoxide is 9-11 mg / mL; the amount of p-dimethylaminopyridine added to dimethyl sulfoxide is 0.2-0.4 mg / mL;

[0022] Preferably, in S7, the amount of phellodendronine hydrochloride added in a phosphate buffered saline solution with a pH of 7.4 is 0.7-0.9 mg / mL; the amount of triethylamine added in a phosphate buffered saline solution with a pH of 7.4 is 0.8-0.85 mg / mL.

[0023] The beneficial effects achieved by the present invention are as follows: extracting platen Spirulina protein and enzymolyzing it to prepare polypeptides, modifying the polypeptide amino group with 3,3-diselenodipionic acid to obtain modified Spirulina polypeptides, extracting hydrangea polysaccharide and linking it to the modified Spirulina polypeptides to obtain amphiphilic macromolecules linked by diselenide bonds, and then loading phellodendron alkaloids in a self-assembly manner to obtain ROS-responsive drug-loaded micelles; Spirulina contains a large amount of protein, and the enzymolyzed Spirulina peptides have the function of inhibiting tumors, hydrangea polysaccharides have good scavenging ability for hydroxyl radicals and superoxide anions, and phellodendron alkaloids have good scavenging ability for ABTS+, DPPH free radicals, O2 - Taking advantage of the effect of ROS, spirulina peptide and hydrangea polysaccharide were linked with 3,3-diselenodipionic acid to load phellodendron pine to obtain drug-loaded micelles, so that the water-insoluble drugs were encapsulated inside the micelles, increasing the solubility of the drugs, reducing the premature release of the drugs, protecting the drugs and prolonging the blood circulation time; the ROS environment can convert the hydrophobic diselenide into hydrophilic selenic acid, thereby allowing phellodendron pine to be quickly released from the micelles and improving the targeting efficiency. The polypeptides and polysaccharides used to construct the drug delivery system have the effect of inhibiting ROS in preventing and treating intracranial aneurysms, and realize the integration of drug and excipient, reduce the use of ineffective ingredients, and improve the prevention and treatment effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The results of the cytotoxicity tests of Examples 1-3 and Comparative Example 1 are shown;

[0025] Figure 2 The results of the ROS response drug release test in Example 1-3 are shown in FIG.

[0026] Figure 3 The results of the intracranial aneurysm formation test of Examples 1-3, Comparative Examples 1-2 and the saline group are shown.

[0027] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0029] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only and are not intended to limit the content of this application.

[0030] The experimental methods in the following examples are conventional methods unless otherwise specified; the experimental materials and test strains used in the following examples are purchased from commercial channels unless otherwise specified.

[0031] Example 1

[0032] The pharmaceutical composition for preventing and treating intracranial aneurysms based on inhibiting ROS includes the following components in parts by weight: 60 parts of Spirulina platensis powder, 130 parts of Hydrangea macrophylla powder, 4.5 parts of 3,3-diselenodipionic acid and 8 parts of phellodendron chinense.

[0033] The preparation method of the pharmaceutical composition for preventing and treating intracranial aneurysms based on inhibiting ROS specifically comprises the following steps:

[0034] S1. Add 6 g of Spirulina platensis powder to 120 mL of ultrapure water, dissolve in a 37°C water bath with 60 rpm magnetic stirring for 30 min, then freeze at -20°C for 170 min, thaw in a 30°C water bath, freeze and thaw repeatedly for 3 times, homogenize for 2 min, then ultrasonicate for 7 s at an interval of 17 s under an ice bath condition at a power of 300 W, for a total of 165 times to obtain a protein mixture;

[0035] S2, centrifuging the mixed solution obtained in S2 at 8000-10000 rpm for 30 min, taking the supernatant, freeze-drying to obtain spirulina protein powder, preparing the spirulina protein powder into a protein solution with a mass fraction of 2%, adding trypsin, the enzyme addition amount is 3% of the mass fraction of the spirulina protein powder, adjusting the pH to 8, bathing in a 42°C water bath for 9 h, inactivating in a 100°C oil bath for 10 min, centrifuging at 6000 rpm for 15 min, taking the supernatant, and freeze-drying to obtain spirulina peptide;

