A kind of Macleaya powder and flavonoids natural supramolecular hydrogel and its application as antibacterial and anti-inflammatory drug

Abstract

The present invention provides a type of supramolecular hydrogel compound formed by self-assembly of effective ingredients of Macleaya cordata powder and flavonoid active molecules, a structure, a preparation method and an application. The supramolecular hydrogel compound has excellent selective antibacterial ability. While selectively killing pathogenic bacteria such as Staphylococcus aureus, it does not affect the activity of probiotics such as Bacillus subtilis and Enterococcus faecium in the intestine; it is particularly sensitive to methicillin-resistant Staphylococcus aureus (MRSA); it also has good anti-inflammatory effects and promotes wound healing. Among them, the natural supramolecular hydrogel formed by the self-assembly of effective ingredients of Macleaya cordata powder and flavonoid active molecules has a uniform morphology, good dispersion and stability, forms a gel without the need for excipients, and has the value of being developed into a carrier-free drug delivery preparation. The general structural formula of the compound is shown below:

Description

Technical Field

[0001] The invention relates to a preparation method of a Macleaya huixin-flavonoid glycoside supramolecular hydrogel drug and its antibacterial and anti-inflammatory application, and in particular has good inhibitory activity against clinical methicillin-resistant Staphylococcus aureus and promotes the healing of MRSA-infected skin wounds, and belongs to the fields of medicinal chemistry and pharmacy. Background Art

[0002] Bacterial infection is one of the biggest threats facing humanity today. With the abuse of antibiotics, more and more drug-resistant bacteria and even multi-drug-resistant "superbugs" have emerged. Every year, drug-resistant infections cause about 700,000 deaths worldwide, most of which occur in developing countries; by 2050, bacterial resistance will cause 10 million deaths worldwide each year. Moreover, bacteria threaten humans in many ways. Therefore, it is urgent to develop new antibiotics with strong selectivity, low toxicity and few side effects to combat drug-resistant bacteria and develop multi-level antibacterial strategies.

[0003] Among the diseases caused by bacterial infection, wound infection is one of the most common phenomena in the medical field. As the largest organ of the human body, the skin is the first barrier to protect us. Skin wound infection usually makes it difficult for the wound to heal and even causes other infection-related diseases such as pyoderma or even sepsis. Therefore, antibacterial hydrogels that have anti-pollution, anti-secondary trauma, and promote cell regeneration, and that have both hydrogel and antibacterial functions, have become a hot spot in research and development; first of all, its soft characteristics minimize the mechanical damage to the surrounding cell tissues, and the hydrogel has good permeability, which is convenient for the transportation and delivery of metabolites and nutrients. It is precisely because of the unique properties of hydrogels that they have received widespread attention and research in the medical field, such as for drug carriers and wound dressings. However, most of the hydrogels reported in the past are based on high molecular weight polymers, and there are fewer reports on the self-assembly of natural active small molecules to form hydrogels, especially binary natural small molecule self-assembly hydrogels.

[0004] Traditional Chinese medicine is one of the sources of natural antibiotics, and many ingredients in traditional Chinese medicine have clear antibacterial effects. The wild resources of the traditional Chinese medicine Macleaya are widely distributed, and are used to treat trauma, arthritis, pityriasis versicolor, ulcers, bee stings, and anesthesia, analgesia, and swelling. Macleaya extracts have been widely used in animal feed additives and plant fungicides due to their strong antibacterial effects. Macleaya powder is a Class II Chinese veterinary preparation in my country, and has been approved as the first Chinese veterinary drug feed additive (approval number: Veterinary Additive 180415250, Veterinary Additive 180415329). It is a powder made of benzophenanthridine alkaloids (mainly sanguinarine and chelerythrine) extracted from the natural plant Macleaya. It has antibacterial and anti-inflammatory, appetizing and other functions. It is commonly used in the production of pigs, chickens, ducks, freshwater fish, shrimps, crabs, turtles and tortoises, and is widely used as an antibiotic substitute for livestock and poultry feed, but its application form is very single, mainly added in powder form and used orally, lacking rich and flexible application scenarios. The present invention is inspired by the concept of combining medicine with auxiliary medicine. According to the structural characteristics of the main ingredients in Macleaya Powder, it is combined with other Chinese medicinal ingredients such as the natural antibacterial and anti-inflammatory flavonoids contained in Scutellaria baicalensis to develop a natural antibacterial carrier-free supramolecular hydrogel. Summary of the invention

[0005] On the basis of the prior art, the present invention aims to develop a new type of carrier-free hydrogel preparation based on Macleaya huipin and flavonoid glycosides. The inventors surprisingly found that the main components of Macleaya huipin, sanguinarine and chelerythrine, and flavonoid glycosides such as baicalin and scutellaria baicalensis can directly form hydrogels without the aid of carrier molecules, and can develop new application forms such as oral gels and external gels on the basis of the original powdered oral products, and have better antibacterial and anti-inflammatory activities. This invention has great research significance for discovering and developing natural hydrogel drugs with clear structures from natural Chinese medicines.

