Berberine-asiaticoside co-crystal (BBR-AS) for the treatment of infected wounds and a method for its preparation

Abstract

The application discloses a berberine-asiaticoside (BBR-AS) co-crystal as well as a preparation method and application thereof, and belongs to the field of biological medicines. The co-crystal is prepared by a cooling crystallization method with BBR and AS in a 1:1 molar ratio, and the crystal structure is confirmed by X-ray diffraction (XRD) and infrared spectroscopy (FTIR). The co-crystal significantly improves the solubility of asiaticoside and the sustained-release performance of berberine, and in-vitro experiments prove that the co-crystal has a strong inhibitory effect on escherichia coli and staphylococcus aureus, and can promote the proliferation and migration of fibroblasts, and is suitable for the synergistic treatment of infectious wounds.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to a berberine-asiaticoside co-crystal (BBR-AS) for the treatment of infectious wounds and a preparation method thereof. BACKGROUND

[0002] Skin is the largest tissue organ of the human body. Once its normal structure and function are damaged, a wound will occur. Among the many situations leading to the occurrence of wounds, bacterial-induced wound infection is particularly troublesome. It not only makes the wound healing process slow, but also causes the patient to suffer from wound pain, and even causes symptoms such as suppuration. At present, in terms of the treatment of infectious wounds, in addition to the conventional debridement treatment and anti-infection measures, effective treatment methods are relatively scarce. Traditional Chinese medicine external treatment has shown unique advantages in the clinical treatment of infectious wounds. Traditional Chinese medicine believes that "fire toxin" is the key pathogenesis that makes infectious wounds difficult to heal. Based on this, through syndrome differentiation and treatment, the appropriate use of traditional Chinese medicine for fumigation and washing can achieve the effects of clearing heat and resolving toxin, removing necrotic tissue and promoting tissue regeneration, and can achieve the dual effects of antibiosis and promoting wound healing in clinical treatment.

[0003] Berberine (BBR) is a biologically active alkaloid in traditional Chinese medicine Coptis chinensis Franch, which can effectively inhibit the growth and reproduction of common wound infection pathogens such as Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. It plays an antibacterial effect through multiple pathways such as destroying the bacterial cell membrane structure and inhibiting bacterial protein synthesis, thereby reducing the number of bacteria on the wound and reducing the further damage of inflammatory reactions to the wound tissue. Asiaticoside (AS) is a pentacyclic triterpenoid compound derived from Centella asiatica (L.) Urban of the Apiaceae family. It can reduce the damage of excessive inflammation to the wound tissue by regulating the inflammatory response, create favorable conditions for repair. At the same time, it can stimulate the proliferation of fibroblasts and the synthesis of collagen, promote the growth of granulation tissue, accelerate the process of epithelialization, and help improve the microenvironment of the wound and reduce scar formation, thereby promoting the repair and healing of the wound in multiple dimensions when the wound is infected. However, the solubility of BBR and AS is not ideal, which limits their transmembrane absorption and bioavailability.

[0004] Drug co-crystal technology is a technology for improving the physicochemical properties and bioavailability of drugs by combining drug molecules with suitable ligands to form co-crystal structures. BBR and AS can self-assemble to form co-crystals. The hydroxyl and nitrogen atoms in the BBR molecule can act as hydrogen bond donors or acceptors, while the hydroxyl and carbonyl groups in the AS molecule can form a hydrogen bond network with berberine to optimize the physicochemical properties of both through intermolecular hydrogen bonding. The BBR-AS co-crystal form can significantly improve the solubility and dissolution rate of both under the premise of not changing the chemical structure of the drug, thereby enhancing its absorption efficiency and improving its bioavailability. In addition, the co-crystal technology can also improve the stability and synergistic effect of the drug, further optimizing the therapeutic effect of the drug.

[0005] Infection wound treatment needs to consider both antibacterial and healing promotion, but existing drugs such as berberine (BBR) have low solubility, leading to insufficient bioavailability, and asiaticoside (AS) alone has limited repair-promoting effect. Drug co-crystal technology can improve drug performance through intermolecular interactions, but there is no preparation of BBR-AS co-crystals and their application in infected wounds. SUMMARY

[0006] In view of the deficiencies in the prior art, the purpose of the present application is to provide a berberine-asiaticoside co-crystal (BBR-AS) for the treatment of infected wounds and a preparation method thereof. By optimizing the co-crystal preparation process, the defects of single drugs are solved, and the synergistic enhancement of antibacterial and healing promotion is achieved.

