A traditional Chinese medicine molecular compound preparation with high bacteriostatic performance and acoustic immunization activity, a preparation method and application thereof

Abstract

The application discloses a traditional Chinese medicine molecular compound preparation with high efficient bacteriostatic performance and acoustic immunization activity, and a preparation method and application thereof. The traditional Chinese medicine molecular compound preparation is mainly prepared from bismuth nitrate, hypericin and astragalus polysaccharide. First, the bismuth nitrate, the hypericin methanol solution and the astragalus polysaccharide aqueous solution are respectively prepared; the astragalus polysaccharide aqueous solution is added into a reaction medium, under the stirring action, the bismuth nitrate aqueous solution and the hypericin methanol solution are added dropwise, after the dropwise addition is completed, the stirring is continuously carried out, and a reaction mother liquor is obtained; the reaction mother liquor is purified, and the traditional Chinese medicine molecular compound preparation is obtained after the purification. The traditional Chinese medicine molecular compound preparation prepared by the application is self-assembled from a traditional Chinese medicine acoustic sensitizer, a metal ion and a traditional Chinese medicine immunoregulator, does not need to introduce an additional carrier or a surfactant, the proportion of active ingredients reaches 100%, and therefore, the traditional Chinese medicine molecular compound preparation is a full-activity traditional Chinese medicine molecular compound nano preparation.

Description

1. Technical Field:

[0001] The present invention belongs to the technical field of traditional Chinese medicine antibacterial properties, and in particular relates to a traditional Chinese medicine molecular compound preparation with high-efficiency antibacterial properties and sonic immune activity, as well as a preparation method and application thereof. 2. Background technology:

[0002] Bacterial infections have long threatened human health. The rapid evolution and spread of multidrug-resistant bacteria, in particular, has significantly impacted the efficacy of traditional antimicrobial drugs in recent years. As a crucial component of traditional Chinese medicine, Chinese herbal medicines (TCMs) have a long history of use in preventing and treating infectious diseases. Compared with modern antimicrobial drugs, they offer a wide range of sources, diverse varieties, and fewer adverse reactions. No significant pathogen resistance has been observed in long-term clinical use, making them a valuable resource for the development of new antimicrobial agents. Recent studies have shown that certain TCMs contain sound-sensitive components, such as chlorophyll derivatives in silkworm excrement, curcumin in turmeric, hypocrellin B in bamboo fungus, and hypericin in Hypericum perforatum. These components, stimulated by low-frequency ultrasound, interact with surrounding oxygen, water, or hydrogen peroxide to generate large amounts of reactive oxygen species (ROS). These components induce intense and irreversible oxidative stress damage in bacteria, resulting in bacterial inhibition and killing. Because ROS are highly lethal to nearly all bacteria and pose no resistance risk, sonodynamic antimicrobial therapy has garnered significant attention in the treatment of drug-resistant infections. However, it also has certain drawbacks, such as poor water solubility of sonosensitizers, limited ROS production, and low bacterial targeting. These limitations make it difficult for sonodynamic therapy (SDT) alone to eradicate infections promptly and completely, significantly limiting its clinical application. Combining SDT with other treatment modalities has become a commonly used and highly effective antibacterial strategy in recent years.

[0003] Traditional Chinese Medicine (TCM) theory holds that infectious diseases are primarily caused by "exogenous pathogenic factors invading and weakening vital energy." The general principle of "strengthening vital energy and eliminating pathogenic factors" is particularly crucial in combating infectious diseases. From a modern medical perspective, "strengthening vital energy" in TCM primarily refers to modulating the immune microenvironment and enhancing the body's ability to fight foreign pathogens and exotoxins, which aligns closely with the concept of modern immunotherapy. "Exorcising pathogenic factors," on the other hand, involves directly targeting and eliminating invading pathogenic microorganisms. Therefore, guided by the TCM principle of "strengthening vital energy and eliminating pathogenic factors," traditional Chinese medicine immunomodulators and sonosensitizers offer the potential for optimal synergy. This innovative integration of these two approaches allows TCM immunomodulators to regulate immune cells to exert an active immunotherapy effect, thereby "strengthening vital energy." Furthermore, sonosensitizers can generate ROS to directly eliminate invading bacteria, thereby "eliminating pathogenic factors." This two-pronged approach achieves highly effective sonosensitization therapy for drug-resistant infections.

[0004] The rational integration of modern formulation technologies with traditional Chinese medicine (TCM) to maximize its efficacy is a key advantage of my country's unique approach to integrating traditional Chinese and Western medicine for the treatment of bacterial infections. However, guided by TCM theory, TCM treatments are characterized by multiple formulas, multiple ingredients, and multiple targets. Furthermore, traditional Chinese medicine preparations suffer from complex combinations and compositional profiles, unclear active ingredients and mechanisms of action, high dosages, and difficulty in quality control, significantly limiting the application of modern formulations. To address these challenges, researchers in this field have recently developed the innovative "molecular compatibility" theory, building on the pharmacological basis of TCM. This theory, guided by TCM theory of syndrome differentiation and treatment and the principles of monarch, minister, assistant, and envoy, as well as the seven emotions and harmony, optimizes the combination of multiple active TCM molecules with well-defined structures, efficacy, and mechanisms into "molecular compounds." These compounds are then refined into modern TCM formulations using modern formulation methods, such as liposome nanotechnology and controlled-release technologies. However, current research on "molecular compound" formulations focuses primarily on the use of nanocarriers for drug delivery. The introduction of carriers inevitably poses significant challenges to the formulation's biosafety and in vivo metabolism. In addition, there are problems such as cumbersome synthesis, low drug loading, poor reproducibility, and large batch-to-batch variability. Breaking the limitations of current nanocarrier drug delivery systems and designing and developing new, efficient, carrier-free co-delivery platforms is essential to promote the application of traditional Chinese medicine "molecular compound" preparations and promote their clinical translation.