[0006] One of the purposes of the present invention is to provide a hydrogel compound formed by the effective components of Macleaya cordata and its powder and -flavonoid glycoside supramolecules and a preparation method thereof.

[0007] The second object of the present invention is to provide four supramolecular hydrogel compounds formed by combining the alkaloid components sanguinarine and chelerythrine of Macleaya San with the flavonoid glycoside components baicalin and scutellariae in pairs, and a preparation method of the above compounds.

[0008] The third object of the present invention is to provide the application of the obtained supramolecular hydrogel compounds, specifically the in vitro antibacterial, anti-inflammatory, anti-infective and healing-promoting applications of four binary carrier-free supramolecular hydrogel compounds. The hydrogel compounds have very good selective inhibitory effects on common pathogenic bacteria such as Staphylococcus aureus, Escherichia coli, methicillin-resistant Staphylococcus aureus and their biofilms, and do not affect probiotics. They have great potential in replacing traditional antibiotics in clinical applications, and can be used as drugs for preventing or treating infections in the body, wound infections, inflammation, non-healing wounds, etc. caused by common pathogenic bacteria.

[0009] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0010] 1. Determine the preparation process of the supramolecular hydrogel compound formed by Macleaya Glycyrrhiza or its active ingredients and flavonoid components by exploring different pH values, heating temperatures, molar ratios of feed materials, and centrifugal speeds. Use nuclear magnetic resonance, mass spectrometry and other technical means to determine its structural information.

[0011] The preparation method comprises the following steps:

[0012] (1) Heat to dissolve Macleaya Powder or Sanguinarine or Chelerythrine in water.

[0013] (2) Flavonoid glycosides (baicalin, scutellaria baicalensis, wogonin, rutin) are suspended in water, heated, and the pH is adjusted to obtain a clear and transparent solution.

[0014] (3) Mixing the macleya powder or sanguinarine, chelerythrine aqueous solution and flavonoid glycoside aqueous solution prepared in steps (1) and (2), stirring and centrifuging to obtain a precipitate or colloid, washing with water and then centrifuging, repeating three times, and freeze-drying to obtain a brown-yellow powder.

[0015] Preferably, the heating temperature in steps (1) and (2) is 35-80°C, such as 45°C, 55°C, 65°C, or 75°C.

[0016] Preferably, the molar ratio of Macleaya cordata powder or its active ingredients and flavonoid glycosides is 1:0.1 to 1:10, further 1:0.5-1:5, and specifically 1:0.1, 1:0.5, 1:1, 1:2, 1:5, 1:10 and the like.

[0017] Preferably, the mixing temperature in step (3) is room temperature to 35°C.

[0018] Preferably, the water used in steps (1), (2) and (3) is deionized water.

[0019] Preferably, the pH adjuster in step (2) is an organic or inorganic base, such as sodium hydroxide, sodium carbonate, sodium bicarbonate, or ammonia water.

[0020] Preferably, in step (2), the pH is 7-9, for example, pH=7, pH=8, pH=9, etc.

[0021] Preferably, the centrifugal speed during the centrifugal purification in step (3) is 5000-13000 rpm, for example, 5000 rpm, 5500 rpm, 6000 rpm, 6500 rpm, 7000 rpm, 7500 rpm, 8000 rpm, 8500 rpm, 9000 rpm, 9500 rpm or 13000 rpm.

[0022] 2. The antibacterial effect of the obtained peptide molecule hydrogel compound was evaluated by measuring the MIC values ​​of different bacterial species.

[0023] The evaluation of antibacterial effect includes the following steps:

[0024] The MIC values ​​of the compounds and the control group against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Enterococcus faecium, and methicillin-resistant Staphylococcus aureus were detected by the broth dilution method.

[0025] 3. Taking the sanguinarine-baicalin supramolecular hydrogel compound as an example, the rheological study of the supramolecular hydrogel compound obtained by the present invention was carried out; taking MRSA, which is the most difficult to inhibit and eliminate, as an example, the in vitro antibacterial effect of the supramolecular hydrogel compound on various pathogens such as MRSA and biofilms was observed by scanning electron microscopy. Taking MRSA, which is the most difficult to inhibit and eliminate, as an example, by establishing a mouse skin wound infection bacterial animal model, the wound healing rate, bacterial growth and skin pathological tissue changes of the mice after treatment were observed to evaluate its antibacterial effect on bacteria such as Staphylococcus aureus, Escherichia coli, and methicillin-resistant Staphylococcus aureus (MRSA).

[0026] The safety of supramolecular hydrogel compounds was evaluated through cell safety experiments, in vitro hemolysis experiments, and pathological sections of mouse heart, spleen, lungs, and kidneys.

[0027] Taking the sanguinarine-baicalin hydrogel compound as an example, the rheology of the supramolecular hydrogel compound of the present application and the evaluation of its in vitro and in vivo antibacterial effect on bacteria such as MRSA include the following steps:

[0028] (1) Scanning electron microscopy was used to observe the morphological effects of 1 / 3 MIC concentration on bacteria.

[0029] (2) The XTT method was used to evaluate the inhibitory effect of supramolecular hydrogel compounds on bacterial biofilms.