[0007] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions.

[0008] The present application discloses a berberine-asiaticoside (BBR-AS) co-crystal, characterized in that it is formed by BBR and AS in a molar ratio of 1:1.

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

[0010] The present application also discloses a bacteriostatic product, characterized in that it contains the above-mentioned berberine-asiaticoside co-crystal and is used for inhibiting Escherichia coli and Staphylococcus aureus.

[0011] The present application also discloses a preparation method of the above-mentioned berberine-asiaticoside (BBR-AS) co-crystal, characterized in that the preparation steps of the drug preparation are as follows:

[0012] (1) accurately weigh an appropriate amount of BBR and AS and mix them;

[0013] (2) dissolve the mixture obtained in (1) in a 95% ethanol solution at 45°C to form a uniform solution;

[0014] (3) keeping the solution obtained in (2) under constant temperature conditions for 48 hours, during which the crystals are gradually precipitated by slow cooling.

[0015] Further, the BBR and AS in (1) are mixed at a mass ratio of 1:1.

[0016] The application further discloses a pharmaceutical composition, characterized by comprising the berberine-asiaticoside co-crystal and pharmaceutical excipients.

[0017] Further, the pharmaceutical composition is applied to the preparation of an infectious wound treatment drug.

[0018] Further, the application accelerates wound healing by promoting the proliferation and migration of fibroblasts.

[0019] The application further discloses an in-vitro antibacterial product, characterized by comprising an effective amount of the pharmaceutical composition.

[0020] The application further discloses a wound repair product, characterized by comprising an effective amount of the pharmaceutical composition.

[0021] Compared with the prior art, the application has the following beneficial effects.

[0022] The application evaluates the antibacterial effect of the BBR-AS co-crystal in the treatment of infectious wounds through in-vitro antibacterial experiments and investigates the influence of the BBR-AS co-crystal on the proliferation and migration of fibroblasts through a cell scratch experiment. The experimental results show that the BBR-AS co-crystal is significantly superior to the raw drug and the physical mixture thereof in antibacterial effect, and shows strong antibacterial activity. The co-crystal structure significantly improves the solubility of the raw drug, especially the solubility of AS, thereby enhancing the bioavailability of the drug. In addition, the BBR-AS co-crystal shows good effect in promoting the proliferation and migration of fibroblasts, which is helpful for wound repair. These findings show that the BBR-AS co-crystal has broad application prospects in the treatment of infectious wounds. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The molecular docking results.

[0024] Figure 2 The SEM scanning diagrams of BBR, AS and BBR-AS co-crystal. 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 co-crystal; and F is the BBR-AS co-crystal.

[0025] Figure 3DSC scan graph of BBR, AS and BBR-AS co-crystal.

[0026] Figure 4 TIR scan graph of BBR-AS co-crystal.

[0027] Figure 5 XRD scan graph of BBR, AS, BBR-AS co-crystal.

[0028] Figure 6 Solubility curve of BBR, AS drug substance and BBR-AS co-crystal in water. A is the solubility curve of AS and BBR-AS co-crystal; B is the solubility curve of BBR and BBR-AS co-crystal.

[0029] Figure 7 Inhibition effect of BBR-AS co-crystal and drug substance on E. coli. a is the control group; b is the BBR-AS physical mixture group; c is the BBR-AS co-crystal group; d is the BBR group; e is the AS group.

[0030] Figure 8 Inhibition effect of BBR-AS co-crystal and drug substance on S. aureus. a is the control group; b is the BBR-AS physical mixture group; c is the BBR-AS co-crystal group; d is the BBR group; e is the AS group.

[0031] Figure 9 Effect 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 micrograph of L929 cell migration. DETAILED DESCRIPTION

[0032] The application will be described in further detail below with specific examples. However, it should not be understood that the scope of the above-mentioned subject matter of the application is limited to the following examples, and any technology realized based on the content of the application falls within the scope of the application.

[0033] Unless otherwise specified, the reagents and materials used in the application are commercially available.

[0034] EXAMPLE

[0035] I. Materials and methods

[0036] 1. Experimental materials

[0037] 1.1 Experimental materials

[0038] Table 1 Drug and reagent names

[0039] .

[0040] 1.2 Experimental instruments

[0041] Table 2 Main instruments and consumables

[0042] .

