A self-assembled nanosheet of rhein-baicalin, its preparation method and application

Abstract

This invention relates to the field of pharmaceutical preparation technology, specifically to a rhein-baicalin self-assembled nanosheet, its preparation method, and its application. Single-component rhein or single-component baicalin exhibits extremely low solubility in aqueous phase, resulting in unsatisfactory absorption and utilization after oral administration. To address these issues, this invention provides a rhein-baicalin self-assembled nanosheet. Rhein and baicalin are synergistically transformed into amorphous two-dimensional nanosheets under ultrasonication through π-π stacking and hydrogen bonding. This significantly reduces lattice energy, dramatically increases specific surface area, and the highly hydrated hydrophilic groups on the outer layer provide strong steric hindrance and colloidal kinetic stability, thereby achieving high-concentration stable dispersion in aqueous phase.

Description

Technical Field

[0001] This invention relates to the field of drug preparation technology, specifically to a rhein-baicalin self-assembled nanosheet, its preparation method, and its application. Background Technology

[0002] Inflammation is a ubiquitous pathological basis in the occurrence and progression of many diseases. A normal inflammatory response, as a defensive mechanism, effectively resists the invasion of foreign pathogens and initiates tissue repair processes. However, when the inflammatory response becomes uncontrolled or excessively expressed, it can induce pathological changes in tissues, thereby causing serious damage to the body. Therefore, effectively controlling the inflammatory process has become a key intervention strategy for treating many diseases.

[0003] Currently, numerous studies have confirmed that extracts from various natural drugs possess significant anti-inflammatory activities. Among them, rhein and baicalein, as core active monomers of traditional Chinese medicine, have shown good therapeutic potential in scavenging free radicals and antioxidation. Rhein is an anthraquinone compound, and baicalein is a flavonoid compound; both have multi-target anti-inflammatory effects, and their mechanisms of action can regulate inflammatory signaling pathways at multiple stages. Their anti-inflammatory effects are superior to some existing anti-inflammatory drugs, and the incidence of toxic side effects is low. However, these two active monomers have significant inherent limitations in drug delivery. Rhein has a highly hydrophobic anthraquinone core structure, while baicalein has a planar polyphenol ring structure, which directly leads to extremely low solubility in aqueous systems. This poor physical solubility, coupled with undesirable pharmacokinetic behavior, results in low absorption and utilization rates after oral administration, making it difficult to maintain effective therapeutic concentrations in systemic circulation, severely restricting their clinical translation and practical application.

[0004] Carrier-free nanotechnology has attracted widespread attention in recent years. This technology relies on non-covalent forces such as hydrogen bonding and π-π stacking to achieve the self-assembly of drug molecules. This strategy not only avoids the potential toxicity and immunogenicity risks associated with traditional polymeric excipients or inorganic carriers, but also significantly improves the apparent solubility, formulation stability, and in vivo bioavailability of poorly soluble drugs without altering the chemical structure of the active pharmaceutical ingredient, providing a new solution for the efficient delivery of poorly soluble active monomers. Summary of the Invention

[0005] The existing technology has the problem that single-component rhein or baicalein have extremely low solubility in aqueous phase, resulting in unsatisfactory absorption and utilization after oral administration. To address these issues, this invention provides a rhein-baicalein self-assembled nanosheet, the preparation method of which includes the following steps: (1) Dissolve rhein completely in an organic solvent to obtain a rhein solution; (2) Dissolve baicalein completely in an organic solvent to obtain a baicalein solution. The baicalein solution has the same molar concentration as the rhein solution obtained in step (1). (3) Mix the rhein solution and the baicalein solution evenly according to the molar ratio of rhein to baicalein of 1:1 or 1:2 to obtain a mixed solution; (4) At 30°C, the mixture obtained in step (3) is added dropwise to ultrapure water at a volume ratio of 1:10. No more than 50 μL of the mixture is added every 0.5 hours. During the addition process, the ultrapure water is continuously sonicated. After the addition is completed, sonication is continued for at least 1 hour to obtain a light yellow clear mixture. (5) After dialysis purification and freeze-drying of the pale yellow clear mixture obtained in step (4), rhein-baicalin self-assembled nanosheets are obtained.

[0006] Preferably, the organic solvent is dimethyl sulfoxide.

[0007] Preferably, the concentration of rhein solution is 10-20 mmol / L.

[0008] Preferably, the concentration of the baicalein solution is 10-20 mmol / L.

