Pharmaceutical composition containing brazilin as well as preparation method and application of pharmaceutical composition

Abstract

The invention relates to the technical field of medicine, in particular to a brazilin-containing pharmaceutical composition and a preparation method and application thereof, and preparation raw materials comprise brazilin and a zinc preparation. Through the synergistic effect of the brazilin and the zinc preparation, the fibrosis area after myocardial infarction can be remarkably reduced, the cardiac function after myocardial infarction can be improved, and the cardiac fibrosis degree can be effectively relieved by inhibiting the expression of fibrosis-related genes and proteins at transcription and protein levels.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a pharmaceutical composition containing brassinolide, its preparation method, and its application. Background Technology

[0002] Cardiac fibrosis is a common pathophysiological remodeling process that occurs in various cardiovascular diseases, significantly impacting cardiac structure and function and gradually driving the progression of heart failure. The main characteristic of cardiac fibrosis is the excessive accumulation of extracellular matrix, caused by the abnormal proliferation, differentiation, and migration of cardiac fibroblasts.

[0003] In recent years, the use of natural products to treat cardiac fibrosis has attracted attention. For example, Chinese invention patent application CN121129831A discloses the application of triscutellarin in cardiac fibrosis, which shows a potential anti-fibrotic effect by reducing the area of ​​cardiac fibrosis and downregulating the mRNA level and protein expression of fibrosis-related indicators.

[0004] However, there is currently little research on the application of natural products combined with metal ions in the preparation of drugs for the treatment of cardiac fibrosis. For example, although quercetin and selenium have fibrotic effects, the narrow therapeutic window of selenium limits their further research and application. Curcumin and zinc easily form insoluble complexes, resulting in oral bioavailability of less than 1%, which cannot effectively exert anti-fibrotic effects. Summary of the Invention

[0005] To address the problems in the prior art, the first aspect of the present invention provides a pharmaceutical composition containing brassinolide, wherein the raw materials for preparation include brassinolide and zinc preparations.

[0006] In some embodiments, the zinc preparation includes at least one of anhydrous zinc chloride and zinc gluconate.

[0007] In some embodiments, the mass ratio of the brassinolide to the zinc preparation is (10-15):(3-5). For example, 10:3, 10:4, 10:5, 12:3, 12:4, 12:5, etc., or any value within the range of (10-15):(3-5).

[0008] In some embodiments, the raw materials for preparing the pharmaceutical composition further include dimethyl sulfoxide and double-distilled water.

[0009] A second aspect of the present invention provides a method for preparing a pharmaceutical composition, comprising at least the following steps: Brazilian hematoxylin was dissolved in dimethyl sulfoxide to obtain a Brazilian hematoxylin stock solution; Zinc preparations were dissolved in double-distilled water to obtain a zinc ion stock solution; The pharmaceutical composition is obtained by mixing the Brazilian hematoxylin stock solution with the zinc ion stock solution.

[0010] In some embodiments, the molar concentration of the Brazilian hematoxylin stock solution is 10 mM. The molar concentration of the zinc ion stock solution is 40 mM.

[0011] A third aspect of the present invention provides the use of a pharmaceutical composition in the preparation of a drug for treating cardiac fibrosis.

[0012] In some embodiments, the drug inhibits the expression of α-SMA protein.

[0013] In some embodiments, the drug inhibits the expression of the Postn, Fn1, Acta2, Tgfb1, Tgfbr1, and Smad3 genes.

[0014] In some embodiments, the volume percentage of dimethyl sulfoxide (DMSO) in the drug is less than 1%.

[0015] In this invention, the polyphenolic hydroxyl-containing chemical structure of Brazilian hematoxylin (BX) forms a soluble coordination complex with zinc ions, and the volume percentage of DMSO in the final drug is strictly controlled to <1% when DMSO is used as a solvent. This further avoids interference with the stability of the complex by high concentrations of DMSO, ensuring that zinc ions are effectively released in vivo and participate in synergistic effects.

[0016] In some embodiments, the dosage of the drug is determined based on the subject's weight, with the following conditions: 10-15 mg / kg of brassinolide and 3-5 mg / kg of zinc preparations.