[0043] II. Experimental methods

[0044] 2.1 Molecular docking

[0045] First, the three-dimensional structure files of BBR and AS small molecules were determined, and then the hydrogen atoms were added, protonated / deprotonated, and other treatments were performed. Finally, the DOCK molecular docking software was used for docking. In the docking process, BBR was used as the "receptor" and AS was used as the "ligand". By defining the docking pocket, the interaction and binding mode of the two molecules were simulated. Finally, according to the docking score and the generated complex conformation, the possibility of forming a co-crystal and the binding stability of the two small molecules were evaluated.

[0046] 2.2 Preparation of BBR-AS co-crystal

[0047] The cooling crystallization method was used to prepare BBR-AS co-crystal (Wang Y Y, Yuan P H, Yang D Z, et al. Design and preparation methods of drug-drug co-crystals and research progress in application[J]. Medical Journal of National Defense, 2023, 42(07):977-983.). An appropriate amount of BBR and AS was accurately weighed and mixed in a 1:1 molar ratio. Then, the mixture was dissolved in a 95% ethanol solution at 45°C to form a uniform solution. The solution was kept at a constant temperature (45°C) for 48 h, and the crystals were gradually precipitated by slowly reducing the temperature (5°C per hour, until the temperature dropped to 25°C). After the reaction was completed, the precipitated crystals were separated from the mother liquor by filtration, and the crystals were dried. The BBR-AS co-crystal was collected and its structure was detected by transmission electron microscopy (SEM), X-ray single crystal diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR).

[0048] 2.3 Solubility determination

[0049] An excess amount of BBR, AS raw material, and BBR-AS co-crystal was weighed and placed in a dissolution cup containing 0.005% Tween-80 in water solution. The instrument speed was set to 250 r / min, and the temperature was set to (37.0±0.5) °C. The timing started when the drug came into contact with the dissolution medium. Samples were taken at 6, 12, 18, 24, 30, 45, 60, 120, and 240 min, 150 μL of each (while supplementing the same temperature and equal amount of dissolution medium), filtered, and 20 μL of the filtrate was taken for HPLC determination.

[0050] Chromatographic conditions of BBR: Waters C18 column (4.6 mm x 250 mm, 5 μm), acetonitrile-0.05 mol / L potassium dihydrogen phosphate solution (50:50) (0.4 g of sodium dodecyl sulfate was added to 100 mL, and the pH value was adjusted to 4.0 with phosphoric acid) as the mobile phase; the detection wavelength was 345 nm. The column temperature was 37 °C; the flow rate was 1 mL / min; the injection volume was 20 μL.

[0051] Chromatographic conditions of AS: Waters C18 column (4.6 mm x 250 mm, 5 μm), acetonitrile-2 mmol / L betacyclodextrin solution (24:76) as the mobile phase; the detection wavelength was 205 nm, the column temperature was 30 °C; the flow rate was 1 mL / min; the injection volume was 20 μL.

[0052] 2.4 Anti-bacterial experimental study of BBR-AS eutectic.

[0053] (1) Preparation of culture medium.

[0054] Prepare LB broth medium and LB agar medium according to the requirements of the culture medium instruction, adjust the pH value to 7.2±0.1, and sterilize by high pressure; prepare MH agar medium according to the requirements of the instruction, then sub-pack and sterilize by high pressure, and reserve for use.

[0055] (2) Preparation of bacterial suspension.

[0056] Take the stored Staphylococcus aureus and Escherichia coli and inoculate them on LB solid medium, and cultivate them at 37 °C for 24 h. Take 3-5 colonies and inoculate them in LB liquid medium, and cultivate them at 37 °C in a constant temperature incubator for 24 h, and then count.

[0057] (3) In vitro antibacterial experiment.

[0058] Pour the high-temperature sterilized solid medium into a six-well plate and cool it, and 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, and set up three parallels for each group.

[0059] Add 100 μL of purified Staphylococcus aureus and Escherichia coli bacterial suspension to the BBR group, the AS group, the BBR-AS group, the blank group, and the positive control group.

[0060] After 18 h of cultivation in a 37 °C constant temperature incubator, take pictures of the bacterial colonies on the plate and count them.

[0061] (4) Observation of bacterial morphology,

[0062] The morphology of bacteria after different drug treatments was observed by SEM. The bacterial suspension of different treatment groups was centrifuged (5000 rpm) at 25°C for 20 min and the bacteria at the bottom were collected. 4% paraformaldehyde was added and the bacteria were fixed overnight at 4°C. The paraformaldehyde was aspirated and the fixed samples were washed with PBS multiple times, and then gradient dehydrated with different concentrations of ethanol solution (20%, 40%, 60%, 80% and 100%) for 10 min for each gradient. Finally, 10 μL of the obtained bacterial sample was dropped on a silicon wafer and naturally dried in air, and then observed by SEM after gold spraying.