[0036] S3, adding 0.45g 3,3-diselenodipronic acid to 15mL dimethyl sulfoxide, then adding 0.3g N,N'-dicyclohexylcarbodiimide and 0.17g N-hydroxysuccinimide under nitrogen protection, stirring at room temperature for 60min to obtain a mixed solution, adding the spirulina peptide obtained in S2 to the mixed solution, stirring at room temperature for 24h, filtering, dialyzing with deionized water for 3d, and freeze-drying to obtain the modified spirulina peptide;

[0037] S4, adding 13g of hydrangea powder to 200mL of a 2.3% complex enzyme solution, wherein the mass ratio of cellulase to papain is 1:0.55, performing enzymolysis at 40°C for 55min, and performing ultrasound at 80w for 20min to obtain a polysaccharide mixture;

[0038] S5, adding 3 times the volume of anhydrous ethanol solution to the polysaccharide mixture obtained in S4, centrifuging at 3200 rpm for 8 min, filtering, washing with anhydrous ethanol 3 times, freeze-drying to obtain crude sugar, preparing the crude sugar into a crude sugar solution with a mass fraction of 5%, and removing the protein by Sevage method to obtain hydrangea polysaccharide;

[0039] S6, add 0.4g 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, 0.015g p-dimethylaminopyridine, the modified spirulina peptide obtained in S2, and the hydrangea polysaccharide obtained in S5 into 50mL of dimethyl sulfoxide, stir at room temperature for 48h, dialyze with deionized water for 3d to remove impurities, and freeze-dry to obtain ROS-responsive drug-excipient integrated nanomicelles;

[0040] S7. Add the ROS-responsive drug-excipient integrated nanomicelles obtained in S6 to 1000 mL of phosphate buffered saline solution with a pH of 7.4, add 0.8 g of phellodendron hydrochloride and 0.83 g of triethylamine, perform ultrasound at 100 W for 30 min in a 30°C water bath, dialyze with deionized water until the phellodendron hydrochloride is undetectable, and freeze-dry to obtain a pharmaceutical composition for preventing and treating intracranial aneurysms based on inhibiting ROS.

[0041] Example 2

[0042] The pharmaceutical composition for preventing and treating intracranial aneurysms based on inhibiting ROS includes the following components in parts by weight: 54 parts of Spirulina platensis powder, 120 parts of Hydrangea macrophylla powder, 4.2 parts of 3,3-diselenodipionic acid and 7 parts of phellodendron chinense.

[0043] Preferably, the method for preparing the pharmaceutical composition for preventing and treating intracranial aneurysms based on inhibiting ROS specifically comprises the following steps:

[0044] S1. Add 5.4 g of Spirulina platensis powder into 120 mL of ultrapure water, dissolve in a 37°C water bath with magnetic stirring at 60 rpm for 30 min, then freeze at -20°C for 150 min, thaw in a 30°C water bath, freeze and thaw three times, homogenize for 2 min, then sonicate for 6 s at 300 W of ultrasonic power in an ice bath with an interval of 15 s for a total of 150 times to obtain a protein mixture;

[0045] S2, centrifuging the mixed solution obtained in S2 at 8000rpm for 25min, taking the supernatant, freeze-drying to obtain spirulina protein powder, preparing the spirulina protein powder into a protein solution with a mass fraction of 2%, adding trypsin, the enzyme addition amount is 2.8% of the mass fraction of the spirulina protein powder, adjusting the pH to 8, bathing in 42℃ water for 8h, inactivating in 100℃ oil bath for 10min, centrifuging at 6000rpm for 15min, taking the supernatant, and freeze-drying to obtain spirulina peptide;

