Discovery of a natural baicalin-zinc ion supramolecular hydrogel and its application as an antibacterial and antioxidant drug

Abstract

The application provides a structure of a carrier-free supramolecular hydrogel formed by self-assembly of baicalin and metal zinc ions, preparation and application. The supramolecular hydrogel can form a gel without adding accessories, has excellent gel properties such as injectability, heat sensitivity and stability, and can be applied to the clinic as an ideal wound dressing. The supramolecular hydrogel also has selective bacteriostatic ability, can selectively kill pathogenic bacteria such as Staphylococcus aureus, and does not affect the activity of probiotic bacteria such as Bacillus subtilis and Enterococcus faecium. In addition, the supramolecular hydrogel also has good antioxidant effect. Through a large number of tests, it is found that when baicalin reacts with many metal ions, only zinc ions can self-assemble to form a hydrogel, and reactions with other metal ions are all precipitates. The structure of the baicalin-zinc supramolecular hydrogel is shown in the following formula:

Description

TECHNICAL FIELD

[0001] The application is a preparation method of baicalin-zinc supramolecular hydrogel and its antibacterial application, especially good inhibitory activity to Staphylococcus aureus in clinic; it has self-assembly performance and can be prepared into carrier-free hydrogel drugs, belonging to the field of medicinal chemistry. BACKGROUND

[0002] The coordination chemistry theory of traditional Chinese medicine believes that the complexes formed by organic components and trace elements in traditional Chinese medicine are one of the effective substance bases of traditional Chinese medicine, and the existing form is closely related to biological activity. Coordination chemistry mainly studies the compounds formed by coordination bonds between metal atoms or ions and molecules or ions containing lone pair electrons. The coordination assembly between metal ions and organic ligands has dual advantages of inorganic and organic compounds, and has unique advantages in drug delivery, catalysis, nuclear magnetic resonance imaging, etc., which has attracted widespread attention from scholars all over the world.

[0003] Flavonoids are a kind of natural polyphenols, and their structures are mostly 3-hydroxy-4-carbonyl and 5-hydroxy-4-carbonyl, which are easy to complex with metal ions. The research on the complexes formed by natural flavonoids organic small molecules in traditional Chinese medicine and metal ions has been widely reported, for example, luteolin reacts with metal iron ions, calcium ions, magnesium ions, zinc ions and copper ions to form precipitates, and chrysin, apigenin and genistein also react with copper ions to form precipitates. Although there have been a large number of reports on the reaction of flavonoids and metal ions to form precipitates, there are few reports on the research on the formation of hydrogel by flavonoids and metal ions. Therefore, the present application has great innovation and can enrich the field of metal-organic small molecule complex carrier-free hydrogel research.

[0004] Staphylococcus aureus can be detected in the environment, widely exists in nature, can parasitize in human skin, oropharynx, gastrointestinal tract and other parts, causes various infections, including wound infection, bloodstream infection, pneumonia and osteomyelitis, etc. It is the main pathogenic bacteria causing community and hospital infection. With the wide use of antibacterial drugs, the detection rate of multi-drug resistant bacteria is increasing, especially the multi-drug resistant Staphylococcus aureus has remained at a certain level, which can easily cause hospital infection epidemic or outbreak, and is difficult to treat with high mortality, which has become a big problem in the prevention and control of hospital infection. For many years, antibiotics have played a huge role in promoting the development of medical, food and animal husbandry fields, especially for the human health and the improvement of livestock production. However, the overuse and improper use of antibiotics have also brought various problems, such as the emergence of drug-resistant bacteria or even "super bacteria", or long-term and disordered application leading to antibiotic residues in livestock products, thereby endangering human health. In the literature, various research reports that the improper use of antibiotics leads to the emergence of new drug resistance mechanisms in bacteria, which leads to global spread. The number of multi-drug resistant bacteria is growing at an alarming rate, that is, bacteria have developed resistance to all known natural and synthetic antibiotics, so new treatment alternatives are urgently needed. Therefore, finding efficient and low-toxicity, environmentally friendly antibacterial drugs is one of the research hotspots for scientists at home and abroad.

