Honeysuckle carbon dot nanozymes and their application in the preparation of free radical scavenging products

Abstract

The present invention relates to the technical field of antioxidant materials, and relates to a honeysuckle carbon dot nanozyme and its application in the preparation of a product for scavenging free radicals. The honeysuckle carbon dot nanozyme of the present invention is obtained by hydrothermal carbonization of honeysuckle, and the hydrothermal carbonization is a hydrothermal reaction at 185°C to 205°C for 8h to 12h; the present invention finds through research that the honeysuckle carbon dot nanozyme not only has a high-efficiency free radical scavenging property, but also has the effect of treating lung inflammation. The present invention provides a new candidate drug for treating lung injury and lung ischemia-reperfusion.

Description

Technical Field

[0001] The invention relates to the technical field of antioxidant materials, and in particular to a honeysuckle carbon dot nanozyme and application thereof in preparing a product for removing free radicals. Background Art

[0002] Nanozymes are a class of enzyme mimics that have both the unique properties of nanomaterials and catalytic functions. They can catalyze enzyme substrates under physiological or extreme conditions, have enzymatic reaction kinetics similar to those of natural enzymes, and can be used as enzyme substitutes for human health.

[0003] The advent of nanozymes has changed the traditional concept that inorganic nanomaterials are biologically inert substances. It has revealed the intrinsic biological effects and new properties of nanomaterials, enriched the research on enzyme mimics, expanded it from organic complexes to inorganic nanomaterials, and expanded the application range of nanomaterials. Since nanozymes have both the high catalytic activity of natural enzymes and the stability and economy of enzyme mimics, since the report of HRP-like enzyme activity nanozymes in 2007, the research on nanozymes has risen rapidly, and the scope of research has gradually expanded to include different fields such as materials science, physics, chemistry, biology, medicine and environment.

[0004] Artificially synthesized nanozymes have the properties of mimicking natural enzymes because of their specific enzymatic activities. At the same time, nanozymes also have high stability and adjustable catalytic activity, and are expected to replace natural enzymes for wider applications in antibacterial, antioxidant, and therapeutic applications. Currently developed artificial nanozymes include MOF-based nanozymes and single-atom nanozymes, which show high activity in removing reactive oxygen and nitrogen species. However, complex synthesis methods, high production costs, and single enzyme activity have seriously hindered the practical application of these nanozymes. Summary of the invention

[0005] In order to address the deficiencies of the prior art, the purpose of the present invention is to provide a honeysuckle carbon dot nanozyme and its application in the preparation of free radical scavenging products. The present invention has found through research that honeysuckle carbon dots not only have efficient free radical scavenging properties, but also have the effect of treating pulmonary inflammatory diseases including lung injury and pulmonary ischemia-reperfusion.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides a honeysuckle carbon dot nanozyme, which is obtained by hydrothermal carbonization of honeysuckle, wherein the hydrothermal carbonization is a hydrothermal reaction at 185° C. to 205° C. for 8 h to 12 h.

[0008] Preferably, after the hydrothermal reaction is completed, impurities are removed by centrifugation, and then the honeysuckle carbon dot nanozyme is obtained by purification by dialysis.

[0009] Preferably, the molecular weight cutoff for the dialysis is 900Da to 1100Da.

[0010] Preferably, the average particle size of the honeysuckle carbon dot nanozyme is between 3 nm and 5 nm, and the fluorescence lifetime is between 2.5 ns and 3.5 ns.

[0011] The second aspect of the present invention provides an application of the honeysuckle carbon dot nanozyme in the preparation of a product for scavenging free radicals.

[0012] Preferably, the free radicals include ABTS free radicals, hydroxyl free radicals and superoxide anions.

[0013] The third aspect of the present invention provides the use of the honeysuckle carbon dot nanozyme in the preparation of drugs for treating inflammation-related diseases.

[0014] Preferably, the inflammation-related disease is a lung inflammation-related disease.

[0015] Preferably, the pulmonary inflammation-related diseases include lung injury and lung ischemia-reperfusion.

