Berberine-asiaticoside eutectic (BBR-AS) for infectious wound treatment and preparation method thereof

Abstract

The invention discloses a berberine-asiaticoside (BBR-AS) eutectic crystal as well as a preparation method and application thereof, and belongs to the field of biological medicines. A eutectic crystal is prepared from BBR and AS according to the molar ratio of 1: 1 through a cooling crystallization method, and the crystal structure of the eutectic crystal is represented and confirmed through X-ray diffraction (XRD) and infrared spectroscopy (FTIR). According to the eutectic crystal, the solubility of asiaticoside and the slow release performance of berberine are remarkably improved, in-vitro experiments prove that the eutectic crystal has a strong inhibition effect on escherichia coli and staphylococcus aureus, can promote proliferation and migration of fibroblasts and is suitable for collaborative treatment of infectious wounds.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and specifically relates to a berberine - asiaticoside eutectic (BBR - AS) for the treatment of infectious wounds and a preparation method thereof. Background Art

[0002] The skin is the largest tissue organ of the human body. Once its normal structure and function are damaged, wounds will follow. Among the many situations that lead to the appearance of wounds, wound infections caused by bacteria are particularly intractable. It not only slows down the process of wound healing but also subjects patients to the pain of the wound, and even symptoms such as suppuration may occur. Currently, in the treatment of infectious wounds, in addition to using conventional debridement and anti - infection measures, effective treatment methods are relatively scarce. External treatment methods of traditional Chinese medicine have shown unique advantages in the clinical treatment of infectious wounds. Traditional Chinese medicine believes that "fire toxin" is the key pathogenesis that makes it difficult for infectious wounds to heal. Based on this, through syndrome differentiation and treatment, and reasonably selecting traditional Chinese medicine for fumigation, washing, and wet compress, the effects of clearing heat and detoxifying, removing necrosis and promoting granulation can be achieved, and it can play a dual role of antibacterial and promoting wound healing in clinical treatment.

[0003] Berberine (BBR) is an alkaloid with broad - spectrum antibacterial activity present in the traditional Chinese medicine Coptis chinensis. It can effectively inhibit the growth and reproduction of common wound - infecting pathogenic bacteria such as Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, and exerts its antibacterial effect through multiple pathways such as destroying the bacterial cell membrane structure and inhibiting bacterial protein synthesis, thereby reducing the number of bacteria in the wound and reducing the further damage of the inflammatory reaction to the wound tissue. Asiaticoside (AS) is a pentacyclic triterpenoid compound derived from the plant Centella asiatica of the Umbelliferae family. It can regulate the inflammatory reaction, reduce the damage of excessive inflammation to the wound tissue, and create favorable conditions for repair. At the same time, it can also stimulate the proliferation of fibroblasts and the synthesis of collagen, promote the growth of granulation tissue, accelerate the epithelialization process, and help improve the micro - environment of the wound and reduce scar formation, thus promoting the repair and healing of the wound in multiple dimensions during wound infection. However, the solubility of BBR and AS is not ideal, which limits their transmembrane absorption and bioavailability.

[0004] Drug cocrystal technology is a technique that forms a cocrystal structure by combining drug molecules with suitable ligands, thereby improving the physicochemical properties and bioavailability of drugs. BBR and AS can self-assemble to form a cocrystal. Groups such as hydroxyl and nitrogen atoms in the BBR molecule can act as hydrogen bond donors or acceptors, while groups such as hydroxyl and carbonyl in the AS molecule can form a hydrogen bond network with berberine, optimizing the physical and chemical properties of both through intermolecular hydrogen bonding. The BBR-AS cocrystal form can significantly improve the solubility and dissolution rate of both without changing the chemical structure of the drug, thereby enhancing its absorption efficiency and further improving bioavailability. In addition, cocrystal technology can also improve the stability and synergy of drugs, further optimizing the therapeutic effect of drugs.

[0005] The treatment of infectious wounds requires consideration of both antibacterial and wound-healing promotion. However, existing drugs such as berberine (BBR) have insufficient bioavailability due to low solubility, and the effect of asiaticoside (AS) alone in promoting repair is limited. Drug cocrystal technology can improve drug properties through intermolecular interactions, but there is currently no report on the preparation of BBR-AS cocrystal and its application in infectious wounds. Summary of the Invention

[0006] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a berberine-asiaticoside (BBR-AS) cocrystal for the treatment of infectious wounds and its preparation method. By optimizing the cocrystal preparation process, the defects of single drugs are solved, and the synergistic enhancement of antibacterial and wound-healing promotion is achieved.