[0005] Compared with modern anti-infective drugs, traditional Chinese medicines have wide sources, complex components, multiple action targets and are not easy to produce drug resistance, which are important sources for developing new anti-infective drugs. At the same time, the research and development of antibacterial hydrogels are a research hotspot in recent years. However, the previously reported hydrogels are mainly based on high molecular polymers, and the reports on self-assembly of natural active small molecules to form hydrogels are less. Based on this, the present application is based on the theory of coordination chemistry of traditional Chinese medicine, and a natural antibacterial carrier-free supramolecular hydrogel is successfully developed through specific self-assembly of baicalin-zinc. SUMMARY

[0006] On the basis of the theory of coordination chemistry of traditional Chinese medicine, the inventors innovatively found that baicalin and zinc ions can directly form a hydrogel without the aid of auxiliary ingredients, and compared with the monomers of the two, the formed supramolecular hydrogel has better antibacterial activity and antioxidant performance. The present application has great research significance for discovering and developing natural hydrogel drugs with clear structure from natural traditional Chinese medicines. One of the purposes of the present application is to provide a baicalin-zinc supramolecular hydrogel.

[0007] The second purpose of the present application is to provide structure information of the baicalin-zinc supramolecular hydrogel. Specifically, the complexing site is a glucuronic acid carboxyl group of baicalin.

[0008] The third purpose of the present application is to provide a preparation method of the baicalin-zinc supramolecular hydrogel.

[0009] The fourth object of the present application is to provide the excellent gel properties of baicalin-zinc supramolecular hydrogel.

[0010] The fifth object of the present application is to provide the application of baicalin-zinc supramolecular hydrogel in the field of antibacterial.

[0011] The sixth object of the present application is to provide the application of baicalin-zinc supramolecular hydrogel in the field of antioxidant.

[0012] The seventh object of the present application is to provide the application of baicalin-zinc supramolecular hydrogel in the field of promoting the healing of bacterial infection wound.

[0013] To achieve the object of the present application, the following technical solutions are adopted:

[0014] 1. The preparation process of baicalin-zinc supramolecular hydrogel compound is determined by exploring different pH values, heating temperatures, molar ratio of raw materials, and stirring speed. Nuclear magnetic resonance and mass spectrometry are used to determine the structure information.

[0015] (1) Baicalin is suspended in water, heated, and the pH is adjusted to obtain a clear and transparent solution.

[0016] (2) Metal zinc ions are dissolved in water.

[0017] (3) The prepared baicalin aqueous solution and zinc ion solution in steps (1) and (2) are mixed and heated and stirred.

[0018] Preferably, the molar ratio of baicalin and zinc ion solution is 1:0.1 to 1:10, for example, 1:0.1, 1:0.5, 1:1, 1:2, 1:5, and 1:10.

[0019] Preferably, the water in steps (1) and (2) is deionized water.

[0020] Preferably, the pH adjusting agent in step (1) is an organic or inorganic base, such as sodium hydroxide, sodium carbonate, sodium bicarbonate, and ammonia.

[0021] Preferably, the pH in step (1) is 6-10, for example, pH=6, pH=7, pH=8, pH=9, and pH=10.

[0022] Preferably, the zinc ion in step (2) is an inorganic salt, such as zinc chloride and zinc sulfate.

[0023] Preferably, the heating temperature in step (3) is 25-100℃, for example, 25℃, 30℃, 50℃, 80℃, and 100℃.

[0024] Preferably, the stirring speed in step (3) is 0-1500rpm, for example, 100rpm,

[0025] 200 rpm, 300 rpm, 400 rpm, 500 rpm, 600 rpm, 700 rpm, 800 rpm,

[0026] 900 rpm, 1000 rpm, or 1500 rpm.

[0027] 2. Rheological study of baicalin-zinc supramolecular hydrogel; the main research steps are as follows:

[0028] The material characterization of baicalin-zinc supramolecular hydrogel is carried out by frequency scanning and amplitude scanning.

[0029] 3. Evaluate the bacteriostatic effect by determining the MIC value of different strains; observe the influence on the microscopic morphology of Staphylococcus aureus by scanning electron microscopy; evaluate the bactericidal effect on Staphylococcus aureus by live and dead bacteria staining.

[0030] The evaluation of the antibacterial and antioxidant effects of baicalin-zinc supramolecular hydrogel includes the following steps:

[0031] (1) The MIC values of baicalin-zinc supramolecular hydrogel on Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Enterococcus faecium, and multi-drug resistant Staphylococcus aureus are detected by broth dilution method.