[0005] Research has shown that nanostructured products are commonly present in traditional Chinese medicines throughout their harvesting, processing, decoction, systemization, and ingestion. These nanoaggregates, formed by drug molecules through non-covalent bonds such as π-π stacking, electrostatic interactions, hydrogen bonding, and coordination, offer a new avenue for the development of TCM "molecular compounds," potentially expanding beyond traditional carrier-based drug delivery systems to carrier-free co-assembled nanosystems. Due to its advantages such as simplicity, uniformity, stability, and high drug content, this carrier-free drug co-assembly strategy has become one of the most active and promising frontiers in the field of modern TCM formulations. 3. Summary of the invention:

[0006] The technical problem to be solved by the present invention is: based on the existing technical difficulties in the treatment of bacterial infections by combining modern preparation technology with traditional Chinese medicine, the present invention provides a Chinese medicine molecular compound preparation with high-efficiency antibacterial properties and acoustic immune activity, as well as a preparation method and application. The present invention is guided by the general anti-infection treatment principle of "strengthening the body's resistance and eliminating evil" in traditional Chinese medicine, follows the compatibility principle of monarch, minister, assistant and envoy in traditional Chinese medicine, utilizes metal-drug coordination assembly technology, and simultaneously carries Chinese medicine sonosensitizers, Chinese medicine immunomodulators, and bismuth ions to construct a fully active Chinese medicine "molecular compound" preparation that integrates low cost, high efficiency, strong targeting, and stable transportation, thereby achieving efficient acoustic immunotherapy for drug-resistant bacterial infections.

[0007] In order to solve the above problems, the technical solution adopted by the present invention is:

[0008] The invention provides a traditional Chinese medicine molecular compound preparation with high-efficiency antibacterial performance and sonic immune activity. The traditional Chinese medicine molecular compound preparation is mainly prepared from bismuth nitrate, hypericin and astragalus polysaccharide.

[0009] According to the above-mentioned traditional Chinese medicine molecular compound preparation, the mass ratio of the bismuth nitrate, hypericin and astragalus polysaccharide is 0.04-1.455:0.2525:10.

[0010] In addition, a method for preparing the above-mentioned traditional Chinese medicine molecular compound preparation is provided, the preparation method comprising the following steps:

[0011] a. First, prepare bismuth nitrate aqueous solution, hypericin methanol solution and astragalus polysaccharide aqueous solution respectively;

[0012] b. Add the prepared aqueous solution of astragalus polysaccharide to the reaction medium, and then add the prepared aqueous solution of bismuth nitrate and methanol solution of hypericin dropwise under stirring, continue stirring after the addition is completed, and obtain a reaction mother liquor after sufficient stirring;

[0013] c. Purifying the reaction mother liquor to obtain a Chinese medicine molecular compound preparation.

[0014] According to the preparation method of the above-mentioned traditional Chinese medicine molecular compound preparation, the preparation method of the bismuth nitrate aqueous solution is as follows: 4.85 mg of bismuth nitrate pentahydrate is added to 2 mL of ultrapure water, and the mixture is ultrasonicated in a water bath at room temperature for 1 minute to uniformly dissolve the bismuth nitrate to obtain a 5 mM bismuth nitrate aqueous solution;

[0015] The preparation method of the hypericin methanol solution is as follows: 5.04 mg of hypericin is added to 2 mL of methanol, and ultrasonicated in a water bath at room temperature for 1 minute to uniformly dissolve the hypericin to obtain a 5 mM hypericin methanol solution;

[0016] The astragalus polysaccharide aqueous solution is prepared by adding 10 mg of astragalus polysaccharide into 1 mL of ultrapure water, and performing water bath ultrasonication for 1 minute at room temperature to uniformly dissolve the astragalus polysaccharide to obtain a 10 mg / mL astragalus polysaccharide aqueous solution.

[0017] According to the above-mentioned preparation method of the traditional Chinese medicine molecular compound preparation, in step b, the feeding volume ratio between the hypericin methanol solution and the astragalus polysaccharide aqueous solution is 1:10, and the feeding volume ratio between the hypericin methanol solution and the bismuth nitrate aqueous solution is 1:6 to 6:1.

[0018] According to the above-mentioned method for preparing the traditional Chinese medicine molecular compound preparation, the reaction medium in step b is water, phosphate buffer (pH 7.4) or boric acid buffer (pH 7.4).

[0019] According to the above-mentioned preparation method of the traditional Chinese medicine molecular compound preparation, the stirring in step b is stirred at 1-5°C in the dark, and the stirring speed is 800-1200 rpm / min (preferably 1000 rpm / min); the stirring time is continued for 48 hours.