[0007] In order to achieve the above invention purpose, the present invention provides the following technical solutions.

[0008] The present invention discloses a berberine-asiaticoside (BBR-AS) cocrystal, which is characterized by being formed by BBR and AS with a molar ratio of 1:1.

[0009] The present invention also discloses the application of the above-mentioned berberine-asiaticoside (BBR-AS) cocrystal in the preparation of drugs for the treatment of infectious wounds.

[0010] The present invention also discloses an antibacterial product, which is characterized by containing the above-mentioned berberine-asiaticoside cocrystal and is used for inhibiting Escherichia coli and Staphylococcus aureus.

[0011] The present invention also discloses a preparation method of the above-mentioned berberine-asiaticoside (BBR-AS) cocrystal, which is characterized in that the preparation steps of the pharmaceutical preparation are as follows: (1) Accurately weigh appropriate amounts of BBR and AS and mix them. (2) Dissolve the mixture obtained in (1) in a 95% ethanol solution at 45°C to form a uniform solution. (3) Keep the solution obtained in (2) at a constant temperature for 48 hours, and during this period, gradually precipitate the crystals by slowly cooling the temperature.

[0012] Further, the mixing ratio of BBR and AS in (1) is: mixing in a molar ratio of 1:1.

[0013] The present invention also discloses a pharmaceutical composition, which is characterized by comprising the above-mentioned berberine - asiaticoside eutectic and pharmaceutical excipients.

[0014] Further, the application of the above-mentioned pharmaceutical composition in the preparation of a drug for treating infectious wounds.

[0015] Further, it accelerates wound healing by promoting the proliferation and migration of fibroblasts.

[0016] The present invention also discloses an in vitro antibacterial product, which is characterized by comprising an effective amount of the pharmaceutical composition described in any one of the above.

[0017] The present invention also discloses a wound repair product, which is characterized by comprising an effective amount of the pharmaceutical composition described in any one of the above.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0019] The present invention evaluated the antibacterial effect of BBR-AS eutectic in the treatment of infectious wounds through in vitro antibacterial experiments and investigated its effect on the proliferation and migration of fibroblasts through cell scratch experiments. The experimental results show that the antibacterial effect of BBR-AS eutectic is significantly better than that of the raw material drugs and their physical mixtures, showing strong antibacterial activity. The eutectic structure significantly improves the solubility of the raw material drugs, especially the solubility of AS has been greatly increased, thereby enhancing the bioavailability of the drugs. In addition, BBR-AS eutectic shows good effects in promoting the proliferation and migration of fibroblasts, which is helpful for wound repair. These findings indicate that BBR-AS eutectic has broad application prospects in the treatment of infectious wounds. Description of the Drawings

[0020] Figure 1 It is the result of molecular docking.

[0021] Figure 2 It is the SEM scanning images of BBR, AS, and BBR-AS eutectic. A is the chemical structure of BBR; B is the SEM characterization result of BBR; C is the chemical structure of AS; D is the SEM characterization result of AS; E is the reactant of BBR-AS eutectic; F is BBR-AS eutectic.

[0022] Figure 3 It is the DSC scanning curves of BBR, AS, and BBR-AS eutectic.

[0023] Figure 4 TIR scanning image of BBR-AS eutectic

[0024] Figure 5 XRD scanning images of BBR, AS, and BBR-AS eutectic

[0025] Figure 6 Solubility curves of BBR, AS API, and BBR-AS eutectic in water. A is the solubility curve of AS and BBR-AS eutectic; B is the solubility curve of BBR and BBR-AS eutectic

[0026] Figure 7 Inhibitory effects of BBR-AS eutectic and API on Escherichia coli. a is the control group; b is the BBR-AS physical mixture group; c is the BBR-AS eutectic group; d is the BBR group; e is the AS group