[0030] (3) Scanning electron microscopy was used to observe the effects of supramolecular hydrogel compounds on the biofilms of MRSA and other fungi.

[0031] (4) An animal model of MRSA infection in mouse skin wounds was established. The mice were divided into a negative control group (Control), a positive control group (MRSA), and a treatment group. The treatment group was specifically given sanguinarine-baicalin hydrogel (BA-SAN).

[0032] (5) After a 12-day treatment cycle, the wound healing status during the treatment period was compared with the pathological tissue status of the mouse skin 12 days later to evaluate the ability of the supramolecular hydrogel compound to prevent / treat / inhibit infection-induced inflammation and promote wound healing in MRSA-infected patients.

[0033] (6) The MTT method was used to evaluate the survival rate of MDCK cells after 24 h and 48 h of drug culture, and to evaluate the cytotoxicity of the supramolecular hydrogel compounds.

[0034] (7) The drug was incubated with rat erythrocytes, and the hemolysis rate of each drug-treated group was measured at 570 nm using an ELISA reader to evaluate the hemolytic activity of the supramolecular hydrogel compound.

[0035] (8) The heart, liver, spleen, lung, and kidney tissues of the mice treated for 12 days were collected and the pathological conditions of the tissues of the normal mice and the treated mice were compared to evaluate the in vivo safety of the supramolecular hydrogel compound.

[0036] The specific research results of the present invention are described as follows:

[0037] 1. Formation of hydrogel

[0038] The inventors found that: Macleaya powder or the effective ingredients of Macleaya powder can form supramolecular hydrogels with some flavonoid compounds. The raw material compound combinations that can form hydrogels include Macleaya powder-baicalin, Macleaya powder-scutellarin, sanguinarine-baicalin, sanguinarine-scutellarin, chelerythrine-baicalin, chelerythrine-scutellarin; while Macleaya powder-wogonoside, Macleaya powder-rutin, sanguinarine-wogonoside, chelerythrine-wogonoside, sanguinarine-rutin, chelerythrine-rutin cannot form hydrogels, but can only form precipitation. The selected 4 kinds of flavonoid compounds (baicalin, scutellarin, wogonoside, rutin) are similar in structure, but slight changes in functional groups will lead to different forms of products. In addition, the inventors also found that other alkaloids with similar structures, such as jatrorrhizine and bamipine, cannot form hydrogels with flavonoid glycosides such as baicalin and scutellarin. Although the structures of jatrorrhizine and bamipine are highly similar to those of sanguinarine and chelerythrine, they cannot form hydrogels with flavonoid components according to the preparation method. After structural analysis and comparison, it can be seen that sanguinarine has two methylenedioxy rings, chelerythrine has one methylenedioxy ring, and both have methyl structures on nitrogen atoms, which are not possessed by jatrorrhizine and bamipine. It is speculated that the formation of the hydrogel described in the present invention requires the alkaloid components to have specific methylenedioxy rings or nitrogen methyl groups, and it is the first time to propose and preliminarily confirm the view that the formation of carrier-free supramolecular hydrogels requires the presence of certain key functional groups.

[0039] In summary, only specific structural types of the two types of components used in the present invention can be used to prepare hydrogels, and whether the hydrogel can be successfully prepared has extremely low predictability. Moreover, taking the sanguinarine-baicalin hydrogel as an example, the supramolecular hydrogel compound of the present invention exhibits good rheological properties. Under the changes of frequency and shear stress, the storage modulus is greater than the loss modulus, which proves that the compound can stably maintain the hydrogel state and has good fluidity, and is suitable for various applications including external use.

[0040] 2. Antibacterial, anti-infective, anti-inflammatory, and healing-promoting preventive or therapeutic effects of hydrogel compounds

[0041] In the in vitro experiment, the effects of various supramolecular hydrogel drugs on a variety of pathogenic bacteria and non-pathogenic bacteria are shown in Table 2 of Example 4 and Table 3 of Example 6, which can prove that the supramolecular hydrogel compounds obtained by the present invention have high selective inhibition of pathogenic bacteria, while only have a weak effect on beneficial bacteria. Among them, taking the representative compound sanguinarine-baicalin hydrogel as an example, its antibacterial MIC value for MRSA is 3.75μM; the clearance rate of MRSA biofilm at a drug concentration of 50μM is 81.84%. The results of scanning electron microscopy also show that the hydrogel compounds of the present invention represented by sanguinarine-baicalin can destroy the surface structure of bacteria, promote bacterial deformation and lysis, and have significant ability to remove biofilms. Moreover, as can be seen from Table 2, the supramolecular hydrogel compounds in the present invention also show significantly stronger and more selective antibacterial ability than the raw monomer drugs.

[0042] In the in vivo animal experiment, during the 12-day treatment period, the skin regeneration rate of the sanguinarine-baicalin hydrogel group at different time points was greater than that of the MRSA group and the Control group. Histopathological sections after 12 days showed that the sections of the sanguinarine-baicalin hydrogel group were similar to normal skin, with distinct structures of the epidermis and dermis, more hair follicle growth, fewer inflammatory factors, and more collagen fiber deposition; while the skin of the MRSA group was still healing, with visible granulation tissue, no hair follicle recovery, more inflammatory factors, and less collagen fiber deposition. After 12 days, the inflammatory factors TNF-α and IL-1β in the sanguinarine-baicalin hydrogel treatment group were significantly lower than those in the MRSA group, showing significant anti-inflammatory ability.