[0020] According to the above-mentioned preparation method of the traditional Chinese medicine molecular compound preparation, the specific process of purification in step c is: subjecting the obtained reaction mother liquor to ultracentrifugation washing to remove free molecules and residual organic reagents.

[0021] According to the above-mentioned method for preparing the traditional Chinese medicine molecular compound preparation, during the ultracentrifugation process, the temperature is controlled at 25° C., the speed is 15,000 rpm, and the time is 25 minutes.

[0022] The invention relates to the use of the Chinese medicine molecular compound preparation in the preparation of anti-bacterial infection drugs.

[0023] The TCM molecular compound preparation of the technical solution of the present invention includes a "main drug", namely hypericin (Hy) derived from the extract of Hypericum perforatum, which serves as a TCM sonosensitizer; a "minister drug", namely Astragalus polysaccharide (APS) derived from the extract of Astragalus polysaccharides, which serves as a TCM immunomodulator; and an "adjuvant drug", namely the inorganic metal ion "bismuth". The three are assembled into uniform nanoparticles by the antisolvent precipitation method through the coordination effect of metal ions, electrostatic adsorption, π-π stacking or hydrophobic interaction. Therefore, the fully active TCM "molecular compound" preparation prepared by the technical solution of the present invention is a multi-drug system that does not require a carrier. Each component is fully utilized, and the active drug accounts for 100%. It is a therapeutic agent that combines sonodynamic antibacterial properties and immune microenvironment regulation properties, and has great potential.

[0024] Hypericin Hy, the main active ingredient in the traditional Chinese medicine Hypericum perforatum, belongs to the dianthrone class of compounds. With the rise of sonodynamic therapy in recent years, Hy has been widely used as a sonosensitizer to treat bacterial infections. In the present invention's molecular compound formulation, the sonosensitizer Hy serves as the "primary agent," exerting a sonodynamic bactericidal and pathogenic effect.

[0025] Astragalus polysaccharide APS is the chemical component with the highest content in Astragalus and is also one of the main active ingredients. Studies have shown that APS has a strong immunomodulatory function, which can promote the expression of macrophage surface molecules CD80 and CD86, promote the maturation of dendritic cells, enhance the proliferation and transformation of T lymphocytes, and achieve an efficient immune response. In addition, APS can promote the release of immune active substances such as IL-2, IL-6, TNF-α and IFN-γ, and regulate the body's immunity, so it is usually used as an immunopotentiator. In the traditional Chinese medicine "molecular compound" preparation of the present invention, the traditional Chinese medicine immunomodulator APS is a "minister drug", which "strengthens the body's immunity" by enhancing the body's immunity, and assists the sonodynamic antibacterial therapy mediated by the "king drug" Hy, thereby effectively treating bacterial infections.

[0026] Bismuth-based nanomaterials have been widely used in the biomedical field due to their low toxicity, unique physicochemical properties, and ease of surface chemical modification. In the traditional Chinese medicine "molecular compound" preparation of the present invention, bismuth ions serve as "adjuvants" and are a bridge connecting Hy and APS, prompting the "main drug" and "minister drug" to form a nanosystem. At the same time, bismuth ions have a good sealing ability for glutathione in the bacterial microenvironment, thereby destroying the antioxidant capacity of bacteria and enhancing the sonoimmunological efficacy of the traditional Chinese medicine "molecular compound" preparation. In addition, bismuth ion-mediated nanoformulations can also achieve disease microenvironment-responsive drug release, avoiding drug release in normal tissues and organs, thereby reducing the systemic toxicity of the preparation and achieving attenuation.

[0027] It can be seen from this that the traditional Chinese medicine "molecular compound" preparation of the present invention conforms to the principle of mutual benefit and mutual use, mutual fear and mutual killing of traditional Chinese medicine. The traditional Chinese medicine sonosensitizer Hy and the traditional Chinese medicine immunomodulator APS synergize to produce a 1+1>2 effect; the active oxygen generated by the traditional Chinese medicine sonosensitizer Hy under ultrasound will inevitably cause damage to the immune cells in the microenvironment while being antibacterial, and the traditional Chinese medicine immunomodulator APS can effectively eliminate this toxic side effect of the sonosensitizer; in addition, the "molecular compound" is easily affected by the infected microenvironment and decomposes, achieving specific drug release at the disease site to reduce the toxic side effects on normal tissues and organs.

[0028] Therefore, the TCM "molecular compound" preparation of the present invention has the dual therapeutic effects of excellent sonodynamic antibacterial properties and immunogenicity. When treating lung infections, it can also be delivered directly to the infected lung site via the trachea using TCM spray therapy. TCM spray therapy involves passing a TCM solution through a nebulizer to form a drug mist for localized spraying to treat localized diseases. The TCM "molecular compound" preparation of the present invention treats bacterial pneumonia, combining traditional Chinese medicine with modern science and technology to treat localized diseases, offering numerous advantages. First, TCM spray therapy allows the drug to reach the affected site directly and exert its effects. Combined with a lung liquid quantitative nebulizer, the TCM "molecular compound" preparation can also be more evenly dispersed within the affected lung site. Second, the drug is continuously distributed within the lungs, promoting bacterial killing and immune regulation at the affected site, resulting in a long-lasting effect. Finally, quantitative lung atomization avoids the toxic damage to the infected lung site caused by frequent application of antibacterial drugs, as well as the first-pass effect of the drug in the liver, resulting in higher safety and bioavailability.