[0016] In a third aspect of the present invention, a medicine is provided, which includes the honeysuckle carbon dot nanozyme and a pharmaceutically acceptable excipient or carrier. The medicine can be prepared into an acceptable pharmaceutical preparation after adding suitable excipients, such as granules, capsules, tablets, injections, mixtures, oral liquids, syrups or other liquid preparations. The excipients that can be added vary according to the prepared dosage form. Exemplary, optional excipients are suspending agents, thickeners, flavoring agents, colorants, preservatives, permeabilizing agents, pH regulators, wetting agents, etc. The types of specific excipients can be selected by those skilled in the art according to the properties of the honeysuckle carbon dot nanozyme, the properties of the excipients and the requirements of the preparation during implementation.

[0017] The drug is used to:

[0018] 1) Eliminate free radicals;

[0019] 2) Treat lung damage; or

[0020] 3) Treatment of pulmonary ischemia-reperfusion.

[0021] The beneficial effects of the present invention are:

[0022] (1) The present invention utilizes a hydrothermal method, with water as the medium, to prepare the honeysuckle carbon dot nanozyme under conditions of high temperature and high pressure. The raw materials used are low in price, the synthesis process is convenient and fast, and it has a wide range of applications and great commercial potential.

[0023] (2) The honeysuckle carbon dot nanozyme prepared by the present invention using a one-step hydrothermal method exhibits strong antioxidant capacity in a variety of free radical systems including ABTS free radicals, hydroxyl free radicals, and superoxide free radicals.

[0024] (3) The honeysuckle carbon dot nanozyme prepared by the present invention uses Chinese herbal medicine as a precursor material, has a strong antioxidant capacity, and can achieve the treatment of lung inflammatory diseases in vivo while ensuring good biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a transmission electron microscope observation image of the honeysuckle carbon dots prepared in Example 1 of the present invention, and the inset is the statistical results of the measured lattice and particle size distribution.

[0026] Figure 2 This is the fluorescence lifetime spectrum of the honeysuckle carbon dots prepared in Example 1 of the present invention.

[0027] Figure 3 The free radical scavenging ability of the honeysuckle carbon dots prepared in Example 1 of the present invention in different free radical systems, wherein (a) shows the scavenging effect of ABTS free radicals; (b) shows the scavenging effect of hydroxyl free radicals; (c) shows the scavenging effect of superoxide free radicals.

[0028] Figure 4 This is a pathological section of mouse lung tissue after treating mice with lung injury with the honeysuckle carbon dot nanozyme prepared in Example 1 of the present invention.

[0029] Figure 5 This is a pathological section of mouse lung tissue after treating mice with pulmonary ischemia-reperfusion with the honeysuckle carbon dot nanozyme prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0030] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0031] Currently, existing artificially developed nanozymes have disadvantages such as high cost, complex synthesis method, and relatively single enzyme activity. The present invention proposes a hydrothermal method to synthesize honeysuckle carbon dot nanozymes. The synthesis process is convenient and fast, and it has a wide range of applications and great commercial potential.

[0032] In one or more embodiments, during the hydrothermal method for preparing honeysuckle carbon dot nanozymes, the temperature is 185°C to 205°C, and the time is 8h to 12h.

[0033] In one or more embodiments, the honeysuckle carbon dot nanozyme synthesized by the hydrothermal method is centrifuged to remove impurities, and then dialyzed, and the molecular weight cutoff of the dialysis is 900Da to 1100Da.

[0034] In some embodiments, the average particle size of the honeysuckle carbon dot nanozyme prepared by the hydrothermal method is between 3 nm and 5 nm.

[0035] In some embodiments, the fluorescence lifetime of the honeysuckle carbon dot nanozyme prepared by the hydrothermal method is 2.5ns to 3.5ns.

[0036] A typical embodiment of the present invention provides the use of honeysuckle carbon dot nanozymes in the preparation of free radical scavenging products.

[0037] In some embodiments, the honeysuckle carbon dot nanozyme prepared by the hydrothermal method has significant antioxidant properties and has very good scavenging ability for different free radical systems including ABTS free radicals, hydroxyl free radicals, and superoxide free radicals.

[0038] Another embodiment of the present invention provides the use of honeysuckle carbon dot nanozyme in the preparation of drugs for treating lung inflammation and lung injury.

[0039] Another embodiment of the present invention provides the use of honeysuckle carbon dot nanozyme in the preparation of a drug for treating lung inflammation and pulmonary ischemia-reperfusion.