[0027] Figure 8 Inhibitory effects of BBR-AS eutectic and API on Staphylococcus aureus. a is the control group; b is the BBR-AS physical mixture group; c is the BBR-AS eutectic group; d is the BBR group; e is the AS group

[0028] Figure 9 Effects of drugs on the migration ability of L929 cells. A is the cell proliferation rate of each experimental group; B is the cell migration rate of each experimental group; C is the microscopic image of L929 cell migration Detailed implementation manners

[0029] The present invention will be further described in detail below with specific examples. However, it should not be understood that the scope of the above-mentioned subject matter of the present invention is limited to the following examples. All technologies implemented based on the content of the present invention belong to the scope of the present invention

[0030] Unless otherwise specified, the reagents and materials used in the present invention are all commercially available

[0031] Examples

[0032] I. Materials and methods

[0033] 1. Experimental materials

[0034] 1.1 Experimental materials

[0035] Table 1 Names of drugs and reagents .

[0036] 1.2 Experimental instruments

[0037] Table 2 Main instruments and consumables .

[0038] II. Experimental methods.

[0039] 2.1 Molecular docking.

[0040] First, determine the three-dimensional structure files of asiaticoside and berberine small molecules. After adding hydrogen atoms, protonation / deprotonation, etc., use the DOCK molecular docking software for docking. During the docking process, take the asiaticoside molecule as the "receptor" and berberine as the "ligand", and simulate the interaction and binding mode of the two molecules by defining the docking pocket. Finally, based on the docking score and the generated complex conformation, evaluate the possibility of the two small molecules forming a cocrystal and the binding stability.

[0041] 2.2 Preparation of BBR-AS cocrystal.

[0042] The BBR-AS cocrystal was prepared by the cooling crystallization method (Wang Yeyang, Yuan Penghui, Yang Dezhi, et al. Research progress on the design, preparation method and application of drug-drug cocrystals [J]. Herald of Medicine, 2023, 42 (07): 977-983.). Accurately weigh appropriate amounts of BBR and AS, and mix them in a molar ratio of 1:1. Subsequently, dissolve the mixture in a 95% ethanol solution at 45°C to form a homogeneous solution. This solution was maintained at a constant temperature (45°C) for 48 h, and during this period, the crystals were gradually precipitated by slowly cooling (lowering the temperature by 5°C per hour until the temperature reached 25°C). After the reaction, the precipitated crystals were separated from the mother liquor by filtration, and the crystals were dried. The BBR-AS cocrystal was collected, and its structure was detected by transmission electron microscopy (SEM), X-ray single crystal diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR).

[0043] 2.3 Solubility determination.

[0044] Weigh an excess of BBR, AS raw materials, and BBR-AS cocrystal, and place them in a dissolution cup containing 50 mL of an aqueous solution with 0.005% Tween-80. Set the instrument rotation speed to 250 r / min and the temperature to (37.0 ± 0.5)°C. Start timing from the moment the drug contacts the dissolution medium. Take 150 μL of samples at 6, 12, 18, 24, 30, 45, 60, 120, and 240 min (while supplementing the same temperature and equal amount of dissolution medium), filter, and take 20 μL of the subsequent filtrate for HPLC determination.

[0045] Chromatographic conditions for BBR: Waters C18 column (4.6 mm × 250 mm, 5 μm), with acetonitrile - 0.05 mol / L potassium dihydrogen phosphate solution (50:50) (add 0.4 g of sodium dodecyl sulfate to every 100 mL, and adjust the pH value to 4.0 with phosphoric acid) as the mobile phase; the detection wavelength is 345 nm. The column temperature is 37 °C; the flow rate is 1 mL / min; the injection volume is 20 μL.

[0046] Chromatographic conditions for AS: Waters C18 column (4.6 mm × 250 mm, 5 μm), with acetonitrile - 2 mmol / L β - cyclodextrin solution (24:76) as the mobile phase; the detection wavelength is 205 nm, the column temperature is 30 °C; the flow rate is 1 mL / min; the injection volume is 20 μL.

[0047] 2.4 Antibacterial experimental study of BBR - AS eutectic.

[0048] (1) Preparation of culture media.

[0049] Prepare LB broth medium and LB agar medium according to the requirements of the culture medium instructions, adjust the pH value to 7.2 ± 0.1, and autoclave; MH agar medium is prepared according to the instructions, dispensed, and then autoclaved for standby.