[0029] The positive beneficial effects of the present invention are:

[0030] 1. The traditional Chinese medicine "molecular compound" preparation with high antibacterial and immune properties prepared by the present invention is self-assembled by traditional Chinese medicine sonosensitizers, metal ions and traditional Chinese medicine immunomodulators. No additional carriers or surfactants are required, and the active ingredient accounts for 100%. Therefore, it is a fully active traditional Chinese medicine molecular compound nano preparation.

[0031] 2. The present invention prepares the Chinese medicine sonosensitizer into a fully active nanoformulation, which can improve its solubility in water and prevent it from being cleared by the body, thereby improving its bioavailability.

[0032] 3. The Chinese medicine "molecular compound" preparation of the present invention has good ultrasound response to produce active oxygen activity, activity in breaking the bacterial antioxidant microenvironment, antibacterial activity and immunomodulatory activity. Its good synergistic bactericidal effect has great application potential in the field of bacterial infection treatment.

[0033] 4. The traditional Chinese medicine "molecular compound" preparation of the present invention has simple synthesis steps, simple ingredients, and simple structure, achieving ultra-high drug loading and having no obvious toxicity in the body.

[0034] 5. The present invention combines sonodynamic antibacterial technology and immunotherapy to solve the problem of antibiotic inactivation, improve the sensitivity of bacterial treatment, and enhance the body's immune function. It is a synergistic treatment technology that effectively kills bacteria.

[0035] 6. The present invention provides a fully active Chinese medicine "molecular compound" preparation that is simple to prepare, green and safe; the Chinese medicine "molecular compound" preparation of the present invention is a drug co-delivery platform that has both sonodynamic bactericidal activity and immune microenvironment regulation activity, thereby achieving efficient treatment of drug-resistant bacterial infections.

[0036] 7. The present invention utilizes traditional Chinese medicine theory to guide the design of modern preparations, draws on the general anti-infection treatment principle of TCM of "strengthening the body's resistance and eliminating pathogenic factors", and follows the principles of the material basis of TCM efficacy, monarch, minister, assistant and envoy, and the seven emotions and harmony, combines modern nanotechnology with TCM preparations, and develops a new type of TCM preparation. 4. Description of the accompanying drawings:

[0037] Figure 1 Schematic diagram of the process for preparing the Chinese medicine molecular compound preparations described in Examples 1 to 5 of the present invention.

[0038] Figure 2 This is a visual representation of the reaction mother liquor of the Chinese medicine molecular compound preparation obtained during the preparation of Examples 1 to 5 of the present invention.

[0039] Figure 3 These are the transmission microscope images, particle size images and potential images of the traditional Chinese medicine "molecular compound" preparation prepared by the present invention.

[0040] Figure 4 The ultraviolet absorption spectrum, fluorescence absorption spectrum and infrared absorption spectrum of the traditional Chinese medicine "molecular compound" preparation BiHANDs and Bi(NO3)3, Hy and APS prepared in the embodiment of the present invention.

[0041] Figure 5 This is a diagram showing the efficiency of ROS production in vitro by the traditional Chinese medicine "molecular compound" preparation prepared in the present invention under ultrasound.

[0042] Figure 6 This is a graph showing the experimental results of the consumption of glutathione (GSH) in vitro by the traditional Chinese medicine "molecular compound" preparation prepared by the present invention.

[0043] Figure 7 This is a diagram showing the release of APS from a traditional Chinese medicine "molecular compound" preparation.

[0044] Figure 8 This is a thermodynamic analysis diagram of the blood routine test of each group of mice in the in vivo antibacterial experiment of the traditional Chinese medicine "molecular compound" preparation prepared in Example 2 of the present invention.

[0045] Figure 9 This is a diagram showing the in vivo antibacterial activity of the traditional Chinese medicine "molecular compound" preparation prepared in Example 2 of the present invention.

[0046] Figure 10 This is a diagram showing the in vivo immunomodulatory activity of the traditional Chinese medicine "molecular compound" preparation prepared in Example 2 of the present invention. V. Specific implementation methods:

[0047] The present invention is described in more detail below through specific implementation methods to facilitate understanding of the technical solution of the present invention, but is not intended to limit the scope of protection of the technical solution of the present invention.

[0048] The traditional Chinese medicine "molecular compound" preparation of the present invention has high bactericidal performance and immune activity and good biocompatibility. The three components Bi(NO3)3, Hy and APS do not need to be further purified, and ultrapure water is used in the whole process.

[0049] Example 1:

[0050] The preparation method of the Chinese herbal medicine molecular compound preparation having high-efficiency antibacterial performance and sonicoimmunoactivity of the present invention comprises the following detailed steps:

[0051] a. First, prepare bismuth nitrate aqueous solution, hypericin methanol solution and astragalus polysaccharide aqueous solution respectively; the specific preparation process of the three solutions is as follows:

[0052] A 5 mM bismuth nitrate pentahydrate solution was prepared by adding 4.85 mg of bismuth nitrate pentahydrate to 2 mL of ultrapure water and sonicating the solution in a water bath at room temperature for 1 minute to uniformly dissolve the solution, thereby obtaining a 5 mM bismuth nitrate aqueous solution.