[0009] Preferably, the dialysis purification method is as follows: At room temperature, the pale yellow clear mixture obtained in step (4) is placed into a dialysis bag with a molecular weight cutoff of ≥500 Da. After the bag is tied tightly, it is placed in a beaker containing deionized water and dialyzed with magnetic stirring for at least 6 hours. The deionized water is replaced every 2 hours. After the dialysis is completed, the liquid in the bag is collected.

[0010] Preferably, the freeze-drying method is as follows: The product obtained after dialysis was pre-cooled at -80℃ for at least 4 hours, and then freeze-dried at a vacuum of 8 Pa and a cold trap temperature of -55℃ to remove the solvent, resulting in a loose, light yellow coarse powder, which is the rhein-baicalin self-assembled nanosheet.

[0011] Beneficial effects: (1) Rhein and baicalein are transformed into amorphous two-dimensional nanosheets through π-π stacking and hydrogen bonding under ultrasound. Their lattice energy is significantly reduced and their specific surface area is greatly increased. The highly hydrated hydrophilic groups on the outer layer provide strong steric hindrance and colloidal dynamics stability, thus achieving high-concentration stable dispersion in the aqueous phase.

[0012] (2) Based on the molecular structural characteristics of rhein and baicalein, this invention utilizes the different functional groups they contain (hydroxyl, carboxyl, carbonyl, and benzene rings in rhein molecules, and hydroxyl, carbonyl, and benzene rings in baicalein molecules) to achieve self-assembly through non-covalent interactions, forming carrier-free nanosheets. This method eliminates the need for exogenous excipients such as polymers and liposomes, thus avoiding the toxicity risks caused by excipients. Furthermore, this invention fills the gap in the field of synergistic self-assembly of rhein and baicalein nanoparticles.

[0013] (3) The rhein and baicalin self-assembled nanosheets of the present invention have a high negative potential, which can enhance the stability of the particles in the solution, reduce the aggregation phenomenon, and improve the drug efficacy. The water solubility is significantly improved compared with free rhein and baicalin, which improves the solubility of the two drugs and lays the foundation for subsequent formulation development and clinical application.

[0014] (4) The rhein-baicalin self-assembled nanosheets of the present invention have the effect of scavenging reactive oxygen species (ROS) and relieving oxidative stress. Attached Figure Description

[0015] Figure 1 Transmission electron microscopy image of the rhein-baicalin self-assembled nanosheets obtained in Example 1.

[0016] Figure 2 Tyndall effect diagram of the nanosheets obtained in this invention.

[0017] Figure 3 : Ultraviolet characterization diagram of the material in this invention.

[0018] Figure 4 Potentiogram of the nanosheets obtained in this invention.

[0019] Figure 5 Examples 1-3 show the hydroxyl radical scavenging effects of the obtained nanosheets, free rhein, and free baicalin.

[0020] Figure 6 : Test graphs of the DPPH free radical scavenging effects of the nanosheets, free rhein and free baicalin obtained in Examples 1-3 respectively. Detailed Implementation

[0021] The present invention will be described in detail below with reference to embodiments. However, it should be understood that the following embodiments are merely illustrative examples of implementation of the present invention and are not intended to limit the scope of the present invention.

[0022] Example 1 A self-assembled nanosheet of rhein-baicalin is prepared as follows: (1) Preparation of rhein solution: Weigh rhein powder, slowly add it to 1 mL of dimethyl sulfoxide, and let it stand at 25°C in the dark to dissolve until the powder is completely dissolved, so as to obtain a rhein dimethyl sulfoxide mother liquor with a molar concentration of 10 mmol / L. Seal and store in the dark for later use. (2) Preparation of baicalein solution: Weigh baicalein powder, slowly add it to 1 mL of dimethyl sulfoxide, and let it stand at 25°C in the dark to dissolve until the powder is completely dissolved, so as to obtain a baicalein dimethyl sulfoxide mother liquor with a molar concentration of 10 mmol / L. Seal and store it in the dark for later use. (3) Take 75 μL of rhein dimethyl sulfoxide mother liquor and add it to 75 μL of baicalein dimethyl sulfoxide mother liquor and mix evenly to prepare rhein-baicalein mixed solution; (4) Self-assembly reaction: Under the ultrasonic power of 550 W and frequency of 40 KHz, at a temperature of 30℃, 50 μL of the mixture was added dropwise every 0.5 hours in 1.5 mL of ultrapure water. After the addition was completed, the ultrasonication was continued for 1 hour. During the process, the state of the solution was observed to ensure that there was no layering or precipitation. Finally, a light yellow clear mixture was obtained. (5) Dialysis purification: The final pale yellow clear mixture was placed into a dialysis bag with a molecular weight cutoff of 500 Da at 25°C. The bag was then tied tightly and placed in a beaker containing 500 mL of deionized water. The mixture was magnetically stirred at room temperature for 6 h, and the deionized water was replaced every 2 h. The liquid in the bag was collected after the dialysis was completed.