[0040] In some embodiments, the drug for treating lung inflammation is administered by intravenous injection. Specifically, the dosage of the honeysuckle carbon dot nanozyme for intravenous injection is 5 mg / kg to 10 mg / kg.

[0041] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0042] The honeysuckle in the following examples is purchased from Fengqiu honeysuckle of Qianxun Flower Tea, a Taobao store, and the manufacturer is Anhui Zhongyutang Biotechnology Co., Ltd. The product specification is 500g of velvety honeysuckle. The water in the following examples is all deionized water.

[0043] Example 1

[0044] A honeysuckle carbon dot nanozyme, the preparation method of which is as follows:

[0045] Honeysuckle was dried and ground into powder, passed through a 100-mesh sieve, 400 mg of honeysuckle powder was weighed and dispersed in 40 mL of water, mixed and transferred to a 100 mL tetrafluoroethylene autoclave, and hydrothermally reacted at 190 ° C for 12 hours. After the reaction was completed, the autoclave was transferred to room temperature for cooling, and the autoclave was opened after complete cooling of the autoclave. The product after the reaction was transferred to a 50 mL centrifuge tube, centrifuged at a speed of 10000 rpm / min for 10 minutes, and the supernatant was taken to remove impurities, and this step was repeated three times. The final supernatant was transferred to a 1000 Da dialysis bag for 72 hours for purification, and the water was changed every 3 hours.

[0046] Example 2

[0047] A honeysuckle carbon dot nanozyme, the preparation method of which is as follows:

[0048] Honeysuckle was dried and ground into powder, passed through a 100-mesh sieve, 400 mg of honeysuckle powder was weighed and dispersed in 40 mL of water, mixed and transferred to a 100 mL tetrafluoroethylene autoclave, and hydrothermally reacted at 205 ° C for 12 hours. After the reaction was completed, the autoclave was transferred to room temperature for cooling, and the autoclave was opened after complete cooling of the autoclave. The product after the reaction was transferred to a 50 mL centrifuge tube, centrifuged at a speed of 10000 rpm / min for 10 minutes, and the supernatant was taken to remove impurities, and this step was repeated three times. The final supernatant was transferred to a 1000 Da dialysis bag for 72 hours for purification, and the water was changed every 3 hours.

[0049] Example 3

[0050] A honeysuckle carbon dot nanozyme, the preparation method of which is as follows:

[0051] Honeysuckle was dried and ground into powder, passed through a 100-mesh sieve, 400 mg of honeysuckle powder was weighed and dispersed in 40 mL of water, mixed and transferred to a 100 mL tetrafluoroethylene autoclave, and hydrothermally reacted at 190 ° C for 8 hours. After the reaction was completed, the autoclave was transferred to room temperature for cooling, and the autoclave was opened after the autoclave was completely cooled. The product after the reaction was transferred to a 50 mL centrifuge tube, centrifuged at a speed of 10000 rpm / min for 10 minutes, and the supernatant was taken to remove impurities, and this step was repeated three times. The final supernatant was transferred to a 1000 Da dialysis bag for 72 hours for purification, and the water was changed every 3 hours.

[0052] Example 4

[0053] A honeysuckle carbon dot nanozyme, the preparation method of which is as follows:

[0054] Honeysuckle was dried and ground into powder, passed through a 100-mesh sieve, 400 mg of honeysuckle powder was weighed and dispersed in 40 mL of water, mixed and transferred to a tetrafluoroethylene autoclave with a volume of 100 mL, and hydrothermally reacted at 190 ° C for 12 hours. After the reaction was completed, the autoclave was transferred to room temperature for cooling, and the autoclave was opened after the autoclave was completely cooled. The product after the reaction was transferred to a 50 mL centrifuge tube, centrifuged at a speed of 10000 rpm / min for 10 minutes, and the supernatant was taken to remove impurities, and this step was repeated three times. The final supernatant was transferred to a 900 Da dialysis bag for 72 hours for purification, and the water was changed every 3 hours.

[0055] Example 5

[0056] A honeysuckle carbon dot nanozyme, the preparation method of which is as follows:

[0057] Honeysuckle was dried and ground into powder, passed through a 100-mesh sieve, 400 mg of honeysuckle powder was weighed and dispersed in 40 mL of water, mixed and transferred to a tetrafluoroethylene autoclave with a volume of 100 mL, and hydrothermally reacted at 190 ° C for 12 hours. After the reaction was completed, the autoclave was transferred to room temperature for cooling, and the autoclave was opened after the autoclave was completely cooled. The product after the reaction was transferred to a 50 mL centrifuge tube, centrifuged at a speed of 10000 rpm / min for 10 minutes, and the supernatant was taken to remove impurities, and this step was repeated three times. The final supernatant was transferred to a 1100 Da dialysis bag for 72 hours for purification, and the water was changed every 3 hours.