[0046] S3, adding 0.42 g of 3,3-diselenodipronic acid to 15 mL of dimethyl sulfoxide, then adding 0.27 g of N,N'-dicyclohexylcarbodiimide and 0.15 g of N-hydroxysuccinimide under nitrogen protection, stirring at room temperature for 60 min to obtain a mixed solution, adding the spirulina peptide obtained in S2 to the mixed solution, stirring at room temperature for 24 h, filtering, dialyzing with deionized water for 3 days, and freeze-drying to obtain the modified spirulina peptide;

[0047] S4, adding 12g of hydrangea powder into 200mL of a 2% compound enzyme solution, wherein the mass ratio of cellulase to papain is 1:0.5, performing enzymolysis at 37°C for 50min, and performing ultrasound at 80w for 20min to obtain a polysaccharide mixture;

[0048] S5, adding 3 times the volume of anhydrous ethanol solution to the polysaccharide mixture obtained in S4, centrifuging at 3000 rpm for 5 min, filtering, washing with anhydrous ethanol 3 times, freeze-drying to obtain crude sugar, preparing the crude sugar into a crude sugar solution with a mass fraction of 5%, and removing the protein by Sevage method to obtain hydrangea polysaccharide;

[0049] S6, add 0.45g 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, 0.01g p-dimethylaminopyridine, the modified spirulina peptide obtained in S2, and the hydrangea polysaccharide obtained in S5 into 50mL of dimethyl sulfoxide, stir at room temperature for 48h, dialyze with deionized water for 3d to remove impurities, and freeze-dry to obtain ROS-responsive drug-excipient integrated nanomicelles;

[0050] S7. Add the ROS-responsive drug-excipient integrated nanomicelles obtained in S6 to 1000 mL of phosphate buffered saline solution with a pH of 7.4, add 0.7 g of phellodendron hydrochloride and 0.8 g of triethylamine, perform ultrasound at 100W for 30 min in a 30°C water bath, dialyze with deionized water until the phellodendron hydrochloride is undetectable, and freeze-dry to obtain a pharmaceutical composition for preventing and treating intracranial aneurysms based on inhibiting ROS.

[0051] Example 3

[0052] The pharmaceutical composition for preventing and treating intracranial aneurysms based on inhibiting ROS includes the following components in parts by weight: 66 parts of Spirulina platensis powder, 150 parts of Hydrangea macrophylla powder, 5.1 parts of 3,3-diselenodipionic acid and 9 parts of phellodendron chinense.

[0053] Preferably, the method for preparing the pharmaceutical composition for preventing and treating intracranial aneurysms based on inhibiting ROS specifically comprises the following steps:

[0054] S1. Add 6.6 g of Spirulina platensis powder into 120 mL of ultrapure water, dissolve in a 37°C water bath with magnetic stirring at 60 rpm for 30 min, then freeze at -20°C for 200 min, thaw in a 30°C water bath, freeze and thaw three times, homogenize for 2 min, then sonicate for 8 s at an interval of 20 s at an ultrasonic power of 300 W in an ice bath for 180 times to obtain a protein mixture;

[0055] S2, centrifuging the mixed solution obtained in S2 at 10000rpm for 35min, taking the supernatant, freeze-drying to obtain spirulina protein powder, preparing the spirulina protein powder into a protein solution with a mass fraction of 2%, adding trypsin, the enzyme addition amount is 3.5% of the mass fraction of the spirulina protein powder, adjusting the pH to 8, bathing in 42℃ water for 10h, inactivating in 100℃ oil bath for 10min, centrifuging at 6000rpm for 15min, taking the supernatant, freeze-drying to obtain spirulina peptide;

[0056] S3, adding 0.51g 3,3-diselenodipronic acid to 15mL dimethyl sulfoxide, then adding 0.33g N,N'-dicyclohexylcarbodiimide and 0.195g N-hydroxysuccinimide under nitrogen protection, stirring at room temperature for 60min to obtain a mixed solution, adding the spirulina peptide obtained in S2 to the mixed solution, stirring at room temperature for 24h, filtering, dialyzing with deionized water for 3d, and freeze-drying to obtain the modified spirulina peptide;