[0023] (6) Freeze-drying: The liquid collected after dialysis was pre-cooled at -80℃ for 4 h, and then freeze-dried for 24 h while maintaining a vacuum of 8 Pa and a freezing temperature of -55℃ to obtain a loose, light yellow coarse powder, which is called rhein-baicalin self-assembled nanosheets, denoted as rhein-baicalin self-assembled nanosheets 1-1. Transmission electron micrographs of the rhein-baicalin self-assembled nanosheets 1-1 obtained in Example 1 are attached to the instruction manual. Figure 1 As shown in the figure; the test results show that the particle size of the rhein-baicalin self-assembled nanosheets is in the range of 100-1000 nm and they exhibit a uniform sheet-like shape.

[0024] Example 2 is the same as Example 1, except that in Example 2, step (3) involves mixing 100 μL of rhein dimethyl sulfoxide mother liquor with 50 μL of baicalein dimethyl sulfoxide mother liquor until homogeneous. The obtained rhein-baicalein self-assembled nanosheets are designated as rhein-baicalein self-assembled nanosheets 2-1.

[0025] Example 3 is the same as Example 1, except that in Example 3, step (3) involves mixing 50 μL of rhein dimethyl sulfoxide mother liquor with 100 μL of baicalein dimethyl sulfoxide mother liquor until homogeneous. The obtained rhein-baicalein self-assembled nanosheets are designated as rhein-baicalein self-assembled nanosheets 1-2.

[0026] The Tyndall effect refers to the phenomenon where light is scattered when passing through a colloidal medium, making the path of the light beam visible. The Tyndall effect test results for free rhein, free baicalin, and the nanosheets obtained in Examples 1-3 are shown in the attached instructions. Figure 2 As shown, the results indicate that no visible light beam was generated in the solutions of free rhein and free baicalein. The nanosheet solutions obtained in Examples 1-3 all exhibited a typical Tyndall effect after laser irradiation, indicating that rhein and baicalein were successfully assembled. The solvent used in the Tyndall effect tests for free rhein, free baicalein, and the nanosheets obtained in Examples 1-3 was deionized water, with the same mass concentration.

[0027] Physicochemical property testing: The specific experimental methods and results are as follows: (1) Dissolve the free rhein, free baicalein and the nanosheets obtained in Examples 1-3 in deionized water and prepare solutions with a concentration of 0.5 mg / mL. Scan them in the wavelength range of 200-600 nm using an ultraviolet spectrophotometer.

[0028] Test results are as per the instruction manual. Figure 3 As shown.

[0029] Rhein raw material exhibits distinct characteristic absorption peaks at 225 nm, 255 nm, and 430 nm, while baicalein raw material exhibits characteristic absorption peaks at 270 nm and 320 nm. Analysis of the spectra of the self-assembled products in Examples 1-3 reveals a highly positive correlation between the intensity of each characteristic absorption peak of rhein and baicalein in the self-assembled nanosheets and the ratio of the two components in the self-assembly feed ratio.

[0030] (2) Zeta potential detection: The free rhein, free baicalein and the nanosheets obtained in Examples 1-3 were dissolved in deionized water to prepare solutions with a concentration of 0.5 mg / ml. The zeta potential was detected at 25°C using a potentiometer.