[0058] Examples 1 to 5 all prepared honeysuckle carbon dot nanozymes with expected effects, and the performance of the honeysuckle carbon dot nanozymes prepared in each example is basically the same. Therefore, the following only takes the honeysuckle carbon dot nanozyme prepared in Example 1 as an example to illustrate it.

[0059] Figure 1 The transmission electron microscope of honeysuckle carbon dots was used to observe the morphological characteristics of honeysuckle carbon dots. The results showed that the honeysuckle carbon dot nanozymes were spherical structures of 2nm to 3nm under the transmission electron microscope. The small size indicated that the honeysuckle carbon dots had a higher specific surface area and could play a more powerful role. In addition, the high-resolution transmission microscope can observe that the lattice of honeysuckle carbon dots is about 0.21nm, which is consistent with the (100) crystal plane of graphite carbon.

[0060] Figure 2 It shows that the honeysuckle carbon dot nanozyme has fluorescent properties and a fluorescence lifetime of 3.018ns. The fluorescence lifetime of the honeysuckle carbon dot nanozyme prepared in other examples is 2.5-3.5ns.

[0061] Experimental Example 1

[0062] Free radical scavenging effect of honeysuckle carbon dot nanozymes

[0063] Honeysuckle carbon dot nanozymes were used to test their activity in scavenging different free radicals. The specific steps are as follows:

[0064] Mix 0.2 mL of 7.4 mmol / L 2,2'-azobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and 0.2 ml of 2.6 mmol / L K2S2O8, and place at room temperature for 12 hours in the dark. Dilute the above solution 45 times to A with pH 7.4 phosphate buffer. 734nm =0.7±0.02, constructed the ABTS free radical generation system, used 20mmol / L ferrous sulfate and 100mmol / L hydrogen peroxide to produce hydroxyl free radicals by Fenton reaction to construct the hydroxyl free radical generation system, used xanthine oxidase to oxidize xanthine to generate superoxide anion to construct the superoxide anion generation system, prepared 0, 2.5, 5, 10, 15, 20μg / mL different concentrations of honeysuckle carbon dot nanozymes and incubated them with different free radical systems respectively, and detected the free radical scavenging situation by UV absorption spectroscopy, EPR and microplate reader according to the time kinetics.

[0065] The results of scavenging ABTS free radicals are as follows Figure 3 As shown in (a), ABTS has a characteristic ultraviolet absorption peak at 750nm. As the concentration of honeysuckle carbon dot nanozyme continues to increase, the characteristic absorption peak of ABTS at 750nm gradually decreases, indicating that honeysuckle carbon dot nanozyme scavenges ABTS free radicals.

[0066] The scavenging experiment of hydroxyl free radicals is as follows Figure 3 As shown in (b), using the Fenton reaction Fe 2+ It reacts with H2O2 to generate a large number of toxic hydroxyl radicals. In aqueous solution, the hydroxyl radicals are captured by the capture agent DMPO. The captured products can be detected by the electron self-selected resonance wave instrument EPR. From the spectrum, it can be seen that the intensity of hydroxyl radicals detected by EPR is the highest when no honeysuckle carbon dot nanozyme is added. With the increase of honeysuckle concentration, the signal of hydroxyl radicals begins to decrease, which indicates the scavenging activity of honeysuckle carbon dot nanozyme on hydroxyl radicals.

[0067] The superoxide free radical scavenging experiment Figure 3As shown in (c), a superoxide radical generation system is constructed by simulating the generation of superoxide radicals in vivo using xanthine and xanthine oxidase, and superoxide radicals are captured in aqueous solution using BMPO. The captured products can be detected by EPR. From the spectrum, it can be seen that the intensity of superoxide radicals detected by EPR is the highest when honeysuckle carbon dot nanozyme is not added. As the concentration of honeysuckle increases, the signal of superoxide radicals begins to decrease, which indicates that honeysuckle carbon dot nanozyme has scavenging activity against superoxide radicals.