[0050] (2) Preparation of bacterial suspensions.

[0051] Take the preserved Staphylococcus aureus and Escherichia coli and inoculate them onto LB solid medium, culture at 37 °C for 24 h, take 3 - 5 colonies and inoculate them into LB liquid medium, and count after culturing in a 37 °C constant temperature shaker for 24 h.

[0052] (3) In vitro antibacterial experiment.

[0053] Pour the autoclaved solid medium into six - well plates and let it cool. Add 2 mL of solid medium to each well. Set up a blank group, a control group, a BBR group, an AS group, a BBR - AS group, and a positive control group, with three parallels in each group.

[0054] Add 100 μL of purified Staphylococcus aureus and Escherichia coli bacterial suspensions to the BBR group, AS group, BBR - AS group, blank group, and positive control group.

[0055] Invert the petri dishes and culture them in a 37 °C constant temperature incubator for 18 h, then photograph the bacterial colonies on the petri dishes and count.

[0056] (4)Observation of bacterial morphology The morphology of bacteria treated with different drugs was observed by SEM. The bacterial suspensions of different treatment groups were centrifuged at 25 °C (5000 rpm) for 20 min, and the bacteria at the bottom were collected. 4% paraformaldehyde was added thereto and fixed overnight in a refrigerator at 4 °C. The paraformaldehyde was aspirated by centrifugation, and the fixed samples were washed with PBS multiple times, and then dehydrated successively with ethanol solutions of different concentrations (20%, 40%, 60%, 80% and 100%) in gradients for 10 min each. Finally, 10 μL of the obtained bacterial sample was dropped on a silicon wafer and naturally dried in air. After sputtering with gold, it was observed by SEM.

[0057] 2.5 Effects of BBR-AS on the proliferation and migration of fibroblasts.

[0058] (1) Cell culture.

[0059] The L929 mouse fibroblasts were thawed and resuscitated, placed in DMEM medium containing 10% inactivated fetal bovine serum (FBS) + 90% high-glucose, and cultured in a 37 °C, 5% CO2 incubator. The culture medium was changed every 2 - 3 d for subculture.

[0060] (2) Cell scratch assay.

[0061] L929 cells in good growth state were inoculated into 6-well plates at a density of 1×10 6 cells / well, placed in a cell incubator, and cultured overnight. When the cell confluence was about 90% after cell attachment, a sterile 200 μL pipette tip was used to make a scratch, and the cells were washed 3 times with sterile PBS. The experimental groups were added with DMEM medium containing 2% FBS, 1% fetal bovine serum and 0.1 μg / mL of BBR, AS, and BBR-AS solutions, and the control group was added with DMEM medium containing 2% FBS and 1% fetal bovine serum, and continued to be cultured in a cell incubator. Observation and photography were carried out at 0 h and 24 h, and the scratch area was quantified using ImageJ software, and the cell migration rate was calculated according to the following formula: Migration rate (%) = (scratch area at 0 h - scratch area at 24 h) / scratch area at 0 h × 100%.

[0062] III. Experimental results.

[0063] 3.1 Molecular docking results: Molecular docking simulation showed that the binding of berberine and asiaticoside was mainly dominated by hydrophobic interactions: the benzene ring structure of berberine and the pentacyclic triterpene skeleton of asiaticoside were closely arranged by van der Waals forces, forming a continuous hydrophobic structure, and no typical hydrogen bond donor-acceptor pairing mode was observed around the binding site. The binding free energy between the two was -5.0 ± 0.3 kcal / mol, and this value was significantly better than the binding energy levels of most physical mixtures, indicating that berberine and asiaticoside formed a thermodynamically stable supramolecular system through an entropy-driven effect. (Figure 1 ).

[0064] 3.2 Characterization results of BBR-AS eutectic.

[0065] 3.2.1 SEM characterization of BBR-AS eutectic.