[0053] Preparation method of 5 mM hypericin methanol solution: add 5.04 mg of hypericin to 2 mL of methanol, and sonicate in a water bath at room temperature for 1 min to uniformly dissolve the hypericin to obtain a 5 mM hypericin methanol solution;

[0054] Preparation method of 10 mg / mL Astragalus polysaccharide aqueous solution: add 10 mg of Astragalus polysaccharide to 1 mL of ultrapure water, and ultrasonicate in a water bath at room temperature for 1 minute to uniformly dissolve the Astragalus polysaccharide to obtain a 10 mg / mL Astragalus polysaccharide aqueous solution;

[0055] b. 1 mL of the astragalus polysaccharide aqueous solution prepared in step a was added dropwise to 3 mL of ultrapure water, and the mixture was stirred for 5 min at a speed of 1000 rpm / min to uniformly disperse the astragalus polysaccharide in the reaction system. Subsequently, 100 μL of the prepared hypericin methanol solution and 600 μL of the bismuth nitrate aqueous solution were added dropwise to the reaction system under stirring (at a speed of 1000 rpm / min) at a dropping rate of 10 μl / s. After the addition was completed, stirring was continued for 48 h at a speed of 1000 rpm / min to allow the mixture to react fully, and a reaction mother liquor was obtained after the reaction.

[0056] c. The reaction mother liquor was purified by gradient centrifugation. The reaction mother liquor was first centrifuged at 1500 rpm for 10 minutes to remove large particulate matter; then the supernatant was centrifuged at 15000 rpm for 25 minutes to obtain a nano-precipitate with a small and uniform particle size. The centrifugation was repeated three times to remove free molecules and organic solvents to obtain the final purified nano-preparation, i.e., the Chinese medicine molecular compound preparation, which was stored in the dark.

[0057] Example 2: basically the same as Example 1, except that:

[0058] In step b: 1 mL of the astragalus polysaccharide aqueous solution prepared in step a was added dropwise to 3 mL of ultrapure water, stirred for 5 min at a speed of 1000 rpm / min, so that the astragalus polysaccharide was evenly dispersed in the reaction system; then, under stirring (at a speed of 1000 rpm / min), 100 μL of the prepared hypericin methanol solution and 300 μL of the bismuth nitrate aqueous solution were added dropwise to the reaction system at a dropping speed of 10 μl / s; after the addition was completed, stirring was continued for 48 h at a speed of 1000 rpm / min to allow it to react fully, and a reaction mother liquor was obtained after the reaction.

[0059] Example 3: basically the same as Example 1, except that:

[0060] In step b: 1 mL of the astragalus polysaccharide aqueous solution prepared in step a was added dropwise to 3 mL of ultrapure water, stirred for 5 min at a speed of 1000 rpm / min, so that the astragalus polysaccharide was evenly dispersed in the reaction system; then, under stirring (at a speed of 1000 rpm / min), 100 μL of the prepared hypericin methanol solution and 100 μL of the bismuth nitrate aqueous solution were added dropwise to the reaction system at a dropping speed of 10 μl / s; after the addition was completed, stirring was continued for 48 h at a speed of 1000 rpm / min to allow it to react fully, and a reaction mother liquor was obtained after the reaction.

[0061] Example 4: basically the same as Example 1, except that:

[0062] In step b: 1 mL of the astragalus polysaccharide aqueous solution prepared in step a was added dropwise to 3 mL of ultrapure water, stirred for 5 min at a speed of 1000 rpm / min, so that the astragalus polysaccharide was evenly dispersed in the reaction system; then, under stirring (at a speed of 1000 rpm / min), 100 μL of the prepared hypericin methanol solution and 33 μL of the bismuth nitrate aqueous solution were added dropwise to the reaction system at a dropping speed of 10 μl / s; after the addition was completed, stirring was continued for 48 h at a speed of 1000 rpm / min to allow it to react fully, and a reaction mother liquor was obtained after the reaction.

[0063] Example 5: basically the same as Example 1, except that:

[0064] In step b: 1 mL of the astragalus polysaccharide aqueous solution prepared in step a was added dropwise to 3 mL of ultrapure water, stirred for 5 min at a speed of 1000 rpm / min, so that the astragalus polysaccharide was evenly dispersed in the reaction system; then, under stirring (at a speed of 1000 rpm / min), 100 μL of the prepared hypericin methanol solution and 16.7 μL of the bismuth nitrate aqueous solution were added dropwise to the reaction system at a dropping speed of 10 μl / s; after the addition was completed, stirring was continued for 48 h at a speed of 1000 rpm / min to allow it to react fully, and a reaction mother liquor was obtained after the reaction.

[0065] The contents of bismuth, hypericin and astragalus polysaccharide in the traditional Chinese medicine "molecular compound" preparation BiHANDs prepared in Examples 1 to 5 of the present invention are as follows:

[0066] Bismuth content determination method: Disperse the prepared TCM "molecular compound" preparation in 5 mL of deionized water to form a nanopreparation suspension. Remove 4 mL and dilute to 10 mL with digestion solution. Quantify the bismuth ions in the solution using inductively coupled plasma (ICP) emission spectrometry. Calculate the bismuth concentration in the original solution based on the dilution factor.