[0057] S4, adding 15g of hydrangea powder into 200mL of a 2.5% compound enzyme solution, wherein the mass ratio of cellulase to papain is 1:0.6, enzymolysis at 42°C for 60min, and ultrasonication at 80w for 20min to obtain a polysaccharide mixture;

[0058] S5, adding 3 times the volume of anhydrous ethanol solution to the polysaccharide mixture obtained in S4, centrifuging at 3500 rpm for 10 min, filtering, washing with anhydrous ethanol 3 times, freeze-drying to obtain crude sugar, preparing the crude sugar into a crude sugar solution with a mass fraction of 5%, and removing the protein by Sevage method to obtain hydrangea polysaccharide;

[0059] S6, add 0.55g 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, 0.02g p-dimethylaminopyridine, the modified spirulina peptide obtained in S2, and the hydrangea polysaccharide obtained in S5 into 50mL of dimethyl sulfoxide, stir at room temperature for 48h, dialyze with deionized water for 3d to remove impurities, and freeze-dry to obtain ROS-responsive drug-excipient integrated nanomicelles;

[0060] S7. Add the ROS-responsive drug-excipient integrated nanomicelles obtained in S6 to 1000 mL of phosphate buffered saline solution with a pH of 7.4, add 0.9 g of phellodendron hydrochloride and 0.85 g of triethylamine, perform ultrasound at 100 W for 30 min in a 30°C water bath, dialyze with deionized water until the phellodendron hydrochloride is undetectable, and freeze-dry to obtain a pharmaceutical composition for preventing and treating intracranial aneurysms based on inhibiting ROS.

[0061] Comparative Example 1

[0062] This comparative example provides a composition, which is different from Example 1 only in that the composition does not contain 3,3-diselenodipronic acid, and the remaining components and component contents are the same as those in Example 1.

[0063] Comparative Example 2

[0064] This comparative example provides a composition, which is different from Example 1 only in that the components do not contain Spirulina platensis powder and Hydrangea sphaerocephala powder, and the remaining components and component contents are the same as those in Example 1.

[0065] Experimental example

[0066] 1. Cytotoxicity Test

[0067] The zebrafish embryos fertilized for 3 hours were randomly divided and transferred to a 24-well plate, with 50 eggs in each well. The volume of the culture medium was 2 mL. Examples 1-3 and Comparative Example 1 were added as the experimental group, and the concentration of phellodendronine reached 500 μg / mL. The same concentration of physiological saline was added as the blank group. Each group had 3 wells, and the embryos were treated and cultured in a constant temperature and humidity incubator at 28.5°C for 3 days. Then, the survival rate of the eggs on the third day was calculated using the following formula:

[0068] Survival rate = number of surviving eggs in the experimental group / number of surviving eggs in the blank group × 100%.

[0069] Figure 1The results of the cytotoxicity test of Examples 1-3 and Comparative Example 1 are shown in the figure. As shown in the figure, after 3 days, the survival rates of Examples 1-3 and Comparative Example 1 are 82%, 83%, 86% and 51%, respectively; the survival rate of Comparative Example 1 is significantly lower than that of Examples 1-3, indicating that the use of 3,3-diselenodipropionic acid effectively reduces the cytotoxicity of the drug, and the micelles formed by linking polypeptides and polysaccharides with 3,3-diselenodipropionic acid have a sustained release effect, thereby reducing the toxicity of high-concentration drugs to cells.

[0070] 2. ROS-responsive drug release test

[0071] 5 mg of Example 1-3 loaded with the drug was placed in 5 mL of 0.1% H2O2 and deionized water, and quickly transferred into a dialysis bag with a Mw of 3.5 kDa, and then the dialysis bag was immersed in 50 mL of deionized water, and oscillated at 100 rpm in a constant temperature oscillator at 37°C. After 24 hours, the release amount of phellodendrine in deionized water was measured by a spectrophotometer, and the release rate was calculated by the following formula:

[0072] Release rate = release amount / initial drug loading × 100%.