[0063] 2.5 Effects of BBR-AS on fibroblast proliferation and migration.

[0064] (1) Cell culture.

[0065] L929 mouse fibroblasts were thawed and recovered, and then placed in a DMEM medium containing 10% inactivated fetal bovine serum (FBS) + 90% high-sugar, and cultured in a 37°C, 5% CO2 incubator. The culture solution was replaced every 2-3 days for subculture.

[0066] (2) Cell scratch test.

[0067] L929 cells in good growth state were inoculated in a 6-well plate at a density of 1×10 6 cells / well, and placed in a cell culture incubator for overnight culture. After the cells adhered, the density was about 90%, a sterile 200 mL gun head was used for scratching, and the cells were washed with sterile PBS for 3 times. The test group was added with a DMEM medium containing 2% FBS, 1% fetal bovine serum and 0.1 μg / mL of BBR, AS and BBR-AS solution, and the control group was added with a DMEM medium containing 2% FBS and 1% fetal bovine serum. They were placed in a cell culture incubator for continuous culture. At 0 h and 24 h, observation and photography were performed, the scratch area was quantified using ImageJ software, and the cell migration rate was calculated according to the following formula: migration rate (%) = (0 h scratch area-24 h scratch area) / 0 h scratch area×100%.

[0068] III. Experimental results.

[0069] 3.1 Molecular docking results: The molecular docking simulation showed that the combination of berberine and asiaticoside was mainly dominated by hydrophobic interaction: the benzene ring structure of berberine and the pentacyclic triterpenoid skeleton of asiaticoside were closely arranged by van der Waals force to form a continuous hydrophobic structure, and no typical hydrogen bond donor-acceptor pairing mode was observed around the action site. The binding free energy of the two was -5.0±0.3 kcal / mol, which was significantly better than the binding energy level of most physical mixtures, indicating that berberine and asiaticoside formed a thermodynamically stable supramolecular system through an entropy-driven effect.Figure 1 ).

[0070] 3.2 Characterization results of BBR-AS eutectic.

[0071] 3.2.1 Characterization by BBR-AS eutectic SEM.

[0072] The morphology of the co-crystal of berberine (BBR) and asiaticoside (AS) was characterized by scanning electron microscopy (SEM). The results showed that the crystal structure of the co-crystal differed significantly from that of the single drug. SEM images of berberine (BBR) showed an irregular blocky or plate-like crystal structure with a rough surface and blurred edges. The crystal size distribution was wide, and some crystal surfaces showed obvious cracks and pores, indicating a relatively loose crystal structure. Figure 2 As shown in B. The SEM image of asiaticoside (AS) alone shows a plate-like or needle-like crystal structure with a relatively smooth surface and clear edges. The crystal size is relatively uniform, and some crystals exhibit regular geometric shapes, such as rhombuses or elongated shapes, as shown in Figure B. Figure 2 As shown in D.

[0073] SEM images of the BBR-AS eutectic showed that its crystal morphology was more uniform, with a smooth surface and dense structure, and a uniform crystal size distribution. No obvious drug phase separation was observed. Figure 2 As shown in E and 2F, the morphology of the co-crystal differs from both the irregular blocky structure of BBR and the plate-like or needle-like structure of AS, exhibiting a novel, regular crystal morphology. This regular crystal structure may be due to a new crystal arrangement formed between BBR and AS molecules through non-covalent interactions such as hydrogen bonding and π-π stacking. Furthermore, no cracks or pores commonly found in single drug crystals were observed in the SEM images of the BBR-AS co-crystal, indicating that the co-crystal structure is more stable and dense. This dense structure may contribute to improved drug stability and solubility, providing an advantage for its application in drug delivery systems.

[0074] 3.2.2 Characterization of BBR-AS eutectic DSC.

[0075] The thermodynamic properties of berberine (BBR), asiaticoside (AS) and their co-crystal (BBR-AS) were characterized by differential scanning calorimetry (DSC). The results showed that single BBR and AS exhibited sharp and symmetrical endothermic peaks at 180.88 °C and 239.81 °C, respectively, indicating that both of them have high crystallinity and thermal stability, and high purity. In contrast, the DSC curve of BBR-AS co-crystal 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 indicates that BBR and AS form a new crystal phase through intermolecular interactions (such as hydrogen bonding or π-π stacking), rather than a simple physical mixture. In addition, the melting peaks of single drugs were not observed in the DSC curve of the co-crystal, further confirming the successful formation of the co-crystal, as shown in Figure 3 This new thermodynamic behavior indicates that BBR-AS co-crystal has higher thermal stability, providing important thermodynamic basis for its application in drug delivery systems. In summary, the DSC characterization results not only reveal the melting behavior and thermal stability characteristics of BBR-AS co-crystal, but also confirm the formation of the co-crystal from a thermodynamic perspective.