[0067] Astragalus polysaccharide and hypericin content determination method: Prepare 0.05μg / mL, 0.1μg / mL, 0.2μg / mL, 0.4μg / mL, 0.6μg / mL, 0.8μg / mL, and 1.0μg / mL hypericin methanol solutions, measure their fluorescence intensity at 450nm excitation and 596nm detection, and generate a standard curve for hypericin. Use the same method to obtain the fluorescence intensity of BiHANDs at 450nm excitation and 596nm, and substituting it into the standard curve to determine the concentration of hypericin in the TCM "molecular compound" preparation.

[0068] Aqueous solutions of astragalus polysaccharide at 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, and 70 μg / mL were prepared. The phenol-sulfuric acid method was used to mix 200 μL of the astragalus polysaccharide aqueous solution, 100 μL of a 6% phenol solution, and 500 μL of concentrated sulfuric acid. The mixture was incubated in a boiling water bath for 20 minutes, and the ultraviolet absorbance was measured at 490 nm to obtain a standard curve for the phenol-sulfuric acid method of astragalus polysaccharide. The same method was used to obtain the ultraviolet absorbance of BiHA NDs treated with the phenol-sulfuric acid method at 490 nm. This absorbance was then applied to the standard curve to determine the concentration of astragalus polysaccharide in the traditional Chinese medicine "molecular compound" preparation.

[0069] The recovery rates and particle size potential data of the Chinese herbal molecular compound preparations with high-efficiency antibacterial performance and sonicoimmunoactivity prepared in Examples 1 to 5 of the present invention are shown in Table 1.

[0070] Table 1 Recovery rate and particle size potential data of each component of the Chinese medicine molecular compound preparation prepared in Examples 1 to 5

[0071]

[0072] From the data in Table 1, it can be seen that when the feed amount of Astragalus polysaccharide is fixed and the feed volume ratio of hypericin and bismuth nitrate is from 1:6 to 6:1, the three can be co-assembled into a Chinese medicine molecular compound preparation. However, the recovery rate of hypericin varies under different feed ratios, among which the recovery rate is the highest when the feed ratio is 1:3. During the preparation process, when the feed ratio is 1:3, the reaction mother liquor has an obvious color change during the reaction, that is, from dark purple to bright orange (see Figure 2 Although Table 1 shows that the recovery rate of hypericin is relatively good when the feed ratio is 6:1, the nanoprecipitate obtained at this feed ratio is difficult to resuspend and prone to coagulation during actual experiments. In addition, since the sonodynamic therapy of hypericin in the traditional Chinese medicine molecular compound preparation plays a major antibacterial role, considering the actual economic benefits of the preparation process, the feed ratio of 1:3 in Example 2 is the optimal technical solution.

[0073] The Chinese medicine "molecular compound" preparation obtained in Example 2 of the present invention was subjected to the following test and analysis. The analysis results are detailed in the attached Figures 1 to 7 .

[0074] Figure 1 Schematic diagram of the process for preparing the Chinese medicine molecular compound preparations described in Examples 1 to 5 of the present invention.

[0075] Figure 2 This is a visual representation of the reaction mother liquor of the Chinese medicine molecular compound preparation obtained during the preparation of Examples 1 to 5 of the present invention.

[0076] Depend on Figure 2 It can be seen that during the reaction process, the color of the solution of Example 2 changed most significantly, from the initial purple-red to a brighter orange-red.

[0077] Figure 3 These are the transmission microscope images, particle size images and potential images of the traditional Chinese medicine "molecular compound" preparation prepared by the present invention.

[0078] Figure 3 a is a transmission microscope image of the traditional Chinese medicine "molecular compound" preparation prepared by the present invention, Figure 3 As can be seen in a, the prepared Chinese medicine "molecular compound" preparation is spherical, and the Chinese medicine immune enhancer APS is wrapped in the outer layer. Figure 3 As can be seen from the particle size diagram b, the synthesized TCM “molecular compound” preparation is evenly dispersed, and the average particle size in aqueous solution is about 109 nm; Figure 3c is the potential diagram of the prepared traditional Chinese medicine "molecular compound" preparation, which is -20.64mV, indicating that the prepared traditional Chinese medicine "molecular compound" preparation has stable properties.

[0079] Figure 4 Ultraviolet absorption spectra, fluorescence absorption spectra and infrared absorption spectra of the traditional Chinese medicine "molecular compound" preparation BiHA NDs and Bi(NO3)3, Hy and APS prepared in the examples of the present invention.