[0031] Test results are as per the instruction manual. Figure 4As shown, the results indicate that all test groups exhibited negative charge characteristics in the aqueous phase. The self-assembled nanosheets of Examples 1-3 all exhibited significant negative charge characteristics in the aqueous phase. Among them, the zeta potential of the nanosheets obtained in Examples 1 and 2 reached approximately -26 mV. This high absolute value of negative charge originates from the deprotonation ionization of the carboxyl groups in the rhein structure in the aqueous environment. The high density of surface negative charge effectively overcomes the van der Waals attraction between hydrophobic cores. When the baicalin ratio was 1:2, the absolute value of the zeta potential of the system significantly increased to approximately -12.5 mV, and the standard deviation increased significantly. This indicates that the excess baicalin monomer had a dilution and shielding effect on the charge density of the nanosheet surface, weakening the electrostatic repulsion between particles and reducing the microscopic uniformity of the system. The zeta potential of the free rhein group was only approximately -16.5 mV. This is mainly because free rhein is extremely insoluble in water and readily forms large amorphous aggregates. As a result, although it contains carboxyl groups, most of them are encapsulated inside the aggregates and cannot be effectively charged and ionized on the surface. In contrast, the free baicalin group contains only weakly ionized phenolic hydroxyl groups in its structure, and its absolute value of the Zeta potential is the lowest, at only about -12.3 mV.

[0032] (4) Hydroxyl radical scavenging: Free rhein, free baicalin and the nanosheets obtained in Examples 1-3 were dissolved in deionized water and prepared into solutions with a concentration of 0.5 mg / ml as sample solutions.

[0033] Seven identical test solutions (each with a volume of 2 mL) were prepared using the Fenton reaction to generate hydroxyl radicals.

[0034] One portion of the test solution was left untreated and used as a blank control group.

[0035] Take another portion of the test solution and add 250 μL of vitamin C at a concentration of 0.5 mg / mL as a positive control group.

[0036] The remaining 5 test solutions were each supplemented with 250 μL of free rhein, free baicalein, and the sample solutions obtained in Examples 1-3, with a concentration of 0.5 mg / mL, to form the experimental groups.

[0037] The preparation method for the test solution is as follows: Under light-protected conditions, add 1.4 mL of pH 4.0 NaAc / Hac buffer solution, 100 μL of 10 mM FeCl2, 100 μL of 10 mM TMB, and 200 μL of 20 mM H2O2 to a centrifuge tube. Finally, bring the total volume to 2 mL with H2O. Mix well and incubate at 37°C for 30 min using a hydrothermal method. After the reaction is complete, place the tube in ice water to terminate the reaction. Divide the reaction mixture into seven equal volumes for testing.

[0038] Test principle: Using the Fenton reaction to generate hydroxyl radicals: H₂O₂ + Fe 2+ =·OH+H2O+Fe 3+ 3,3',5,5'-Tetramethylbenzidine (TMB) was added to the reaction system as a colorimetric agent. The hydroxyl radicals generated in the Fenton reaction reacted with TMB, turning it blue. When 250 μL of a rhein-baicalin self-assembled nanosheet solution was added, the TMB color lightened if the hydroxyl radicals were scavenged. The UV absorbance of the test solution was then measured across the entire wavelength range, showing a decrease in absorbance at 650 nm. This indicates that the rhein-baicalin self-assembled nanosheets possess excellent hydroxyl radical scavenging ability and antioxidant properties, showing promise for inflammation reduction.

[0039] Specific test results are attached to the instruction manual. Figure 5 As shown. The test results show that, Blank control group: A large number of hydroxyl radicals were generated in the system. These highly oxidizing radicals oxidized the substrate and produced a very strong characteristic color peak at 652 nm, which represents the maximum color state when the radicals were not cleared.

[0040] Positive control group: Vitamin C, as a strong antioxidant, almost completely removes hydroxyl radicals in the system, causing the substrate to fail to develop color and the absorbance to drop directly to near 0, which is represented by a flat line close to the baseline.

[0041] The absorption peaks of the free baicalin group and the free rhein group at 652 nm were as high as 0.35 and 0.31, respectively, which were very close to those of the blank control group. This is because the two monomeric raw materials have extremely low solubility in the aqueous system, and are very prone to amorphous aggregation and precipitation in the experimental environment. As a result, their inherent antioxidant active groups are encapsulated inside the aggregates and cannot effectively contact the hydroxyl radicals in the aqueous phase.

[0042] Experimental group: When the prepared nanosheets were added, the large number of antioxidant active groups exposed on their surface preemptively reacted with hydroxyl radicals, thus inhibiting the colorimetric reaction. The lower the absorbance peak, the stronger the free radical scavenging ability.

[0043] (5) DPPH free radical scavenging: Free rhein, free baicalin and nanosheets prepared in Examples 1-3 were dissolved in deionized water and prepared into sample solutions with a concentration of 0.5 mg / mL.

[0044] The scavenging effect was tested using the DPPH free radical scavenging method, as follows: Seven portions of DPPH working solution of the same volume (2 mL) were used as test working solutions.