[0043] In view of the above, compared with the prior art, the present invention has the following advantages:

[0044] In the present invention, the effective ingredients of Macleaya hui san and the specific flavonoid components in the scutellaria baicalensis extract are self-assembled to directly form a supramolecular hydrogel without the aid of auxiliary materials and carriers. The two main components of Macleaya hui san and chelerythrine, which constitute Macleaya hui san, have certain antibacterial effects when used alone, while the flavonoid components baicalin and scutellaria baicalensis have weak nonspecific antibacterial effects respectively. When these two types of components are combined in pairs, a supramolecular hydrogel with a new structure is generated. Not only does the self-assembled synthesized supramolecular drug significantly enhance the specific antibacterial effect relative to the monomer component, and this enhancement is difficult to be predicted in advance without experimental verification, but also after assembly, morphological changes are brought about, providing the possibility of other application forms. Compared with the technical solution of preparing gel by combining the monomer components of traditional Chinese medicine with polymer carrier materials in the prior art, the present invention can prepare a hydrogel preparation without adding auxiliary materials, reduces the use of raw materials, and has good safety and simplicity on the basis of improving the efficacy, and has potential application value in antibacterial, anti-infective, anti-inflammatory and promoting wound healing of bacterial infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The appearance of one of the representative hydrogel compounds (sanguinarine-baicalin hydrogel) prepared in Example 2 of the present invention shows good stability, ductility and fluidity.

[0046] Figure 2 The MRSA status of each test group observed under a scanning electron microscope in Example 4 of the present invention. 1), 2), 3), and 4) are MRSA scanning electron microscope images of the blank group, baicalin group, sanguinarine group, and sanguinarine-baicalin group, respectively.

[0047] Figure 3 The graph is a graph of the rheological properties of a representative hydrogel (sanguinarine-baicalin hydrogel) prepared in Example 5 of the present invention. 1) and 2) are respectively the curves of the storage modulus (G') and the loss modulus (G") changing with frequency and shear stress.

[0048] Figure 4 The MRSA biofilm states of each test group observed under a scanning electron microscope in Example 6 of the present invention. 1), 2), 3), and 4) are scanning electron microscope images of MRSA biofilms of the blank group, baicalin group, sanguinarine group, and sanguinarine-baicalin hydrogel group, respectively.

[0049] Figure 5 The following are the mouse skin wound healing pictures and pathological tissue sections in Example 7 of the present invention. Figure A shows the mouse skin wound healing at different time points. Figure B shows the wound healing rate. Figure C shows the pathological tissue sections stained by HE and Masson.

[0050] Figure 6 This is an immunofluorescence image of inflammatory factors in mouse skin wounds in Example 7 of the present invention. DETAILED DESCRIPTION

[0051] The following examples are intended to further illustrate the present invention. It should be understood by those skilled in the art that the examples are only provided to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0052] Example 1

[0053] Weigh 1:0.1 to 1:10 molar amounts of Macleaya cordata powder (main components are sanguinarine and chelerythrine) and flavonoid components baicalin, scutellaria baicalin, wogonin and rutin, respectively, and dissolve them in water. The pH value of the flavonoid glycoside components is adjusted to 6-10 to obtain a clear and transparent solution. The mixture is stirred at 60° C. with a magnetic stirrer for 20 minutes and then centrifuged to obtain Macleaya cordata powder-baicalin supramolecular hydrogel, Macleaya cordata powder-scutellaria baicalin supramolecular hydrogel, Macleaya cordata powder-wogonin complex precipitate and Macleaya cordata powder-rutin complex precipitate.

[0054] Example 2

[0055] The molar amounts of sanguinarine, chelerythrine and flavonoid components baicalin, scutellarin, wogonoside and rutin in a ratio of 1:0.1 to 1:10 are weighed and dissolved in water respectively, and the pH of the flavonoid glycoside components is adjusted to 6-10 to obtain a clear and transparent solution. The sanguinarine and chelerythrine solutions are mixed with baicalin, scutellarin, wogonoside and rutin solutions at 60° C., respectively, and the mixture is stirred with a magnetic stirrer for 20 minutes and then centrifuged to obtain sanguinarine-baicalin supramolecular hydrogel, sanguinarine-scutellarin supramolecular hydrogel, chelerythrine-baicalin supramolecular hydrogel, chelerythrine-scutellarin supramolecular hydrogel, sanguinarine-wogonoside precipitate, chelerythrine-wogonoside precipitate, sanguinarine-rutin precipitate and chelerythrine-rutin precipitate.