[0066] The morphology of the eutectic of berberine (BBR) and asiaticoside (AS) was characterized by scanning electron microscopy (SEM). The results showed that the crystal structure of the eutectic was significantly different from that of the single drug. The SEM image of single berberine (BBR) showed that it presented an irregular blocky or flaky crystal structure, with a relatively rough surface and blurred edges. The crystal size distribution was wide, and obvious cracks and pores could be seen on the surface of some crystals, indicating that its crystal structure was relatively loose, as shown in Figure 2 Figure B. The SEM image of single asiaticoside (AS) showed that it presented a flaky or needle-like crystal structure, with a relatively smooth surface and clear edges. The crystal size was relatively uniform, and some crystals presented regular geometric shapes, such as rhombus or long strip, as shown in Figure 2 Figure D.

[0067] The SEM image of BBR-AS eutectic showed that its crystal morphology was more uniform, with a smooth surface and a dense structure. The crystal size distribution was uniform, and no obvious drug phase separation was observed, as shown in Figure 2 Figures E and 2F. The crystal morphology of the eutectic was neither the irregular blocky structure of BBR nor the flaky or needle-like structure of AS, but presented a new regular crystal morphology. This regular crystal structure may be due to the formation of a new crystal arrangement mode between BBR and AS molecules through non-covalent interactions such as hydrogen bonds and π-π stacking. In addition, cracks or pores common in single drug crystals were not observed in the SEM image of BBR-AS eutectic, indicating that the crystal structure of the eutectic was more stable and dense. This dense structure may contribute to improving the stability and solubility of the drug, providing advantages for its application in drug delivery systems.

[0068] 3.2.2 DSC characterization of BBR-AS eutectic.

[0069] The thermodynamic properties of berberine (BBR), asiaticoside (AS) and their eutectic (BBR-AS) were characterized by differential scanning calorimetry (DSC). The results showed that single BBR and AS exhibited sharp and symmetric endothermic peaks at 180.88 °C and 239.81 °C respectively, indicating that both had high crystallinity, thermal stability and high purity. In contrast, the DSC curve of BBR-AS eutectic showed a new endothermic peak between the single melting peaks of BBR and AS, and the peak shape was more complex. This new melting peak indicated that BBR and AS formed a new crystal phase through intermolecular interactions (such as hydrogen bonds or π-π stacking), rather than a simple physical mixture. In addition, the melting peaks of the single drugs were not observed in the DSC curve of the eutectic, further confirming the successful formation of the eutectic, as Figure 3 shown. This new thermodynamic behavior indicates that BBR-AS eutectic has higher thermal stability, providing an important thermodynamic basis for its application in drug delivery systems. In summary, the DSC characterization results not only revealed the melting behavior and thermal stability characteristics of BBR-AS eutectic, but also confirmed the formation of the eutectic from a thermodynamic perspective.

[0070] 3.2.3 Characterization of the TIR of BBR-AS eutectic.

[0071] The molecular vibration characteristics of berberine (BBR), asiaticoside (AS) and their eutectic (BBR-AS) were characterized by total internal reflection infrared spectroscopy (TIR). The results showed that BBR and AS exhibited multiple characteristic absorption peaks in the infrared spectrum, reflecting the presence and vibration modes of functional groups such as hydroxyl, aromatic ring, carbonyl, and ether bond in their molecules. The main absorption peaks of BBR were concentrated around 3400 cm⁻¹ (O-H), 2920 cm⁻¹ and 2850 cm⁻¹ (C-H), 1600 cm⁻¹ and 1500 cm⁻¹ (C=C), and 1250 cm⁻¹ and 1050 cm⁻¹ (C-O / C-N); the main absorption peaks of AS were concentrated around 3400 cm⁻¹ (O-H), 2920 cm⁻¹ and 2850 cm⁻¹ (C-H), 1700 cm⁻¹ (C=O), 1600 cm⁻¹ and 1500 cm⁻¹ (C=C), and 1250 cm⁻¹ and 1050 cm⁻¹ (C-O / C-N). In contrast, the TIR spectrum of BBR-AS eutectic showed new characteristic absorption peaks, and the characteristic peaks of some single drugs shifted or changed in intensity. For example, the shift of the O-H stretching vibration peak (near 3400 cm⁻¹) indicated that BBR and AS formed a new interaction through hydrogen bonds, while the changes in the C=O and C=C stretching vibration peaks reflected π-π stacking or other non-covalent interactions between the aromatic ring and the carbonyl, such as Figure 4As shown. These changes indicate that BBR and AS form a new chemical structure in the eutectic, rather than a simple physical mixture. No obvious impurity peaks were observed in the TIR spectrum, further confirming the high purity and chemical stability of the eutectic. In summary, the TIR characterization results not only reveal the molecular vibration characteristics of the BBR-AS eutectic, but also confirm the successful formation of the eutectic from the perspective of chemical structure.