[0017] In some embodiments, the dosage form of the drug includes at least one of injection, tablet, and aerosol.

[0018] Optionally, the dosage form of the drug includes an injection.

[0019] Optionally, the injection includes at least one of an injection solution and a lyophilized powder for injection.

[0020] In some embodiments, the route of administration of the drug is intraperitoneal.

[0021] Optionally, the drug is administered via intraperitoneal injection for three consecutive days on the 4th, 5th, and 6th days after myocardial infarction surgery.

[0022] Beneficial effects 1. In this invention, the polyphenolic hydroxyl group-containing chemical structure of Brazilian hematoxylin (BX) forms a soluble coordination complex with zinc ions. This complex exhibits good stability and solubility, allowing the drug to exert its effects more effectively in vivo. On one hand, the unique chemical structure of Brazilian hematoxylin endows it with a strong binding capacity to zinc ions, ensuring that the complex is not easily dissociated under physiological conditions, thus maintaining the drug's efficacy. On the other hand, this soluble coordination complex is more easily absorbed and transported by the body, improving the drug's bioavailability. 2. The pharmaceutical composition provided by the present invention, wherein Brazilian hematoxylin blocks signal initiation by inhibiting TGF-β receptor phosphorylation; zinc ions inhibit downstream gene transcription by regulating Smad3 nuclear translocation and DNA binding ability; the two work synergistically at different nodes of the TGF-β / Smad pathway to significantly downregulate the expression of fibrosis-related genes (Postn, Fn1, Acta2, Tgfb1, Tgfbr1, Smad3) and proteins (α-SMA, VCP).

[0023] 3. When the pharmaceutical composition containing brassinolide provided in this invention is used in the preparation of drugs for treating cardiac fibrosis, it can effectively reduce the degree of cardiac fibrosis, and the reduction rate of fibrosis area is 10% higher than that of brassinolide alone.

[0024] 4. This invention is the first to combine natural anti-inflammatory components with essential trace elements in the preparation of drugs for cardiac fibrosis, expanding the application scope of natural products in the treatment of cardiac fibrosis and providing new ideas and methods for the treatment of cardiac fibrosis. Attached Figure Description

[0025] Figure 1 The figure shows the effect of the pharmaceutical composition containing Brazilian hematoxylin prepared in this invention on an acute myocardial infarction (MI) model; A in the figure is the experimental design flowchart; BC is the Masson staining result of cardiac tissue; DF is the echocardiography result; G and H are the α-SMA immunofluorescence staining results; I is the mRNA expression level of fibrosis-related genes; J and K are the expression of fibrosis-related proteins detected by Western blotting. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; the reagents, biological materials, and detection kits described are commercially available unless otherwise specified.

[0027] Example 1 The first aspect of this example provides a pharmaceutical composition containing brassinolide, the raw materials of which include brassinolide, zinc preparation, dimethyl sulfoxide and double-distilled water.

[0028] The zinc preparation is anhydrous zinc chloride.

[0029] The mass ratio of the Brazilian hematoxylin to the zinc preparation is 10:3.

[0030] The second aspect of this example provides a method for preparing a pharmaceutical composition, comprising the following steps: Brazilian hematoxylin was dissolved in dimethyl sulfoxide to obtain a Brazilian hematoxylin stock solution; Zinc preparations were dissolved in double-distilled water to obtain a zinc ion stock solution; The pharmaceutical composition is obtained by mixing the Brazilian hematoxylin stock solution with the zinc ion stock solution.

[0031] The molar concentration of the Brazilian hematoxylin stock solution is 10 mM. The molar concentration of the zinc ion stock solution is 40 mM.

[0032] The third aspect of this example provides the use of a pharmaceutical composition in the preparation of a drug for treating cardiac fibrosis.

[0033] The drug inhibits the expression of α-SMA protein. The drug also inhibits the expression of Postn, Fn1, Acta2, Tgfb1, Tgfbr1, and Smad3 genes.

[0034] The drug is in the form of an injection.