[0056] In Example 1 and Example 2, the selected baicalin, baicalin, wogonin and rutin are all common flavonoid compounds, and they have similar structures, but slight functional group changes lead to different forms of products. Baicalin and rutin cannot form hydrogel compounds with Macleaya San or specific effective ingredients sanguinarine and chelerythrine, but only obtain fibrous precipitates, which shows that the preparation of hydrogels is unpredictable and has accidental nature. In addition, the present invention finds that the prepared carrier-free hydrogel requires a specific methylenedioxy ring, sanguinarine has two methylenedioxy rings, chelerythrine has one methylenedioxy ring, and both have methyl structures on nitrogen atoms, while similar alkaloids such as jatrorrhizine and bamipine that do not contain methylenedioxy rings or nitrogen methyl groups cannot form hydrogels with flavonoid components according to the preparation method, which once again proves that whether alkaloid compounds can form hydrogels with flavonoid compounds has extremely low predictability, and creative labor must be paid to obtain the corresponding technical solutions. It was also preliminarily confirmed that structures such as methylenedioxy rings and nitrogen methyl groups may be the key functional groups that promote the formation of carrier-free supramolecular hydrogels.

[0057]

[0058] Example 3

[0059] The structural information of the supramolecular hydrogel compound in Example 2 was confirmed by a method comprising the following steps.

[0060] Each compound was 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 voltage was 40 V, the ion source temperature was 120 ° C, the collision energy was 35 eV, the cone gas flow rate was 50 L / h, the desolvation gas flow rate was 800 L / h, and the mass spectrum acquisition range was 50-2000. Before mass spectrometry analysis, there was no need for chromatographic column separation, and the sample was directly injected for analysis. The molecular ion peaks and molecular structures of the basic unit of supramolecular drugs obtained by mass spectrometry analysis are shown in Table 1.

[0061] Table 1 Structural information of four supramolecular hydrogel compounds prepared in Example 2

[0062]

[0063] Some supramolecular drugs were characterized by nuclear magnetic resonance proton spectroscopy (Avance IIIHD 400MHz spectrometer, Bruker, America), and the results are as follows:

[0064] NMR identification of sanguinarine-baicalin supramolecular hydrogel drug 1 H NMR (400MHz, DMSO-d6): δ (ppm) 6.94 (s, 1H, H-3, BA), 6.99 (s, 1H, H-8, BA) 8.01 (d, J = 7.2Hz, 1H, H-2′, 6′, BA), 7.54-7.53 (m, 3H, H-3′, 4′,5′,BA),5.19(d,J=5.1Hz,1H,H-1″,BA),3.30-3.40(m,3H,H-2″,3″,4″,BA),4.01(d,J=9.6Hz,1H,H-5″,BA),10.12(s,1H,H-6,SA N),8.74(d,J=8.0Hz,1H,H-9,SAN),8.61(d,J=8.0Hz,1H,H-10,SAN),8.29(d,J=8.0Hz,1H,H-10,SAN),8.26(s,1H,H-4,SAN),8.10(d ,J=8.0Hz,1H,H-11,SAN),7.74(s,1H,H-1,SAN),6.60(s,2H,-OCH2-2,3,SAN),6.35(s,2H,-OCH2-7,8,SAN),4.92(s,3H,N-CH3,SAN).

[0065] NMR characterization of sanguinarine-scutellarin supramolecular hydrogel drug 1H NMR(400MHz,DMSO-d6):10.12(s,1H,H-6,SAN),8.74(d,J=8.0Hz,1H,H-9,SAN),8.61(d,J=8.0Hz,1H,H-10,SAN),8.29(d,J=8.0Hz,1H,H-10,S AN),8.26(s,1H,H-4,SAN),8.10(d,J=8.0Hz,1H,H-11,SAN),7.74(s,1H,H-1,SAN),6.60(s,2H,-OCH2-,SAN),6.35(s,2H,-OCH2-,SAN),4.92( s,3H,N-CH3,SAN),6.82(s,1H,H-3,SCU),7.00(s,1H,H-8,SCU),7.94(d,J=7.9Hz,2H,H-2′,6′,SCU),6.95(d,J=7.9Hz,2H,H-3′,5′,SCU),12. 75(s,1H,5-OH,SCU),8.61(s,1H,6-OH,SCU),10.38(s,1H,4′-OH,SCU),5.23(d,J=7.1Hz,1H,H-1″,SCU),3.30-3.40(m,3H,H-2″,3″,4″,SCU).

[0066] NMR characterization of chelerythrine-baicalin supramolecular hydrogel drug 1 H NMR (400MHz, DMSO-d6): δ (ppm) 9.89 (s, 1H, H-6, CHE), 8.53 (d, J = 8.0Hz, 1H, H-11, CHE), 8.52 (d, J = 8.0Hz, 1H, H-12, CHE), 8.57 (d, J = 8.0 Hz,1H,H-9,CHE),8.13(s,1H,H-10,CHE),8.12(s,1H,H-4,CHE),7.46(s,1H,H-1,CHE),6.24(s,2H,-OCH2-,CHE),4.25(s,3H,-OCH3-7,C HE),4.10(s,3H,-OCH3-8,CHE),4.93(s,3H,N-CH3,CHE),6.94(s,1H,H-3,BA),6.99(s,1H,H-8,BA),8.01(d,J=7.2Hz,1H,H-2′,6′,BA), 7.54-7.53(m,3H,H-3′,4′,5′,BA), 5.19(d,J=5.1Hz,1H,H-1″,BA), 3.30-3.40(m,3H,H-2″,3″,4″,BA), 4.01(d,J=9.6Hz,1H,H-5″,BA).