[0076] 3.2.3 Characterization of BBR-AS co-crystal by TIR.

[0077] The molecular vibration characteristics of berberine (BBR), asiaticoside (AS) and their co-crystal (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 of functional groups such as hydroxyl, aromatic ring, carbonyl, ether bond, etc. in their molecules and their vibration modes. The main absorption peaks of BBR were concentrated near 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 near 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 co-crystal showed new characteristic absorption peaks, and some characteristic peaks of single drugs were shifted or changed in intensity, for example, the shift of O-H stretching vibration peak (near 3400 cm⁻¹) indicates that BBR and AS form new interactions through hydrogen bonding, while the changes in C=O and C=C stretching vibration peaks reflect the π-π stacking or other non-covalent interactions between aromatic rings and carbonyl groups, as shown in Figure 4These changes indicate that BBR and AS form a new chemical structure in the cocrystal, 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 cocrystal. In summary, the TIR characterization results not only reveal the molecular vibration characteristics of the BBR-AS cocrystal, but also confirm the successful formation of the cocrystal from a chemical structure perspective.

[0078] 3.2.4 Characterization of BBR-AS cocrystal by XRD.

[0079] The crystal structure of the berberine (BBR) and asiaticoside (AS) cocrystal was characterized by X-ray diffraction (XRD), and the results showed that the diffraction pattern of the cocrystal was significantly different from the crystal structure of single drugs, indicating that BBR and AS form a new crystal phase in the cocrystal. The XRD pattern of single berberine (BBR) showed typical crystal diffraction peaks in the 2θ angle range, with main peak positions at 8.5°, 12.3°, 15.7°, 17.2°, 20.5°, 22.8°, and 25.4°, etc. These sharp and high-intensity 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, with main peak positions at 9.2°, 13.5°, 16.8°, 18.4°, 21.2°, 23.6°, and 26.8°, etc. Similarly, it indicates that AS has high crystallinity, as shown in Figure 5

[0080] The XRD pattern of the BBR-AS cocrystal showed some new diffraction peaks, while some characteristic diffraction peaks of single drugs disappeared or shifted. The diffraction peaks of the cocrystal can appear at new 2θ angle positions (such as 10.5°, 14.2°, 19.1°, 24.3°, etc.), which indicates that BBR and AS molecules form a new crystal arrangement in the cocrystal. In addition, the diffraction peak intensity distribution and peak shape of the cocrystal are also different compared to single drugs, reflecting the influence of intermolecular interactions on the crystal structure. No obvious amorphous diffuse peak was observed in the XRD pattern, further confirming the high purity and crystallinity of the BBR-AS cocrystal. These results indicate that BBR and AS in the cocrystal are not a simple physical mixture, but form a new crystal structure through intermolecular interactions.

[0081] 3.3 Solubility test results of raw materials and cocrystal.

[0082] The solubility results of BBR and AS raw materials and BBR-AS cocrystal in water at 37°C are shown in Figure 5 ​As shown, the solubility rate of AS active pharmaceutical ingredient (API) is slow, with a maximum apparent solubility of only (4.10 ± 0.23) μg / mL. However, the solubility rate of AS in the prepared BBR-AS cocrystal is significantly higher than that of the API, reaching a maximum apparent solubility of (20.62 ± 0.35) μg / mL at 2 hours, which is 5 times that of the AS API. This indicates that cocrystalization can significantly improve the solubility of AS. Compared to the AS API, the maximum apparent solubility of the BBR API is somewhat reduced. Figure 6 It is evident that the maximum apparent solubility of BBR in the eutectic is reduced to 0.2 mg / mL, which may lead to a more sustained release of BBR, thereby improving bioavailability and presenting a potential advantage.

[0083] 3.4 Antibacterial properties of BBR-AS eutectic.

[0084] BBR-AS inhibits the growth of Escherichia coli.