[0035] Example 2 The specific implementation method in this example is the same as in Example 1, except that the zinc preparation is zinc gluconate.

[0036] Performance testing 1. Experimental Design (see...) Figure 1 A): Adult (10-week-old) male C57BL / 6J mice were selected to establish an acute myocardial infarction (MI) model by ligating the left anterior descending coronary artery (LAD). The drug was administered intraperitoneally for three consecutive days on days 4, 5, and 6 post-operatively. There were four experimental groups. One day after the last administration, the efficacy of the drug was assessed in each of the four groups using different methods, including echocardiography, Masson staining, immunofluorescence, qPCR, and Western blotting.

[0037] 2. Experimental Groups: Sham surgery group: only open chest surgery, without ligation of coronary arteries.

[0038] Model control group (MI+Vehicle): Myocardial infarction model + solvent control (physiological saline containing DMSO).

[0039] BX+ZnCl2 group (Example 1): Myocardial infarction model + combined treatment with Brazilian hematoxylin (BX, 10 mg / kg) and zinc chloride (ZnCl2, 3 mg / kg).

[0040] BX+ZG group (Example 2): Myocardial infarction model + combined treatment with Brazilian hematoxylin (BX, 10 mg / kg) and zinc gluconate (ZincGluconate, ZG, 3 mg / kg).

[0041] 3. Experimental steps: (1) Establishment of an acute myocardial infarction model: The experimental mice were fasted for 12 hours before surgery. The mice were placed in the induction chamber of the anesthesia machine, oxygen was turned on and the flow rate was adjusted (0.25 MPa, 1 L / min), and the concentration of the anesthetic (isoflurane) was adjusted to 5%. Induction anesthesia was completed in approximately 1 minute, after which the anesthetic concentration was adjusted to 2%, and a mask was connected for continuous inhalation. The mice were fixed in a supine position, and the surgical area on the left chest was shaved and disinfected. A longitudinal incision of approximately 1.5 cm was made about 1-2 mm from the left sternal border, and a vertical everted mattress suture was used to leave sutures. The chest wall muscles were bluntly dissected layer by layer, and the thoracic cavity was quickly entered through the 3rd intercostal space. The intercostal space was opened with hemostatic forceps, and the heart was gently squeezed with the left hand to expel it from the opening in coordination with the heartbeat. The left atrial appendage was ligated 1-2 mm below the left atrial appendage and 0.5 mm beside the pulmonary conus with a 6-0 suture needle through the anterior descending coronary artery, with appropriate tightness. After ligation, the heart is gently returned to the chest cavity, and the chest cavity is squeezed to expel air while the sutures at the ligation incision are tightened to complete the surgery. During the operation, the anesthetic concentration is gradually adjusted to zero. After removing the mask, the mouse is placed on a temperature-controlled mat for about 3-5 minutes to recover.

[0042] (2) Treatment process: Drug preparation: Zinc ion stock solution: Weigh anhydrous zinc chloride and prepare a 40 mM stock solution with double-distilled water; weigh zinc gluconate and prepare a 40 mM stock solution with double-distilled water. Prepare fresh solution as needed.

[0043] Brazilian hematoxylin (BX) stock solution: Weigh Brazilian hematoxylin and prepare a 10 mM stock solution with DMSO.

[0044] Combined drug solution: Based on the mouse body weight and the administered dosage (BX 10 mg / kg, zinc ions 3 mg / kg), accurately calculate and measure the corresponding volumes of BX stock solution and zinc ion stock solution, mix them with physiological saline in proportion, and make up to a final volume to ensure that the volume percentage of DMSO in the final injection solution is less than 1%. All drug solutions were freshly prepared before administration.

[0045] Dosage regimen: The drug was administered via intraperitoneal injection for three consecutive days on days 4, 5, and 6 after myocardial infarction surgery. The sham-operated group and the model control group were injected with the same volume of solvent (physiological saline containing the corresponding concentration of DMSO).