[0067] NMR characterization of chelerythrine-scutellarin supramolecular hydrogel drug 1 H NMR (400MHz, DMSO-d6): δ (ppm) 6.82 (s, 1H, H-3, SCU), 7.00 (s, 1H, H-8, SCU) 7.94 (d, J=7.9Hz, 2H, H-2′, 6′, SCU), 6.95 (d, J=7.9Hz, 2H, H-3′, 5′, SCU) ,12.75(s,1H,5-OH,SCU),8.61(s,1H,6-OH,SCU),10.38(s,1H,4′-OH,SCU),5.23(d,J=7.1Hz,1H,H-1″,SCU),3.30-3.40(m,3H,H-2″,3″,4″,SCU),9 .89(s,1H,H-6,CHE),8.53(d,J=8.0Hz,1H,H-11,CHE),8.52(d,J=8.0Hz,1H,H-12,CHE),8.57(d,J=8.0Hz,1H,H-9,CHE),8.13(s,1H,H-10,CHE),8.1 2(s,1H,H-4,CHE),7.46(s,1H,H-1,CHE),6.24(s,2H,-OCH2-,CHE),4.25(s,3H,-OCH3-7,CHE),4.10(s,3H,-OCH3-8,CHE),4.93(s,3H,N-CH3,CHE).

[0068] Example 4

[0069] The in vitro antibacterial activity of the carrier-free supramolecular drugs prepared in Example 1 and Example 2 was determined as follows:

[0070] The inhibitory effect of supramolecular drugs on human pathogenic bacteria Staphylococcus aureus, conditional pathogenic bacteria Escherichia coli, probiotics Bacillus subtilis and Enterococcus faecium, and methicillin-resistant Staphylococcus aureus was observed by turbidimetry. Staphylococcus aureus, Escherichia coli, probiotics Bacillus subtilis and Enterococcus faecium, and methicillin-resistant Staphylococcus aureus were all from the School of Life Sciences, Beijing University of Chinese Medicine. The supramolecular hydrogel drug powder prepared in Example 1 was sealed and stored at 4°C. When used, it was dissolved in LB medium to 1×10 4 μg / mL stock solution (DMSO content 1%) is used for later use.

[0071] Bacterial recovery and preparation of bacterial stock solution: The bacteria were dispersed in LB medium and cultured overnight at 37°C and 200 rpm. Then, LB medium was used to prepare a bacterial stock solution with a volume of 2×10 6CFU / mL of bacterial stock solution was counted using the plate count method.

[0072] The MIC of supramolecular drugs was determined by the double dilution method. In a 48-well plate, 1 mL of supramolecular drug solution with concentrations of 320, 240, 160, 120, 80, 60, 40, 30, 20, 15, 10, 7.5, 5, 3.75, and 1.875 μM was prepared using LB medium. Then 30 μL of bacterial stock solution was added and cultured in a 37°C constant temperature and saturated humidity incubator for 12 h. The OD value was measured at 600 nm using an enzyme marker. The experiment was repeated three times, and the drug concentration with a bacterial survival rate greater than 80% was calculated as the minimum inhibitory concentration of the sample. The group without drug or bacteria was set as the blank control group; the group with bacteria but no drug was set as the blank bacteria group. Bacterial survival rate (%) = (absorbance value of the sample group - absorbance value of the blank group) / (absorbance value of the blank bacteria group - absorbance value of the blank group) × 100%.

[0073] See Table 2 for specific results.

[0074] Table 2: Antibacterial effects of the six supramolecular drugs prepared in Example 1 and Example 2 on different bacteria

[0075]

[0076] Bacteria were obtained by centrifugation (3000rpm / min, 10min). Then they were washed three times with PBS. The obtained samples were fixed with 2.5% glutaraldehyde at 4°C for 4 hours and washed with PBS. Then different concentrations of ethanol were used for gradient dehydration (30, 50, 70, 80, 90, 95, 100%) for 10 minutes each time. 2.5μL of the final ethanol sample was dropped onto a single-sided polished silicon wafer with a pipette, and the water was naturally evaporated at room temperature. The sample was treated with gold spraying and placed in FESEM with an operating voltage of 15kV for observation and photography. The photography results are attached. Figure 2 .

[0077] The results show that the antibacterial activity of the supramolecular drug based on the active ingredients of Macleayahui powder, alkaloids and flavonoid glycosides, is significantly enhanced compared with the precursor raw materials, and has excellent selective anti-Staphylococcus aureus activity, and still has excellent sensitivity to methicillin-resistant Staphylococcus aureus; at the same time, it has moderate antibacterial activity against the conditional pathogen Escherichia coli in the intestine, effectively inhibits the active reproduction of Escherichia coli flora, and maintains the balance of intestinal flora. It also has weak inhibitory activity against the probiotics Bacillus subtilis and Enterococcus faecium in the intestine, effectively avoiding accidental damage to beneficial flora. In particular, the compound baicalin-sanguinarine is significantly better than many current first-line antibacterial drugs against methicillin-resistant Staphylococcus aureus (MIC is 3.75μM), such as norfloxacin, oxacillin, tetracycline, and ciprofloxacin (MIC is greater than 400μM), and has the value of in-depth research and further clinical development.