[0080] Figure 4 a is the ultraviolet absorption spectra of the traditional Chinese medicine "molecular compound" preparation BiHA NDs and Bi(NO3)3, Hy, and APS prepared in this example; Figure 4 b is the fluorescence absorption spectra of BiHA NDs and Bi(NO3)3, Hy, and APS; Figure 4 c is the infrared absorption spectra of BiHANDs, Hy and APS. Figure 4 In a, BiHA NDs and Hy have absorption peaks at 550nm and 600nm, indicating that BiHA NDs contain Hy. It is not difficult to see from the figure that the peak of BiHANDs is slightly shifted compared with Hy, indicating that in the formation of the "molecular compound" preparation of traditional Chinese medicine, the coordination effect between Hy and Bi and the π-π stacking between Hy cause the absorption peak to move toward the long-wave direction. Figure 4 In b, there is no fluorescence absorption curve of Hy in BiHANDs in aqueous solution. It is possible that the interaction between the components in the process of forming nanoparticles caused fluorescence quenching. When BiHANDs are dissolved in methanol solution, methanol destroys the structure of the nanoparticles, allowing Hy to be released and thus recovering fluorescence. Figure 4 From c, we can see that 2926cm -1 、1023cm -1 The absorption peak of 1263 cm is the characteristic absorption peak of APS. -1 The absorption peak at 2923 cm is the characteristic peak of Hy, and the absorption peak of BiHANDs at 2923 cm -1 、1259cm -1 、1026cm -1 The absorption peak at indicates the presence of Hy and APS in the system.

[0081] Figure 5 This is a diagram showing the efficiency of ROS production in vitro by the traditional Chinese medicine "molecular compound" preparation prepared in the present invention under ultrasound.

[0082] During the experiment, 9,10-dimethylanthracene (DMA) fluorescent probe was used and mixed with the traditional Chinese medicine "molecular compound" preparation by ultrasound. The principle that ROS can trigger DMA fluorescence quenching was used to monitor the generation and production of ROS by detecting changes in DMA fluorescence intensity at 444nm. Figure 5a is the time-dependent graph of ROS production by BiHANDs under ultrasound; Figure 5 b shows the concentration-dependent ROS production of BiHANDs under ultrasound. As can be seen from the figure, BiHANDs alone do not cause fluorescence quenching of DMA. However, after ultrasound stimulation, BiHANDs exhibit concentration- and time-dependent DMA fluorescence quenching and exhibit good ultrasound controllability, demonstrating that BiHANDs-mediated SDT effectively generates ROS and is a promising nanotherapeutic agent for antibacterial infections.

[0083] Figure 6 This is a graph showing the experimental results of the consumption of glutathione (GSH) in vitro by the traditional Chinese medicine "molecular compound" preparation prepared by the present invention.

[0084] Figure 6 a is the time-dependent graph of GSH consumption in vitro by the TCM “molecular compound” preparation. Figure 6 b is a graph showing the concentration dependence of the consumption of GSH by the TCM "molecular compound" preparation in vitro. During the experiment, the 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) colorimetric reaction was used to evaluate the consumption performance of BiHANDs on GSH. The principle is that after the reaction of DTNB with GSH, there is an obvious characteristic absorption peak at 412nm, and the corresponding absorption intensity is used as the relative content of GSH, and the difference in absorption intensity represents the consumption of GSH. Figure 6 It can be seen that the relative content of GSH gradually decreases with increasing BiHANDs concentration and reaction time. This result demonstrates that BiHANDs have a strong ability to block GSH. When further applied in antibacterial experiments, this property of BiHANDs can disrupt the antioxidant system at the site of bacterial infection, thereby enhancing the bactericidal effect of SDT.

[0085] Figure 7 This is a diagram showing the release of APS from a traditional Chinese medicine "molecular compound" preparation.

[0086] To investigate the pH-responsiveness of the BiHANDs described in this study, PBS solutions with different pH values ​​(7.4 and 5.5) were prepared to simulate the normal physiological environment of the human body and the microenvironment of bacterial infection. The results showed that the cumulative drug release rate over 24 hours in a phosphate solution with a pH of 5.5 was 70.59±8.425%, while the cumulative drug release rate in a phosphate solution with a pH of 7.4 was only 33.56±4.37%. This demonstrates that the BiHA NDs possess pH-responsiveness. In subsequent in vivo antibacterial experiments, this ensured the substantial release of BiHANDs at the lesion site, resulting in a highly effective antibacterial effect.

[0087] Figure 8This is a thermodynamic analysis diagram of the blood routine test of each group of mice in the in vivo antibacterial experiment of the traditional Chinese medicine "molecular compound" preparation prepared in Example 2 of the present invention.

[0088] from Figure 8 It can be seen that after routine blood analysis, the values ​​of white blood cells (WBC), neutrophils (Gran) and lymphocytes (Lymph) in the blood of mice with lung infection exceeded the normal physiological range. However, after the drug administration combined with ultrasound treatment, the various indicators of the blood routine analysis of the treatment group had dropped to near the normal physiological range on the seventh day. This result shows that the traditional Chinese medicine "molecular compound" preparation prepared in Example 2 of the present invention can effectively treat lung infections caused by bacteria.

[0089] Figure 9 This is a diagram showing the in vivo antibacterial activity of the traditional Chinese medicine "molecular compound" preparation prepared in Example 2 of the present invention.

[0090] Figure 9 The following is a plate image of bacterial colonies in the lungs of mice taken three days after administration. It clearly shows that the treatment group effectively reduced the number of bacteria in the infected lungs of mice, demonstrating a stronger antibacterial effect than the other groups. These results demonstrate that the traditional Chinese medicine "molecular compound" preparation prepared by the present invention has highly effective antibacterial activity.