[0032] (2) The morphological influence of 1 / 3 MIC drug concentration on Staphylococcus aureus is observed by scanning electron microscopy.

[0033] (3) The bactericidal effect of 300 μM drug concentration on Staphylococcus aureus is observed by live and dead bacteria staining.

[0034] 4. Evaluate the antioxidant activity by DPPH scavenging test and ROS staining. Evaluate the safety by cell safety experiment and in vitro hemolysis experiment.

[0035] The evaluation of the antibacterial and antioxidant effects of baicalin-zinc supramolecular hydrogel includes the following steps:

[0036] (1) Evaluate the antioxidant activity by DPPH scavenging test and ROS staining.

[0037] (2) Evaluate the cell survival rate of human immortalized epidermal cells (Hacat) and human umbilical vein endothelial cells (Huvec) after 24 hours of drug addition by MTT method, and evaluate the cytotoxicity of the drug.

[0038] (3) Incubate baicalin-zinc supramolecular hydrogel with rat red blood cells, measure the hemolysis rate of each drug administration group at 570 nm by enzyme marker, and evaluate the hemolytic property of the drug BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 The baicalin-zinc supramolecular hydrogel prepared in Example 1 of this invention.

[0040] Figure 2 This is the precipitate of baicalin-other metal ion complex prepared in Example 1 of the present invention.

[0041] Figure 3 The rheological data are for the baicalin-zinc supramolecular hydrogel obtained in Example 4 of this invention.

[0042] Figure 4 This diagram illustrates the effect of the baicalin-zinc supramolecular hydrogel obtained in Example 6 of this invention on the microstructure of Staphylococcus aureus.

[0043] Figure 5 The results show the bactericidal activity of the baicalin-zinc supramolecular hydrogel obtained in Example 7 of this invention against Staphylococcus aureus.

[0044] Figure 6 The staining results of the baicalin-zinc supramolecular hydrogel obtained in Example 9 of this invention on cell safety (live and dead cells).

[0045] Figure 7 This is the result of the baicalin-zinc supramolecular hydrogel obtained in Example 10 of the present invention promoting the healing of bacterial infection wounds. Detailed Implementation

[0046] The following embodiments are intended to further illustrate the present invention. Those skilled in the art should understand that these embodiments are merely illustrative of the invention and should not be construed as limiting the invention.

[0047] Example 1

[0048] Baicalin was weighed in molar ratios of 1:0.5 to 1:10 and dissolved separately in water with different metal ions (zinc chloride, calcium chloride, silver nitrate, ferric chloride, magnesium chloride, copper chloride, manganese chloride, cadmium chloride, barium chloride, europium chloride, and ferrous chloride). The pH was adjusted to 6-10 with baicalin to obtain a clear, transparent solution. The solutions were then mixed at 60°C and stirred with a magnetic stirrer for 2 hours. The reaction of baicalin with zinc yielded a baicalin-zinc supramolecular hydrogel, while the reactions of other metal ions (zinc, calcium, silver, iron, magnesium, copper, manganese, cadmium, barium, and europium) with baicalin resulted in precipitation. This demonstrates that the formation of hydrogels from different raw materials is unpredictable and involves chance. The technical solution of this invention requires creative effort and is therefore innovative.

[0049] Example 2

[0050] The supermolecular hydrogel obtained in Example 1 was further subjected to mass spectrometry analysis (static spray-HRMS, Waters, USA). The mass spectrometry analysis conditions were as follows: the ion source was set to positive ion detection mode, the capillary voltage was 3.5 kV, the cone hole voltage was 40 V, the ion source temperature was 120 °C, the collision energy was 35 eV, the cone hole gas flow rate was 50 L / h, the desolvation gas flow rate was 800 L / h, and the mass spectrometry collection range was 50-2000. No chromatographic column separation was required before mass spectrometry analysis, and direct sample analysis was performed. The complex molecular ion peak and molecular structure obtained by mass spectrometry analysis are shown in Table 1.