[0072] 3.2.4 Characterization of BBR-AS eutectic XRD.

[0073] The crystal structure of the berberine (BBR) and asiaticoside (AS) eutectic was characterized by X-ray diffraction (XRD). The results showed that the diffraction pattern of the eutectic was significantly different from that of the single drug crystal structure, indicating that BBR and AS formed a new crystal phase in the eutectic. The XRD pattern of single berberine (BBR) showed typical crystal diffraction peaks in the 2θ angle range, and the main peak positions were concentrated at 8.5°, 12.3°, 15.7°, 17.2°, 20.5°, 22.8°, and 25.4°, etc. These sharp and relatively strong diffraction peaks indicate that BBR has high crystallinity. The XRD pattern of single asiaticoside (AS) also showed typical crystal diffraction peaks in the 2θ angle range, and the main peak positions were concentrated at 9.2°, 13.5°, 16.8°, 18.4°, 21.2°, 23.6°, and 26.8°, etc., also indicating that AS has high crystallinity, as Figure 5 shown.

[0074] The XRD pattern of the BBR-AS eutectic showed some new diffraction peaks, while some characteristic diffraction peaks of the single drug disappeared or shifted. The diffraction peaks of the eutectic may appear at new 2θ angle positions (such as 10.5°, 14.2°, 19.1°, 24.3°, etc.). These new diffraction peaks indicate that the BBR and AS molecules form a new crystal arrangement in the eutectic. In addition, the intensity distribution and peak shape of the diffraction peaks of the eutectic are also different from those of the single drug, reflecting the influence of intermolecular interactions in the eutectic on the crystal structure. No obvious amorphous diffuse peaks were observed in the XRD pattern, further confirming the high purity and crystallinity of the BBR-AS eutectic. These results indicate that BBR and AS are not a simple physical mixture in the eutectic, but form a new crystal structure through intermolecular interactions.

[0075] 3.3 Solubility test results of the raw materials and eutectic.

[0076] The solubility results of BBR and AS raw materials and the BBR-AS eutectic in 37°C aqueous solution are as Figure 5As shown in the figure, the solubility rate of AS raw material drug is slow, and the maximum apparent solubility is only (4.10±0.23) μg / mL. However, the solubility rate of AS in the prepared BBR-AS eutectic is significantly higher than that of the raw material drug, reaching the maximum apparent solubility of (20.62±0.35) μg / mL at 2 h, which is 5 times that of AS raw material drug. This indicates that the eutectic can significantly improve the solubility of AS. Compared with AS raw material drug, the maximum apparent solubility of BBR raw material drug decreases, from Figure 6 It can be seen that the maximum apparent solubility of BBR in the eutectic decreases to 0.2 mg / mL, which may lead to a more sustained release of BBR, thereby improving the bioavailability and having potential advantages.

[0077] 3.4 Antibacterial properties of BBR-AS eutectic.

[0078] Inhibitory effect of BBR-AS on Escherichia coli.

[0079] In this experiment, Escherichia coli and Staphylococcus aureus were used as representative bacteria to explore the in vitro antibacterial efficacy of BBR-AS eutectic. Figure 7 As shown in the figure, in the experiment of anti-Escherichia coli, Escherichia coli multiplied in large numbers in the control group. Compared with the control group, the number of colonies in the BBR group, AS group and their physical mixture group decreased, indicating that the raw drugs have a certain inhibitory effect on Escherichia coli. The number of colonies in the BBR-AS eutectic group and the positive group decreased significantly compared with the control group, and the effect was significantly better than that of the raw material drug and the physical mixture group, indicating that the inhibitory effect of BBR-AS eutectic on Escherichia coli is significantly better than that of the raw material drug.