[0078] Example 5

[0079] In this example, the sanguinarine-baicalin hydrogel prepared in Example 2 was subjected to rheological testing as follows:

[0080] The sanguinarine-baicalin hydrogel was placed on the rheometer measuring plate, the gap was set to 5 mm, and the temperature was set to 25°C. Frequency sweep: the strain was constant at 0.1%, and the measurement frequency range was 0.1Hz-10Hz. Amplitude sweep: the frequency was set to a constant value of 1Hz, and the strain range was 0.001%-10%, and the storage modulus (G′) and loss modulus (G″) that varied with strain and frequency were obtained. Under the changes in frequency and shear stress, the storage modulus was greater than the loss modulus, indicating that it has good rheological properties.

[0081] Example 6

[0082] Taking the sanguinarine-baicalin hydrogel prepared in Example 2 as an example, the in vitro bacterial biofilm clearance rate of the supramolecular hydrogel compound of the present invention was tested as follows:

[0083] First, the bacterial suspension was injected into a 96-well plate and cultured at 37°C for 24 hours. After 24 hours, the culture medium was aspirated to obtain a mature drug-resistant Staphylococcus aureus biofilm attached to the bottom of the 96-well plate. Then, BA-SAN Gel and SAN solutions with concentrations of 6.25μM, 12.5μM, 25μM and 50μM were added respectively, and the culture was continued in a 37°C constant temperature incubator for 24 hours. The supernatant was aspirated, and the bacterial biofilm adhered to the bottom was visible. The biofilm was washed 3 times with PBS buffer, 200μL XTT dye solution was added to each well, and incubated at 37°C in a constant temperature incubator for 2 hours, and then the absorbance value was measured at a wavelength of 490nm. Using blank culture medium as the solvent control, culture medium plus bacteria as the blank bacteria control, the sample clearance rate of the biofilm was calculated according to the following formula:

[0084] Clearance rate (%) = 100-(OD sample-OD solvent) / (OD blank bacteria-OD solvent) × 100%, see Table 3 for specific results

[0085] Table 3: Clearance rate of MRSA biofilm by different drug groups

[0086]

[0087] Place silicon wafers of the same size (5mm×5mm) in a 24-well plate, add bacterial suspension, and culture at 37°C for 24 hours. After 24 hours, add 50μM BA, BA-SAN Gel, and SAN, respectively, and continue to culture for 24 hours. Wash the biofilm three times with PBS buffer and fix it with 2.5% glutaraldehyde at 37°C for 4 hours. Then dehydrate the samples with a gradient of ethanol solutions with increasing concentrations (30%, 50%, 70%, 80%, 90%, 95%, 100%) and observe them under a scanning electron microscope. The photographic results are attached. Figure 4 .

[0088] Example 7

[0089] Taking the sanguinarine-baicalin hydrogel prepared in Example 2 as an example, and taking MRSA, which is the most difficult to inhibit and eliminate after infection, as an example, the supramolecular hydrogel compound of the present invention was evaluated for its in vivo antibacterial, anti-inflammatory, anti-infective and healing-promoting pharmacodynamics, and the method is as follows:

[0090] Female Balb / c mice aged 6-8 weeks and weighing 16-18g were used as experimental animals. The hair on the back of the mice was trimmed with electric clippers, and Shutai 50 was extracted at an anesthetic dose of 50mg / kg, and the mice were anesthetized by intraperitoneal injection. After the mice became slow in movement and their breathing became deeper and slower, the model was established. The skin of the depilated area on the back of the mice was wiped with a cotton ball dipped in 75% alcohol for disinfection. After the alcohol evaporated completely and the skin became dry again, a hole was punched on the back of the mouse with an 8mm circular punch to shape it, and then a circular skin with a diameter of 8mm was cut off with scissors, and then 100μL 2×10 6 CFU of MRSA bacterial liquid was inoculated into the wound of each mouse. After 1 hour of infection with the bacterial liquid, the mouse MRSA bacterial infection model was established.

[0091] The modeled mice were divided into 3 groups, namely, blank group (Control), model group (MRSA), and sanguinarine-baicalin hydrogel treatment group (BA-SAN Gel). The control group was not infected with bacteria, and the MRSA bacterial infection was not treated. The BA-SAN Gel group applied 3mM BA-SAN hydrogel on the mouse wound. Five mice (n=5) were set up in each group as parallel controls, and the treatment cycle was 12 days. The wound size was recorded every 3 days, and the wound healing rate was calculated.

[0092] After the 12-day treatment cycle, the mice were killed by dislocating the cervical vertebrae, and the wound skin on the back of the mice was cut with sterile surgical scissors and placed in a paraformaldehyde fixative to flatten and fix.