[0051] Table 1 Structure information of baicalin-zinc supermolecular hydrogel prepared in the application

[0052]

[0053] Example 3

[0054] In this example, the supermolecular hydrogel prepared in Example 1 was subjected to nuclear magnetic resonance (Avance III HD400MHz spectrometer, Bruker, America) characterization, and the results were as follows:

[0055] NMR assignment of baicalin-zinc supermolecular hydrogel: 1 H NMR (400 MHz, DMSO-d6): δ (ppm) 12.53 (s, 1H, 5-OH, BA), 8.70 (s, 1H, 6-OH, BA), 8.08 (d, J = 8 Hz, 2H, H-2', 6', BA), 7.55-7.62 (m, 3H, H-3', 4', 5', BA), 7.05 (s, 1H, H-8, BA), 7.01 (s, 1H, H-3, BA), 5.06 (d, J = 8 Hz, 1H, H-1", BA), 3.72 (d, J = 12 Hz, 1H, H-5", BA), 3.23-3.36 (m, 3H, H-2", 3", 4", BA).

[0056] Example 4

[0057] In this example, the baicalin-zinc supermolecular hydrogel prepared in Example 1 was subjected to rheological test, and the method was as follows:

[0058] The baicalin-zinc supramolecular hydrogel was placed on the measurement plate of a rheometer, and the gap was set to 5 mm and the temperature was set to 25 DEG C. Frequency sweep: the constant strain was 0.1%, and the frequency range was 0.1 Hz-10 Hz. Amplitude sweep: the constant frequency was set to 1 Hz, and the strain range was 0.001%-10%, and the storage modulus (G') and loss modulus (G'') were obtained with the change of strain and frequency. Under the change of frequency and shear stress, the storage modulus was greater than the loss modulus, indicating that it had good rheological properties.

[0059] Example 5

[0060] In this example, the antibacterial activity of the complex prepared in Example 1 was determined as follows:

[0061] The turbidimetry method was used to observe the inhibition effect of the complex on pathogenic bacteria Staphylococcus aureus, conditional pathogenic bacteria Escherichia coli, probiotic bacteria Bacillus subtilis and Enterococcus faecium, and multiple drug-resistant Staphylococcus aureus obtained by clinical isolation. Staphylococcus aureus, Escherichia coli, probiotic bacteria Bacillus subtilis and Enterococcus faecium were from the School of Life Sciences of Beijing University of Chinese Medicine. Multiple drug-resistant Staphylococcus aureus was from the Department of Clinical Laboratory of Dongzhimen Hospital of Beijing University of Chinese Medicine (sample number: 19PXTH0119). The complex powder prepared in Example 1 was sealed and stored at 4 DEG C. When used, it was dissolved in LB medium to prepare a 10 mM storage solution (DMSO content 1%).

[0062] Bacterial recovery and preparation of bacterial stock solution: bacteria were dispersed in LB medium and cultured at 37 DEG C and 200 rpm overnight. Then, a bacterial stock solution with a bacterial amount of 2 x 10 6 CFU / mL was prepared with LB medium, and the counting was performed by plate counting method.

[0063] The MIC of the complex was determined by doubling dilution method. In a 48-well plate, 1 mL of complex solution with concentrations of 500, 300, 250, 150 and 125 μmol / L was prepared with LB medium. Then, 20 μL of bacterial stock solution was added, and the mixture was incubated in a constant temperature incubator at 37 DEG C, 5% CO2 and saturated humidity for 16 h. The OD value was determined at 600 nm by using a microplate reader. The test was repeated three times, and the drug concentration at which the bacterial survival rate was greater than 80% was calculated as the minimum inhibitory concentration of the sample. The group without drug and without bacteria was set as a blank control group; the group with bacteria without drug was set as a blank bacteria group. Bacterial survival rate (%) = (absorbance value of sample group - absorbance value of blank group) / (absorbance value of blank bacteria group - absorbance value of blank group) x 100%.

[0064] The specific results are shown in Table 2.

[0065] Table 2: Inhibition effect of the complex prepared in the application on different bacteria

[0066]

[0067] Results show that the antibacterial activity of baicalin-zinc supramolecular hydrogel is significantly enhanced compared to the monomer raw material, and has excellent selective anti-S. aureus activity; at the same time, the inhibitory activity of the intestinal condition pathogenic bacteria E. coli and probiotics B. subtilis and E. faecium is weak, and the clinically isolated multi-drug resistant S. aureus still has excellent sensitivity, which has the value of in-depth research and further clinical development.