[0080] As Figure 8 shown in the figure, in the experiment of anti-Staphylococcus aureus, Staphylococcus aureus multiplied in large numbers in the control group. Compared with the control group, BBR raw material drug and BBR-AS physical mixture showed a certain antibacterial effect on Staphylococcus aureus, and AS had no obvious inhibitory effect on Staphylococcus aureus. Compared with the control group, the number of colonies in the BBR-AS eutectic group and the positive group decreased significantly, showing a significant inhibitory effect, and was better than the raw material drug group and the physical mixture group.

[0081] 3.5 Effects of BBR-AS on the proliferation and migration of fibroblasts.

[0082] Figure 9As shown in A, after treating L929 cells with 0.1 μg / mL of the drug for 24 h, the cell viabilities of the BBR, AS, physical mixture of BBR-AS, and BBR-AS eutectic groups were (80.32 ± 3.15)%, (105.34 ± 4.21)%, (98.62 ± 4.65)%, and (125.34 ± 3.56)%, respectively. The results showed that compared with the control group, the BBR-AS eutectic could significantly promote the proliferation of mouse L929 fibroblasts, and the proliferation-promoting effect was significantly better than that of the physical mixture of BBR-AS (P < 0.05). Figure 9 As shown in B, after treating L929 cells with the drug for 24 h, the migration rates of L929 cells in the BBR, AS, physical mixture of BBR-AS, and BBR-AS eutectic groups were (15.65 ± 2.78)%, (31.24 ± 4.19)%, (32.26 ± 2.84)%, and (45.32 ± 2.53)%, respectively. Compared with the control group, the scratched area of L929 cells in the AS, physical mixture of BBR-AS, and BBR-AS eutectic groups decreased, and the migration rate of L929 cells increased significantly (P < 0.01), and the migration-promoting effect of the BBR-AS eutectic was significantly better than that of the physical mixture of BBR-AS (P < 0.05). The microscopic structure of L929 cell migration in each experimental group is as Figure 9 shown in C.

[0083] In summary, the BBR-AS eutectic of the present invention significantly improves the solubility of AS, realizes the sustained release of BBR, and the eutectic structure is beneficial to the improvement of the bioavailability of BBR and AS. The BBR-AS eutectic has significant inhibitory effects on both Escherichia coli and Staphylococcus aureus, and the antibacterial effect is significantly better than that of the raw material drugs. The BBR-AS eutectic can significantly promote the proliferation and migration of fibroblasts, and the effect is significantly better than that of the BBR and AS raw drugs.

[0084] The above are only the preferred embodiments of the present invention and are not used to limit the patent scope of the present invention. For those skilled in the art, various changes and modifications can be made to the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A berberine - asiaticoside (BBR - AS) cocrystal, characterized in that, Formed from BBR and AS with a molar ratio of 1:

1.

2. Use of the berberine - asiaticoside (BBR - AS) eutectic as described in claim 1 in the preparation of a therapeutic drug for infectious wounds.

3. An antibacterial product, characterized in that, Containing the berberine - asiaticoside eutectic as described in claim 1, for inhibiting Escherichia coli and Staphylococcus aureus.

4. A preparation method of berberine-asiaticoside (BBR-AS) cocrystal according to claim 1, characterized in that, The preparation steps of the pharmaceutical preparation are as follows: (1) Accurately weigh an appropriate amount of BBR and AS and mix them; (2) Dissolve the mixture obtained in (1) in a 95% ethanol solution at 45 °C to form a homogeneous solution; (3) Keep the solution obtained in (2) under constant temperature conditions for 48 hours, and during this period, gradually precipitate crystals by slowly lowering the temperature.

5. The preparation method of the pharmaceutical preparation according to claim 4, characterized in that, The mixing ratio of BBR and AS in (1) is: mixing in a molar ratio of 1:

1.

6. A pharmaceutical composition, characterized in that, Containing the berberine - asiaticoside eutectic as described in claim 1 and pharmaceutical excipients.

7. Use of the pharmaceutical composition as described in claim 6 in the preparation of a therapeutic drug for infectious wounds.

8. The pharmaceutical composition according to claim 6, wherein It accelerates wound healing by promoting fibroblast proliferation and migration.

9. An in vitro antibacterial product, characterized in that, Includes an effective amount of the pharmaceutical composition as described in claims 6 - 8.

10. A wound repair product, characterized in that, Includes an effective amount of the pharmaceutical composition as described in claims 6 - 8.

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