[0046] (2) Heart sections and Masson staining: A whole heart was harvested, embedded in OCT embedding medium, and flash-frozen at -80°C. Using a rotary cryostat, the embedded heart was sectioned starting from the apex, with 16 sections spaced 180 μm apart to uniformly reflect the degree of infarction throughout the heart. Each section was 10 μm thick, mounted on a glass slide, and fixed in 4% PFA at room temperature for 60 min, followed by rinsing with water. Masson staining was then performed: mordanting in Bouin solution for 1 h, followed by rinsing with water; staining with Ponceau S and fuchsin for 15-20 min, followed by rinsing with water; treatment with phosphomolybdic acid solution for approximately 5 min; counterstaining with aniline blue for 5 min, followed by rinsing with water; clearing with xylene; and mounting with neutral resin. Images were observed and acquired under an optical microscope, and ImageJ software was used to quantitatively analyze the proportion of the infarcted area (blue collagen fiber area) to the total myocardial area.

[0047] Results: Masson staining results of cardiac tissue ( Figure 1 (B, 1C) showed that the model control group had large areas of blue collagen fiber deposition (fibrosis) in the heart. Both combination therapy groups significantly reduced the area of ​​fibrosis after myocardial infarction.

[0048] (3) Cardiac ultrasound examination of cardiac function: On postoperative day 7 (24 hours after the last administration), mice were anesthetized with isoflurane gas (3% concentration). After the anesthesia stabilized, the mice were fixed in a supine position on a temperature-controlled operating table. The fur on the chest area was shaved, and ultrasound coupling agent was applied. A small animal high-frequency ultrasound imaging system equipped with a 30 MHz linear probe was used. A short-axis section (papillary muscle level) of the left ventricle was obtained in the parasternal long-axis section. M-mode ultrasound images were recorded in this section. The left ventricular end-diastolic diameter (LVEDD) and end-systolic diameter (LVESD) were measured using the instrument's accompanying analysis software, and the left ventricular ejection fraction (LVEF) and left ventricular shortening fraction (LVFS) were automatically calculated.

[0049] Result: As Figure 1As shown in D, 1E, and 1F, compared with the sham-operated group, the LVEF / LVFS of the model control group mice was significantly decreased. Both the BX+ZnCl2 and BX+ZG combined treatment groups significantly improved cardiac function and increased LVEF / LVFS.

[0050] (4) Heart sampling: After echocardiography, mice were euthanized with an overdose of anesthesia. The hearts were quickly removed via thoracotomy and perfused with pre-cooled PBS through the apex until they turned white. The surface liquid was blotted dry with filter paper and the hearts were weighed. A portion of the heart from each group was longitudinally divided into two parts along the midline at the ligation site. These parts were placed in RIPA and Trizol solutions respectively and stored at -80°C for subsequent protein and RNA extraction. The remaining heart was embedded in OCT and flash-frozen at -80°C for histological analysis.

[0051] (5) Immunofluorescence staining: After collecting the sections for Masson staining, five 5 μm thick sections were cut from each heart without intervals for immunofluorescence staining. After equilibration at room temperature, the sections were fixed with 4% paraformaldehyde for 1 h, washed with PBS, boiled in antigen retrieval solution for 2 min, cooled to room temperature, and washed with PBS. The sections were permeated with 1% Triton X-100 for 10 min, washed again with PBS, blocked with 1% BSA at room temperature for 1 h, the blocking solution was discarded, and α-SMA primary antibody (1:100 dilution) was added and incubated overnight in a humidified chamber at 4°C. The next day, the sections were washed with PBS, and FITC-labeled donkey anti-rabbit fluorescent secondary antibody (1:100 dilution) was added and incubated at room temperature in the dark for 1 h. The sections were washed with PBS and mounted with a DAPI-containing anti-fluorescence quenching mounting medium. Images were observed and acquired under a laser confocal microscope.

[0052] Results: Immunofluorescence staining with α-SMA ( Figure 1 In situ verification of G and 1H in tissues, the α-SMA positive signal (green) in the combined drug group was significantly weakened, which intuitively reflects that the activation of myofibroblasts was effectively inhibited.