[0068] Example 6

[0069] In this example, the effect of the supramolecular hydrogel prepared in Example 1 on the micro-morphology of S. aureus is determined as follows:

[0070] Under 1 / 3 MIC concentration, the supramolecular hydrogel is incubated with bacteria for 6 hours, then centrifuged to collect the bacteria, 2.5% glutaraldehyde is added for fixation for 4 hours, then eluted with gradient ethanol, and finally scanned by scanning electron microscope. The results show that the self-assembly of supramolecular hydrogel can cause the shrinkage or even rupture of bacterial cell wall, while zinc ions and baicalin can only cause slight shrinkage or cannot cause shrinkage.

[0071] Example 7

[0072] In this example, the bactericidal activity of the supramolecular hydrogel prepared in Example 1 on S. aureus is determined as follows:

[0073] The bactericidal effect is evaluated by live and dead bacteria staining method. First, the bacteria are cultured with LB medium for natural growth for 12 hours, then 300 μM supramolecular hydrogel is added and cultured for 6 hours. Then centrifuged, collected bacteria and added live and dead bacteria staining solution to avoid light for 30 minutes, and observed under laser confocal microscope. The results show that the self-assembly of supramolecular hydrogel has good bactericidal effect.

[0074] Example 8

[0075] In this example, the antioxidant activity of the supramolecular hydrogel prepared in Example 1 is determined, and the first-line antibiotics such as penicillin and norfloxacin are selected for comparison, as follows:

[0076] 0.2 mmol / L DPPH solution is prepared with anhydrous ethanol and stored in the dark. Then different concentrations of test samples are prepared with anhydrous ethanol. 2 mL of test sample solution and 2 mL of DPPH solution are added to the same test tube, shaken, and placed in the dark at room temperature for 30 minutes, then the absorbance A at 520 nm is measured. sampleMeanwhile, the absorbance A0 of 2 mL DPPH solution mixed with 2 mL anhydrous ethanol was measured. Finally, the calculation was performed according to the formula:

[0077] DPPH scavenging percentage = (A0-A sample ) / A0*100

[0078] The specific results are shown in Table 3.

[0079] Table 2: DPPH scavenging rate of the supramolecular hydrogel prepared in the application

[0080] Concentration Baicalin-Zinc Supramolecular Hydrogel Baicalin 15 μΜ 90.73±1.06％ 70.83±1.23％ 10 μΜ 80.09±1.36％ 46.83±0.066％ 7.5 μΜ 76.97±0.96％ 23.58±1.33％ 3.75 μΜ 64.31±1.25％ 19.73±2.02％

[0081] The results show that the antioxidant activity of penicillin and norfloxacin is less than 10% at each concentration, while the antioxidant activity of baicalin-zinc supramolecular hydrogel is significantly enhanced compared with the single raw material, which has the value of further research and clinical development.

[0082] Example 9

[0083] In this example, the safety of the supramolecular hydrogel prepared in Example 1 was evaluated, and the first-line antibiotics such as penicillin and norfloxacin were selected for comparison, and the method was as follows:

[0084] After treating Hacat and Huvec cells with samples in the range of 37.5-300 μM for 24 h, 20 μL of MTT mixed solution was added to the cells in the 96-well plate, and incubation was continued for 4 h. Then 150 μL of DMSO was added for dissolution, and the absorbance value was measured at 490 nm. The cell survival rate was calculated according to the following formula:

[0085] Cell survival rate (%) = (OD 给药组 - OD 空白组 ) / (OD 正常组 - OD 空白组 ) x 100%

[0086] The results show that the baicalin-zinc supramolecular hydrogel group still has no cytotoxicity at a drug concentration of 300 μM, while the first-line antibiotics such as penicillin and norfloxacin have shown cytotoxicity at a concentration of more than 100 μM, indicating that the baicalin-zinc supramolecular hydrogel not only has good antibacterial activity, but also has good biological safety.