[0068] Experimental Example 2

[0069] Improvement effect of honeysuckle carbon dot nanozyme on lung injury

[0070] The prepared honeysuckle carbon dot nanozyme was purified by dialysis and freeze-dried to obtain a solid powder, which was used as a reactive oxygen scavenger to verify its effectiveness in an in vivo lung injury improvement experiment.

[0071] Forty C57BL / 6 female mice aged 6 to 8 weeks were selected and randomly divided into four groups: blank control group, model group (labeled as LPS group), low-dose drug group (labeled as LPS+Hy-CDs1 group) and high-dose drug group (labeled as LPS+Hy-CDs2 group), with 10 mice in each group. The mice in the low-dose drug group were injected with 5 mg / kg of honeysuckle carbon dot nanozyme through the tail vein, the mice in the high-dose group were injected with 10 mg / kg of honeysuckle carbon dot nanozyme through the tail vein, and the mice in the blank group were injected with 10 mg / kg of normal saline through the tail vein. The mice in each group were anesthetized by gas anesthesia with isoflurane, and the model group, low-dose drug group and high-dose drug group were all intranasally instilled with 10 μg of LPS to establish a mouse lung injury model. The mice were killed 12 hours later, and the lung organs of the mice were collected. The treatment of lung injury in each group of mice was observed by histopathological sections.

[0072] like Figure 4 As shown, it was observed that both the low-dose drug group and the high-dose drug group could effectively alleviate inflammatory damage in the lungs of mice, inhibit the recruitment of inflammatory cells, and restore the normal structure of lung cells, indicating that the honeysuckle carbon dot nanozyme provided by the present invention can effectively improve lung damage.

[0073] Experimental Example 3

[0074] Improvement effect of honeysuckle carbon dot nanozyme on pulmonary ischemia-reperfusion

[0075] The prepared honeysuckle carbon dot nanozyme was purified by dialysis and then freeze-dried to obtain a solid powder, which was used as a reactive oxygen scavenger for the next in vivo experimental verification.

[0076] Forty C57BL / 6 female mice aged 6 to 8 weeks were selected and randomly divided into four groups: blank control group, model group, low-dose drug group and high-dose drug group. The model group was marked as LPS group, the low-dose drug group was marked as LPS+Hy-CDs1 group, and the high-dose drug group was marked as LPS+Hy-CDs2 group, with 10 mice in each group. The mice in the low-dose drug group were injected with 5 mg / kg of honeysuckle carbon dot nanozyme through the tail vein, the mice in the high-dose group were injected with 10 mg / kg of honeysuckle carbon dot nanozyme through the tail vein, and the mice in the blank group were injected with 10 mg / kg of normal saline through the tail vein. The left hilum of the mice in the model group, low-dose drug group and high-dose drug group were ligated for 60 minutes and reperfused for 240 minutes to establish the mouse lung ischemia-reperfusion model. The mice were killed 12 hours later, and the lung organs of the mice were collected. The treatment of the lungs of each group of mice was observed by histopathological sections.

[0077] like Figure 5 As shown, it was observed that both the low-dose drug group and the high-dose drug group could effectively alleviate the damage caused by reperfusion in the mouse lungs, inhibit the perfusion of blood cells in the lungs, and maintain the normal alveolar cell structure, indicating that the honeysuckle carbon dot nanozyme provided by the present invention can effectively improve lung ischemia-reperfusion.

[0078] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0079] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0080] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. An application of honeysuckle carbon dot nanozyme in the preparation of a drug for the treatment of pulmonary ischemia-reperfusion, characterized in that: The honeysuckle carbon dot nanozyme is obtained by hydrothermal carbonization of honeysuckle, wherein the hydrothermal carbonization is a hydrothermal reaction at 185° C. to 205° C. for 8 h to 12 h. The average particle size of the honeysuckle carbon dot nanozyme is 3 nm to 5 nm, and the fluorescence lifetime is 2.5 ns to 3.5 ns.

2. The use according to claim 1, characterized in that: After the hydrothermal reaction is completed, impurities are removed by centrifugation, and then the honeysuckle carbon dot nanozyme is obtained by purification through dialysis.

3. The use according to claim 2, characterized in that: The molecular weight cutoff of the dialysis is 900Da~1100Da.

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