[0093] As shown in the attached figure of the instruction manual Figure 5As shown, the histopathological sections after 12 days showed that the tissue sections of the BA-SAN Gel group were similar to normal skin, with distinct structures of the epidermis and dermis, more hair follicles growing, fewer inflammatory factors, and more collagen fiber deposition; while the skin of the MRSA group was still healing, with visible granulation tissue, no hair follicle recovery, more inflammatory factors, and less collagen fiber deposition. Moreover, after 12 days, the inflammatory factors TNF-α and IL-1β in the sanguinarine-baicalin hydrogel group were significantly lower than those in the MRSA group, showing significant anti-inflammatory ability. This shows that the hydrogel obtained in the present invention can be effectively used to inhibit inflammation caused by bacterial infection, reduce the level of inflammatory factors, and promote wound healing. In view of the fact that the aforementioned embodiments have verified the inhibitory effects of various hydrogels in the present invention on a variety of bacteria, those skilled in the art will be able to expect that the hydrogel of the present invention has an inhibitory effect on infections and inflammation caused by a variety of bacteria and promotes wound healing.

[0094] Example 8

[0095] Taking the sanguinarine-baicalin hydrogel prepared in Example 2 as an example, the safety of the supramolecular hydrogel compound of the present invention was evaluated as follows:

[0096] MDCK cells were treated with 3.125-50 μM sanguinarine-baicalin hydrogel for 24 and 48 h. After culture, 100 μL of serum-free medium and MTT mixed solution were added to the MDCK cells in the 96-well plate and incubated for another 4 h. Subsequently, 150 μL of DMSO was added to dissolve the water-insoluble MTT dye, and its absorbance was measured at 490 nm. Cell viability was calculated according to the following formula:

[0097] Cell survival rate (%) = (OD 给药组 -OD 空白组 ) / (OD 正常组 -OD 空白组 )×100% The results showed that the cell survival rates of the different concentration drug groups after 24h and 48h incubation were all greater than 90%, indicating that the drug had no significant cytotoxicity.

[0098] Fresh rat blood was used to determine the in vitro hemolytic effect of the sample. First, the red blood cells were collected by centrifugation at a speed of 3000rmp / min for 15 minutes, and the red blood cells were washed 3 times with normal saline. Then 3mL of the red blood cells obtained by centrifugation were mixed into 11mL of normal saline for storage and dispersion. Sanguinarine-baicalin hydrogel was diluted with normal saline to concentrations of 2MIC, 4MIC, 8MIC and 16MIC, respectively. Then the test solution (1mL) was mixed with 100μL of red blood cell stock solution to obtain a 4% red blood cell solution, and incubated at 37°C for 4h. Then centrifuged at 3000rmp / min for 15min, and the absorbance value of the supernatant was measured at 570nm by an enzyme marker. The positive control was deionized water, and the negative control was normal saline. The hemolysis rate was calculated according to the following formula:

[0099] Hemolysis rate (%) = (A 给药组 -A PBS组 ) / (A 去离子水组 -A PBS组 )×100%

[0100] Hemolysis tests showed that sanguinarine-baicalin hydrogel had no obvious hemolytic properties. Even at a concentration as high as 16 times the MIC, the hemolysis rate was still lower than the internationally recognized standard of 5%.

[0101] After the 12-day treatment cycle, the mice were killed by cervical dislocation. The heart, liver, spleen, lung, and kidney of the mice were dissected and fixed in paraformaldehyde fixative. After the samples were fixed and preserved, they were sent to Seville Biotechnology Co., Ltd. for slice making. The prepared slices were placed under 4×10 and 10×10 objective magnifications for tissue observation. The heart, liver, spleen, lung, and kidney of the drug-treated group were similar to those of the normal group, and no pathological changes were observed, indicating that BA-SAN hydrogel has good safety.

[0102] The applicant declares that the present invention uses the above-mentioned embodiments to illustrate the supramolecular pharmaceutical composition of the present invention and its preparation method and application, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A hydrogel, composed of the compounds scutellarin, sanguinarine, and chelerythrine, further having the following structure:

2. The hydrogel according to claim 1, further characterized in that: The hydrogel is prepared without adding excipients and needs to be prepared according to the following method: Take scutellarin and sanguinarine or chelerythrine at a molar ratio of 1:0.1 to 1:10, and suspend them in water respectively; heat sanguinarine and chelerythrine to dissolve, heat scutellarin and adjust the pH to 6-10 to obtain a clear and transparent solution, mix and stir the two solutions, and let them stand to obtain a hydrogel compound.

3. Use of the hydrogel according to any one of claims 1 and 2 in the preparation of antibacterial drugs.

4. The use according to claim 3, characterized in that Used to fight against in vivo infections caused by methicillin-resistant Staphylococcus aureus.

5. Use of the hydrogel according to any one of claims 1 and 2 in the preparation of anti-inflammatory drugs.

6. Use of the hydrogel according to any one of claims 1 and 2 in the preparation of a drug for promoting wound healing.

7. Use of the hydrogel according to any one of claims 1 and 2 in the preparation of a drug for inhibiting bacterial biofilm, wherein the hydrogel is further characterized in that The drug is used to inhibit the biofilm of methicillin-resistant Staphylococcus aureus.

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