[0091] Figure 10 This is a diagram showing the in vivo immunomodulatory activity of the traditional Chinese medicine "molecular compound" preparation prepared in Example 2 of the present invention.

[0092] Figure 10 a and Figure 10 b is a representative flow cytometry analysis of each group; Figure 10 c and Figure 10 d is the CD4 + CD3 + and CD8 + CD3 + The histogram of T cell percentage shows that compared with the PBS group, the treatment group increased the number of CD4 + The proportion of T cells increased from 5.81% to 23.85%, and CD8 + The proportion of T cells increased from 5.19% to 22.81%. These results show that the traditional Chinese medicine "molecular compound" preparation prepared by the present invention can increase CD4 + CD3 + T cells and CD8 + CD3 + The proportion of T cells promotes the proliferation and transformation of T lymphocytes.

[0093] In summary, the traditional Chinese medicine "molecular compound" preparation BiHA NDs prepared by the present invention has safe ingredients, simple synthesis, and achieves extremely high drug loading. Compared with traditional antibiotic antibacterial and single treatment models, the present invention not only solves the problem of bacterial resistance, but also achieves the dual effects of sonodynamic bactericidal and immune enhancement under the guidance of "strengthening the body's resistance and eliminating pathogens", and has broad application prospects in the treatment of lung infections. At the same time, the present invention allows the drug to reach the lung lesion site directly through inhalation administration, achieving large-scale enrichment of the drug at the lesion site, with minimal toxic and side effects on normal tissues, which is of great significance in the field of bacterial infection treatment. Therefore, the traditional Chinese medicine "molecular compound" preparation BiHA NDs prepared by the present invention can be used to prepare antibacterial infection drugs.

[0094] The embodiments described above are only preferred embodiments of the present invention and do not limit the scope of implementation of the present invention. Therefore, any equivalent changes or modifications made according to the structure, characteristics and principles described in the patent scope of the present invention should be included in the scope of the patent application of the present invention.

Claims

1. A Chinese herbal compound preparation having high antibacterial properties and sonicoimmunoactivity, characterized in that: The traditional Chinese medicine molecular compound preparation is mainly prepared from bismuth nitrate, hypericin and astragalus polysaccharide; the mass ratio of the bismuth nitrate, hypericin and astragalus polysaccharide is 0.04-1.455:0.2525:

10.

2. A method for preparing the Chinese medicine molecular compound preparation according to claim 1, characterized in that: The preparation method comprises the following steps: a. First, prepare bismuth nitrate aqueous solution, hypericin methanol solution and astragalus polysaccharide aqueous solution respectively; b. Add the prepared aqueous solution of astragalus polysaccharide to the reaction medium, and then add the prepared aqueous solution of bismuth nitrate and methanol solution of hypericin dropwise under stirring, continue stirring after the addition is completed, and obtain a reaction mother liquor after sufficient stirring; c. Purifying the reaction mother liquor to obtain a Chinese medicine molecular compound preparation.

3. The method for preparing the Chinese medicine molecular compound preparation according to claim 2, characterized in that: The bismuth nitrate aqueous solution is prepared by adding 4.85 mg of bismuth nitrate pentahydrate into 2 mL of ultrapure water, and performing water bath sonication at room temperature for 1 minute to uniformly dissolve the bismuth nitrate to obtain a 5 mM bismuth nitrate aqueous solution. The preparation method of the hypericin methanol solution is as follows: 5.04 mg of hypericin is added to 2 mL of methanol, and ultrasonicated in a water bath at room temperature for 1 minute to uniformly dissolve the hypericin to obtain a 5 mM hypericin methanol solution; The astragalus polysaccharide aqueous solution is prepared by adding 10 mg of astragalus polysaccharide into 1 mL of ultrapure water, and performing water bath ultrasonication for 1 minute at room temperature to uniformly dissolve the astragalus polysaccharide to obtain a 10 mg / mL astragalus polysaccharide aqueous solution.

4. The method for preparing the Chinese medicine molecular compound preparation according to claim 2, wherein: In step b, the volume ratio of the hypericin methanol solution to the astragalus polysaccharide aqueous solution is 1:10, and the volume ratio of the hypericin methanol solution to the bismuth nitrate aqueous solution is 1:6 to 6:

1.

5. The method for preparing the Chinese medicine molecular compound preparation according to claim 2, wherein: The reaction medium in step b is water, a phosphate buffer solution at pH 7.4, or a borate buffer solution at pH 7.

4.

6. The method for preparing the Chinese medicine molecular compound preparation according to claim 2, characterized in that: The stirring in step b is carried out at 1-5° C. in the dark, with a stirring speed of 800-1200 rpm; the stirring time is continued for 48 hours.

7. The method for preparing the Chinese medicine molecular compound preparation according to claim 2, characterized in that: The specific process of purification in step c is: the obtained reaction mother liquor is subjected to ultracentrifugation washing to remove free molecules and residual organic reagents.

8. The method for preparing the Chinese medicine molecular compound preparation according to claim 7, characterized in that: During the ultracentrifugation process, the temperature was controlled at 25° C., the rotation speed was 15,000 rpm, and the time was 25 minutes.

9. Use of the Chinese medicine molecular compound preparation according to claim 1 in the preparation of antibacterial infection drugs.

Images