[0087] Fresh rat blood was used to determine the in vitro hemolytic effect of the samples. First, the red blood cells were collected by centrifugation at 3000 rpm / min for 15 minutes, and washed with normal saline for 3 times. Then, 3 mL of the centrifuged red blood cells were mixed with 11 mL of normal saline for storage and dispersion. The baicalin-zinc hydrogel was diluted with normal saline to the corresponding concentration. Then, 100 μL of the red blood cell stock solution was mixed with 1 mL of the test solution to obtain a 4% red blood cell solution, which was incubated at 37°C for 4 h. Then, the solution was centrifuged at 3000 rpm / min for 15 min, and the absorbance value of the supernatant was determined at 570 nm by a microplate reader. The positive control was deionized water, and the negative control was normal saline. The hemolysis rate was calculated according to the following formula:

[0088] Hemolysis rate (%) = (A 给药组 -A PBS组 ) / (A 去离子水组 -A PBS组 ) x 100%

[0089] The hemolysis test showed that the baicalin-zinc hydrogel had no obvious hemolytic properties, even at a concentration of 500 μM, and the hemolysis rate was still lower than the internationally recognized standard of 5%.

[0090] Example 10

[0091] In this example, the in vivo pharmacodynamic evaluation of the supramolecular hydrogel prepared in Example 1 was carried out as follows:

[0092] 6-8 week old female Balb / c mice weighing 16-18 g were used as test animals. The mice were anesthetized with pentobarbital after the hair on their backs was removed with an electric clipper. After the mice became lethargic and their breathing became deep and slow, the modeling was started. The skin of the depilated area on the back of the mouse was disinfected with a cotton ball dipped in 75% alcohol. After the alcohol completely evaporated and the skin dried, a 8 mm round punch was used to punch and shape the mouse back, and then a pair of scissors was used to cut off the 8 mm diameter circular skin. Then, 100 μL of 2 x 10 6 CFU of Staphylococcus aureus (SA) bacteria solution was inoculated into each mouse wound, and after 1 h of bacterial infection, the mouse bacterial infection model was completed.

[0093] The mice after modeling were randomly divided into 3 groups, namely, a blank group (Control), a model group (SA), and a Baicalin-Zinc Supramolecular Hydrogel treatment group (BA-Zn Gel). The Control was not infected with bacteria, the SA was infected with bacteria but not treated, and the BA-Zn Gel was treated with the Baicalin-Zinc Supramolecular Hydrogel after being infected with the SA bacteria. Five mice (n=5) were set as parallel controls in each group, and the treatment cycle was 12 days. The wound surface size was recorded every 3 days, and the wound healing rate was calculated. It was found that the Baicalin-Zinc Supramolecular Hydrogel could effectively promote wound healing.

[0094] After the 12-day treatment cycle ended, the mice were sacrificed by cervical dislocation. The skin on the back of the mice was cut off with high-pressure sterile surgical scissors and was placed in a paraformaldehyde fixing solution for flat fixation. After 12 days, histopathology sections showed that the tissue sections of the Baicalin-Zinc Supramolecular Hydrogel group were similar to normal skin, the epidermal layer and dermal layer structure was distinct, there were more hair follicle growth, fewer inflammatory cells, and more collagen fiber deposition; while the skin of the SA group was still healing, granulation tissue was visible, and no hair follicle recovery was observed, there were more inflammatory cells, and the amount of collagen fiber deposition was less.

Claims

1. A traditional Chinese medicine-metal ion carrier-free hydrogel compound, the structural formula of which is shown in Formula 1: The aforementioned traditional Chinese medicine-metal ion carrier-free hydrogel compound does not require the addition of excipients; the drug molecules themselves can form a hydrogel.

2. The method for preparing the traditional Chinese medicine-metal ion carrier-free hydrogel compound as described in claim 1, characterized in that, The method is as follows: Includes the following steps: Baicalin and zinc ions were suspended separately in water at a molar ratio of 1:0.1 to 1:

10. The pH of the baicalin solution was adjusted to 6-10 to obtain a clear and transparent solution. The solutions were mixed, heated and stirred to obtain baicalin-zinc supramolecular hydrogel.

3. The application of the traditional Chinese medicine-metal ion carrier-free hydrogel compound as described in claim 1 in the preparation of antibacterial drugs.

4. The application as described in claim 3, characterized in that, The antimicrobial drug is used for external human bacterial infections or internal bacterial infections, or for infections caused by Staphylococcus aureus, Escherichia coli, multidrug-resistant Staphylococcus aureus, or the bacteria that cause them.

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