[0053] (6) Real-time quantitative PCR (qPCR) detection of fibrosis-related genes: Heart samples stored at -80°C in Trizol were thawed and lysed using a tissue homogenizer. After lysis on ice for 10 min, total RNA was extracted. RNA concentration and purity were determined, and 1 μg of total RNA was reverse transcribed to synthesize cDNA. Using the cDNA as a template, amplification was performed using SYBR Green qPCR premixed buffer. The reaction program was: 95°C, 10 min, 4 cycles → 95°C, 15 sec denaturation, 55°C, 15 sec annealing, 72°C, 30 sec extension, 40 cycles. GAPDH was used as an internal reference gene, and the relative expression levels of target genes (Postn, Fn1, Acta2, Tgfb1, Tgfbr1, Smad3) were calculated using the 2^(-ΔΔCt) method.

[0054] Results: The mRNA expression levels of fibrosis-related genes were detected ( Figure 1 I). The results showed that, compared with the model control group, the transcriptional levels of multiple profibrosis genes were significantly downregulated in the combination drug group.

[0055] (7) Western blot detection of fibrosis-related proteins: Heart samples stored at -80℃ in RIPA were thawed and lysed using a tissue homogenizer. Lysis was performed on ice for 10 min. The samples were then centrifuged at 12,000 rpm for 15 min at 4℃, and the supernatant was collected. Protein concentration was determined using the BCA method. An appropriate amount of protein sample from each group was added to 5×SDS loading buffer and heated at 95℃ for 10 min to denature the protein. An equal amount of denatured protein sample from each group was separated by SDS-PAGE gel electrophoresis and transferred to a PVDF membrane. After rapid blocking, primary antibodies (α-SMA, VCP, HSP90) were added and incubated overnight at 4℃. After washing with PBST, HRP-labeled secondary antibody was added and incubated at room temperature for 1 h. After washing with PBST, ECL chemiluminescence imaging was performed, and the target bands were analyzed using ImageJ software.

[0056] Results: Western blot analysis of cardiac tissue ( Figure 1 The results showed that, compared with the model control group, the protein expression level of α-SMA, a key marker of myocardial fibrosis, was significantly reduced in the combination drug group.

[0057] The above in vivo experimental data fully demonstrate that the combined use of zinc preparations (whether in the form of zinc chloride or zinc gluconate) and brassinolide can synergistically improve cardiac function after myocardial infarction, and effectively reduce the degree of cardiac fibrosis by inhibiting the expression of fibrosis-related genes and proteins at the transcriptional and protein levels, resulting in significant therapeutic effects.

Claims

1. A pharmaceutical composition comprising hematoxylin, characterized in that, The raw materials for preparation include hematoxylin and zinc preparation.

2. The pharmaceutical composition of claim 1, wherein, The zinc preparation includes at least one of anhydrous zinc chloride and zinc gluconate.

3. The pharmaceutical composition of claim 1, wherein, The mass ratio of the hematoxylin and zinc preparation is (10-15):(3-5).

4. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that, The raw materials for preparation of the pharmaceutical composition further include dimethyl sulfoxide and double distilled water.

5. A process for the preparation of a pharmaceutical composition according to claim 4, characterized in that, At least the following steps are included: hematoxylin is dissolved in dimethyl sulfoxide to obtain hematoxylin stock solution; zinc preparation is dissolved in double distilled water to obtain zinc ion stock solution; the hematoxylin stock solution and the zinc ion stock solution are mixed to obtain the pharmaceutical composition.

6. Use of the pharmaceutical composition according to any one of claims 1-4 in the preparation of a drug for treating cardiac fibrosis.

7. Use according to claim 6, characterized in that, The drug inhibits the expression of α-SMA protein.

8. Use according to claim 6, characterized in that, The drug inhibits the expression of Postn, Fn1, Acta2, Tgfb1, Tgfbr1 and Smad3 genes.

9. Use according to claim 6, characterized in that, The volume percentage of dimethyl sulfoxide in the drug is less than 1%.

10. Use according to claim 6, characterized in that, The dosage form of the drug includes at least one of injection, tablet and aerosol.

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