[0045] Take one portion of the test working solution without adding any substances as a blank control group.

[0046] Take another portion of the test working solution and add 250 μL of vitamin C at a concentration of 0.5 mg / mL as a positive control group.

[0047] 250 μL of free rhein, free baicalein, and the sample solutions obtained in Examples 1-3 were added to the remaining 5 test working solutions as experimental groups. After mixing and reacting for 20 min, the UV full-wavelength absorption of the DPPH working solution was detected.

[0048] The preparation method for DPPH working solution is as follows: Weigh 1.47 mg of 1,1-diphenyl-2-trinitrophenylhydrazine, dissolve it in 40 mL of anhydrous ethanol, and sonicate to dissolve it to obtain the DPPH working solution.

[0049] It is known that DPPH free radicals are scavenged in the presence of antioxidants, resulting in a lighter solution color and a characteristic absorption at 519 nm. This indicates that the sample solution possesses the ability to scavenge DPPH free radicals, exhibiting antioxidant properties and showing promise for inflammation clearance.

[0050] Test results are as per the instruction manual. Figure 6 As shown, the test results indicate that... Blank control group: Free radicals were not cleared, showing typical strong absorption peaks of organic free radicals, with the highest absorbance value at 515-517nm, representing the maximum color development state when free radicals were not cleared.

[0051] Positive control group: In the positive control group, vitamin C, as a strong reducing antioxidant, can rapidly reduce and consume DPPH free radicals. The absorbance value of the entire curve is almost zero, and the characteristic absorption peak disappears completely, confirming that the sensitivity of this DPPH detection system is reliable.

[0052] In the free rhein and free baicalin experimental groups, both free rhein and free baicalin could reduce the absorbance of the DPPH characteristic peak, indicating that the two monomers themselves have basic antioxidant activity. However, their peak values ​​at 515-517 nm were significantly higher than those of the three groups of self-assembled nanosheets, indicating that the free radical scavenging ability of the free monomers is significantly limited.

[0053] Experimental group: The DPPH removal by nanosheets mainly relies on the hydrogen atom transfer mechanism. The lower the absorbance peak, the stronger the removal ability.

[0054] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A self-assembled nanosheet of rhein-baicalin, characterized in that, The preparation method includes the following steps: (1) Dissolve rhein completely in an organic solvent to obtain a rhein solution; (2) Dissolve baicalein completely in an organic solvent to obtain a baicalein solution. The baicalein solution has the same molar concentration as the rhein solution obtained in step (1). (3) Mix the rhein solution and the baicalein solution evenly according to the molar ratio of rhein to baicalein of 1:1 or 1:2 to obtain a mixed solution; (4) At 30°C, the mixture obtained in step (3) is added dropwise to ultrapure water at a volume ratio of 1:

10. No more than 50 μL of the mixture is added every 0.5 hours. During the addition process, the ultrapure water is continuously sonicated. After the addition is completed, sonication is continued for at least 1 hour to obtain a light yellow clear mixture. (5) After dialysis purification and freeze-drying of the pale yellow clear mixture obtained in step (4), rhein-baicalin self-assembled nanosheets are obtained.

2. The rhein-baicalin self-assembled nanosheet according to claim 1, characterized in that, The organic solvent is dimethyl sulfoxide.

3. The rhein-baicalin self-assembled nanosheet according to claim 1, characterized in that, The concentration of rhein solution is 10-20 mmol / L.

4. The rhein-baicalin self-assembled nanosheet according to claim 1, characterized in that, The concentration of baicalein solution is 10-20 mmol / L.

5. The rhein-baicalin self-assembled nanosheet according to claim 1, characterized in that, The dialysis purification method is as follows: At room temperature, the pale yellow clear mixture obtained in step (4) is placed into a dialysis bag with a molecular weight cutoff of ≥500 Da. After the bag is tied tightly, it is placed in a beaker containing deionized water and dialyzed with magnetic stirring for at least 6 hours. The deionized water is replaced every 2 hours. After the dialysis is completed, the liquid in the bag is collected.

6. The rhein-baicalin self-assembled nanosheet according to claim 1, characterized in that, The freeze-drying method is as follows: The product obtained after dialysis was pre-cooled at -80℃ for at least 4 hours, and then freeze-dried at a vacuum of 8 Pa and a cold trap temperature of -55℃ to remove the solvent, resulting in a loose, light yellow coarse powder, which is the rhein-baicalin self-assembled nanosheet.

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