[0073] Figure 2 This is a result graph of the ROS response drug release test of Example 1-3. As shown in the figure, after 24 hours, the release rates of Example 1-3 in H2O2 solution and water are 70%, 68%, 69% and 26%, 29%, 28%, respectively; the release rate of Example 1-3 in water is significantly lower than the release rate in H2O2 solution, indicating that the prepared drug composition has good responsiveness to ROS.

[0074] 3. Intracranial aneurysm formation test

[0075] 16-week-old C57BL / 6 wild-type mice were selected, and intracranial aneurysm model mice were prepared by administration of angiotensin II combined with elastase. The mice were randomly divided into groups, with 50 mice in each group, and were treated with Example 1-3, Comparative Example 1-2, and intravenous injection of normal saline, respectively. If neurological signs such as hemiplegia, abnormal bending of the front and rear limbs, and a single-day weight loss of 2g or more were present, they were killed immediately, and the others were killed 3 weeks later. After sufficient blood perfusion in the cerebral vessels, 8% bromophenol blue gelatin solution was injected into the left ventricle. The formation of intracranial aneurysms near the Willis artery circle at the skull base of each group of mice was observed, and image acquisition and analysis were performed. The aneurysm formation rate was calculated using the following formula:

[0076] Aneurysm formation rate = number of mice with aneurysm formation / total number of mice × 100%.

[0077] Figure 3The results of the intracranial aneurysm formation test of Examples 1-3, Comparative Examples 1-2 and the saline group are shown in the figure. As shown in the figure, after 3 weeks, the aneurysm formation rates of Examples 1-3, Comparative Examples 1-2 and the saline group were 22%, 24%, 20%, 44%, 60% and 72%, respectively; the aneurysm formation rate of Examples 1-3 was significantly lower than that of Comparative Example 1, indicating that the use of 3,3-diselenodipionic acid effectively reduced the aneurysm formation rate, and the aneurysm formation rate of Examples 1-3 was significantly lower than that of Comparative Example 2, indicating that the use of Spirulina platensis powder and Hydrangea macrophylla powder effectively reduced the aneurysm formation rate.

[0078] While the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that many changes, modifications, substitutions and alterations may be made to the embodiments without departing from the principles and spirit of the invention.

[0079] The present invention and its implementation methods are described above, which is not restrictive. The drawings are only one of the implementation methods of the present invention, and the actual application is not limited thereto. In short, if ordinary technicians in the field are inspired by it and design methods and embodiments similar to the technical solution without creativity without departing from the purpose of the invention, they should all fall within the protection scope of the present invention.