[0085] This experiment used Escherichia coli and Staphylococcus aureus as representative bacteria to investigate the in vitro antibacterial efficacy of BBR-AS cocrystals. Figure 7 As shown, in the anti-E. coli experiment, E. coli proliferated extensively in the control group. Compared with the control group, the colony counts in the BBR group, AS group, and the physical mixture of the two were reduced, indicating that the raw material drug has a certain inhibitory effect on E. coli. The colony counts in the BBR-AS cocrystal group and the positive group were significantly reduced 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 cocrystal on E. coli is significantly better than that of the raw material drug.

[0086] Depend on Figure 8 As shown, in the anti-Staphylococcus experiment, Staphylococcus aureus multiplied significantly in the control group. Compared with the control group, BBR raw material and the BBR-AS physical mixture showed a certain inhibitory effect on Staphylococcus aureus, while AS had no significant inhibitory effect on Staphylococcus aureus. Compared with the control group, the BBR-AS co-crystal group and the positive control group showed a significant reduction in colony count, demonstrating a significant inhibitory effect, which was superior to the raw material group and the physical mixture group.

[0087] 3.5 Effects of BBR-AS on fibroblast proliferation and migration.

[0088] Figure 9As shown in A, after 0.1 μg / mL of drugs treated L929 cells for 24 h, the cell viability of BBR, AS, BBR-AS physical mixture and BBR-AS co-crystal group was (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, BBR-AS co-crystal can significantly promote the proliferation of mouse L929 fibroblasts, and the proliferation-promoting effect is significantly better than that of BBR-AS physical mixture (P < 0.05). Figure 9 As shown in B, after 24 h of drug treatment of L929 cells, the migration rate of L929 cells in BBR, AS, BBR-AS physical mixture and BBR-AS co-crystal groups was (15.65 ± 2.78) %, (31.24 ± 4.19) %, (32.26 ± 2.84) % and (45.32 ± 2.53) % respectively. Compared with the control group, the area of the scratch region of L929 cells in AS, BBR-AS physical mixture and BBR-AS co-crystal groups was reduced, and the migration rate of L929 cells was significantly increased (P < 0.01), and the migration-promoting effect of BBR-AS co-crystal was significantly better than that of BBR-AS physical mixture (P < 0.05). The microstructure of L929 cell migration in each experimental group is shown in Figure 9 C.

[0089] In summary, the BBR-AS co-crystal of the application significantly improves the solubility of AS, realizes the sustained release of BBR, and the co-crystal structure is beneficial to the improvement of the bioavailability of BBR and AS. BBR-AS co-crystal has a significant inhibitory effect on E. coli and Staphylococcus aureus, and the antibacterial effect is significantly better than that of the raw drug. BBR-AS co-crystal can significantly promote the proliferation and migration of fibroblasts, and the effect is significantly better than that of BBR and AS raw drugs.

[0090] The above only describes the preferred embodiments of the application and is not used to limit the patent scope of the application. For those skilled in the art, the application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the application shall be included in the protection scope of the application.

Claims

1. A berberine-asiaticoside (BBR-AS) cocrystal, characterized in that, The eutectic is formed by BBR and AS in a molar ratio of 1:

1. The eutectic is formed by berberine and asiaticoside linked by hydrogen bonds and has an X-ray powder diffraction pattern that is different from that of a single component. The diffraction pattern of the eutectic has characteristic peaks at 2θ angle positions of 10.5°, 14.2°, 19.1° and 24.3°.

2. The use of the berberine-asiaticoside (BBR-AS) cocrystal as described in claim 1 in the preparation of a drug for treating infected wounds.

3. An antibacterial product, characterized in that, The product contains the berberine-asiaticoside co-crystal of claim 1, which is used to inhibit Escherichia coli and Staphylococcus aureus.

4. A method for preparing the berberine-asiaticoside (BBR-AS) eutectic according to claim 1, characterized in that, The preparation steps of the eutectic 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 homogeneous solution; (3) Keep the solution obtained in (2) at a constant temperature for 48 hours, and gradually precipitate the crystals by slowly cooling it during this period.

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

1.

6. A pharmaceutical composition, characterized in that, It comprises the berberine-asiaticoside cocrystal as described in claim 1 and pharmaceutical excipients.

7. The pharmaceutical composition according to claim 6, characterized in that, It accelerates wound healing by promoting fibroblast proliferation and migration.

8. The use of the pharmaceutical composition of claim 6 in the preparation of a treatment for infected wounds.

9. An in vitro antibacterial product, characterized in that, The effective amount of the pharmaceutical composition according to any one of claims 6-7.

10. A wound repair product, characterized in that, The effective amount of the pharmaceutical composition according to any one of claims 6-7.

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