Claims

1. A pharmaceutical composition for preventing and treating intracranial aneurysms based on inhibiting ROS, characterized in that: The pharmaceutical composition for preventing and treating intracranial aneurysms based on inhibiting ROS is prepared by the following method: The method for preparing the pharmaceutical composition for preventing and treating intracranial aneurysms based on inhibiting ROS specifically comprises the following steps: S1. Add 54-66 parts by weight of Spirulina platensis powder to 120 mL of ultrapure water, dissolve in a 37°C water bath with magnetic stirring at 60 rpm for 30 min, then freeze at -20°C for 150-200 min, thaw in a 30°C water bath, freeze and thaw repeatedly for 3 times, homogenize for 2 min, then ultrasonicate for 6-8 s at an interval of 15-20 s at an ultrasonic power of 300 W under ice bath conditions, for a total of 150-180 times to obtain a protein mixture; S2, centrifuge the mixed solution obtained in S1, take the supernatant, freeze-dry, and obtain spirulina protein powder, prepare the spirulina protein powder into a protein solution with a mass fraction of 2%, add trypsin, the enzyme addition amount is 2.8-3.5% of the mass fraction of the spirulina protein powder, adjust the pH to 8, bathe in 42°C water for 8-10h, inactivate in 100°C oil bath for 10min, centrifuge at 6000rpm for 15min, take the supernatant, and freeze-dry to obtain spirulina peptide; S3, adding 4.2-5.1 parts by weight of 3,3-diselenodipronic acid to 15 mL of dimethyl sulfoxide, then adding N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide under nitrogen protection, stirring at room temperature for 60 min to obtain a mixed solution, adding the spirulina peptide obtained in S2 to the mixed solution, stirring at room temperature for 24 h, filtering, dialyzing against deionized water for 3 d, and freeze-drying to obtain a modified spirulina peptide; S4, adding 120-150 parts by weight of hydrangea powder to 200 mL of a composite enzyme solution, wherein the mass ratio of cellulase to papain is 1:0.5-0.6, performing enzymolysis at 37-42° C. for 50-60 min, and performing ultrasound at 80 W for 20 min to obtain a polysaccharide mixture; S5, adding 3 times the volume of anhydrous ethanol solution to the polysaccharide mixture obtained in S4, centrifuging, filtering, washing 3 times with anhydrous ethanol, freeze-drying to obtain crude sugar, preparing the crude sugar into a crude sugar solution with a mass fraction of 5%, and removing the protein by Sevage method to obtain hydrangea polysaccharide; S6, adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, 0.01-0.02 g of p-dimethylaminopyridine, the hydrangea polysaccharide obtained in S5, and the modified spirulina peptide obtained in S2 to 50 mL of dimethyl sulfoxide, stirring at room temperature for 48 h, dialyzing with deionized water for 3 d to remove impurities, and freeze-drying to obtain ROS-responsive drug-excipient integrated nanomicelles; S7. Add the ROS-responsive drug-excipient integrated nanomicelles obtained in S6 to 1000 mL of phosphate buffered saline solution with a pH of 7.4, add 7-9 parts by weight of phellodendron hydrochloride and triethylamine, perform ultrasound at 100W for 30 minutes in a 30°C water bath, dialyze with deionized water until the phellodendron hydrochloride is undetectable, and freeze-dry to obtain a pharmaceutical composition for preventing and treating intracranial aneurysms based on inhibiting ROS.

2. The pharmaceutical composition for preventing and treating intracranial aneurysms based on inhibiting ROS according to claim 1, characterized in that: In S1, the amount of the Spirulina platensis powder added to the ultrapure water is 0.45-0.55 g / mL.

3. The pharmaceutical composition for preventing and treating intracranial aneurysms based on inhibiting ROS according to claim 2, characterized in that: In S2, the mixed solution is centrifuged at a speed of 8000-10000 rpm for 25-35 min.

4. The pharmaceutical composition for preventing and treating intracranial aneurysms based on inhibiting ROS according to claim 3, characterized in that: In S3, the amount of 3,3-diselenodiprolic acid added to dimethyl sulfoxide is 0.028-0.034 g / mL; the amount of N,N'-dicyclohexylcarbodiimide added to dimethyl sulfoxide is 0.018-0.022 g / mL; and the amount of N-hydroxysuccinimide added to dimethyl sulfoxide is 0.01-0.013 g / mL.

5. The pharmaceutical composition for preventing and treating intracranial aneurysms based on inhibiting ROS according to claim 4, characterized in that: In S4, the amount of the hydrangea powder added to the complex enzyme solution with a concentration of 2-2.5% is 0.06-0.075 g / mL.

6. The pharmaceutical composition for preventing and treating intracranial aneurysms based on inhibiting ROS according to claim 5, characterized in that: In S5, the centrifugal speed is 3000-3500 rpm, and the time is 5-10 min.

7. The pharmaceutical composition for preventing and treating intracranial aneurysms based on inhibiting ROS according to claim 6, characterized in that: In S6, the amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride added to dimethyl sulfoxide is 9-11 mg / mL; the amount of p-dimethylaminopyridine added to dimethyl sulfoxide is 0.2-0.4 mg / mL.

8. The pharmaceutical composition for preventing and treating intracranial aneurysms based on inhibiting ROS according to claim 7, characterized in that: In S7, the amount of phellodendronine hydrochloride added to the phosphate buffered saline solution with a pH of 7.4 is 0.7-0.9 mg / mL; the amount of triethylamine added to the phosphate buffered saline solution with a pH of 7.4 is 0.8-0.85 mg / mL.

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