CN113477068A

CN113477068A - Preparation method and addition method of cigarette smoke active oxygen scavenger

Info

Publication number: CN113477068A
Application number: CN202110596356.6A
Authority: CN
Inventors: 李宏; 谭伟龙; 毛应华; 汪春晖; 荣署
Original assignee: Eastern Theater Disease Prevention And Control Center Of Pla
Current assignee: Eastern Theater Disease Prevention And Control Center Of Pla
Priority date: 2021-05-30
Filing date: 2021-05-30
Publication date: 2021-10-08
Anticipated expiration: 2041-05-30
Also published as: CN113477068B

Abstract

The invention discloses a preparation method and an adding method of a cigarette smoke active oxygen scavenger, wherein the preparation method comprises the following steps: nanometer cerium oxide CeAL modified by using alendronic acid is used as a raw material, and an active oxygen scavenger for cigarette smoke is formed by adjusting the surface valence state so as to be suitable for cigarettes of different brands; the adding method comprises the following steps: adding a CeAL aqueous solution to the hookah; dripping CeAL mixed liquid into the tobacco leaves or the filter tip; adding CeAL powder to the junction of the filter tip and the tobacco leaf; add CeAL powder to the mouthpiece filter. The nano cerium oxide CeAL is a nano particle with good dispersibility, the nano particle is used as a metal enzyme, exerts superoxide dismutase imitating activity, hydroxyl free radical scavenging activity and catalase imitating activity, has thermal stability and is influenced by the valence state of the nano particle and the external pH value to a certain extent; the CeAL product can be added into cigarette product to effectively remove active oxygen in cigarette smoke and reduce harm of smoking to health.

Description

Preparation method and addition method of cigarette smoke active oxygen scavenger

Technical Field

The invention relates to the field of tobacco product additives, in particular to a preparation method and an addition method of a cigarette smoke active oxygen scavenger based on nano cerium oxide.

Background

Tobacco prevalence seriously threatens human Health, and smoking is recognized as an important risk factor for eight major death diseases worldwide (Lancet oncol.2019,20, e208, Public Health 2016,37, 149). During the burning process of the cigarette, a large amount of reactive oxygen species (ROS, every time of smoking) is generated>10¹⁶Molecules), such as oxygen radicals (. O)₂ ^-) OH, hydrogen peroxide (H)₂O₂) Etc., current cigarette filtration technology has not been able to effectively scavenge the above ROS (Acad sci.1993,686,12, Nanoscale 2017,9, 3952). These ROS are inhaled into the respiratory system through smoke, directly damage cells, and induce oxidative stress, and take biomacromolecules as substrates to generate other ROS with higher activity and toxicity, thereby causing damage to lung tissues and immune system (Environ Health Perscope. 1997,105, 875). Research shows that ROS in cigarette smoke is an important reason for causing various diseases such as lung cancer, chronic obstructive pulmonary disease, pulmonary heart disease and the like (Thromb Vasc biol.2014,34,509). Therefore, the development of the efficient and nontoxic ROS scavenger for cigarette smoke has great significance for reducing the harm of smoking.

At home and abroad, researches are carried out to improve the cigarette filter by adding antioxidants, such as pycnogenol, grape seed extract, lycopene, vitamin C and other natural plant extracts, so that ROS in cigarette smoke is reduced, and certain progress is achieved. The binary composite cigarette filter tip based on ferulic acid and activated carbon is prepared by Changsha cigarette factories (patent CN1656952A), and the ROS content in smoke can be reduced by 20.6% -29.4%; zhengzhou tobacco institute (patent CN102860583A) adds the sesame leaf extract to the cigarette filter, and the ROS clearance rate of cigarette smoke is 63.8%; the tobacco industry company in Hubei (patent CN101606751A) uses lycopene as an antioxidant, and the ROS clearance reaches 50.5%. However, these antioxidants have the disadvantages of poor thermal stability, poor antioxidant capacity and single ROS scavenging species. A new cigarette filter additive has been recently reported, which can effectively improve the scavenging efficiency of smoke ROS by catalyzing the catalase reaction with manganoporphyrin derivatives, however, the application of the additive is still limited by the poor heat resistance of the reaction in hot smoke gel (Funct mater.2015,25,5159). Therefore, the development of ROS scavenger for cigarette smoke with strong activity and high thermal stability is particularly important.

In recent years, some metal nanomaterials, such as nano vanadium oxide, nano gold, nano manganese, nano copper, etc., have been gradually reported and confirmed to have antioxidant enzyme mimic activity (nanoenzyme) (Chem commu.2007, 1056, angelw Chem Int ed.2015,54,1832, J Mater Chem b.2016,4,7423). The nano materials have large surface area and high density of active sites, so that the nano materials have higher catalytic and antioxidant activities. Compared with natural biological enzyme, the nano enzyme has higher stability and better tolerance to the external environment, and provides a foundation for exploring a novel cigarette smoke ROS scavenger.

Surface Ce of nano cerium oxide³⁺/Ce⁴⁺Coexisting and mutually transforming to ensure that the nano-enzyme has multiple antioxidant enzyme mimic activities (nanoscale.2011,3, 1411-. Research reports that the nano cerium oxide can effectively remove O₂ ^-，H₂O₂OH, NO, and other ROS, and has high efficiency, long-lasting effect and reproducibility [ Antioxidants,2016,5(2): 15)]So that the ROS scavenging agent can be applied to the scavenging of cigarette smoke. However, most of the previous related researches are established on the basis of the insufficiently modified nano cerium oxide, and have the defects of poor dispersibility, stability and biocompatibility of nano particles and the like, so that the biological and catalytic activities of the nano particles are influenced to a certain extent. Therefore, the nano cerium oxide is subjected to surface modification, and the stability of the nano cerium oxide is improvedAnd the enzyme activity, explores a novel efficient and stable ROS scavenger for cigarette smoke, and has important significance for reducing the harm of smoking to health.

Disclosure of Invention

Aiming at the defects of the existing ROS removing technology of cigarette smoke and the key problems of poor thermal stability, low removing efficiency, single removing type and the like of the existing natural antioxidant, the invention provides a preparation method and an adding method of a cigarette smoke active oxygen scavenger based on nano cerium oxide, thereby reducing the harm of smoking to health.

In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:

a preparation method of a cigarette smoke active oxygen scavenger based on nano cerium oxide comprises the following steps:

the method is characterized in that nanometer cerium oxide CeAL modified by alendronic acid is used as a raw material, and a cigarette smoke active oxygen scavenger is formed through surface valence state adjustment and is added into a cigarette product.

As a further improved technical scheme of the invention, the synthesis method of the raw materials comprises the following steps:

synthesizing nano cerium oxide by a microemulsion method;

preparing nanometer cerium oxide CeAL modified by alendronic acid by adopting a two-phase ligand exchange experiment.

As a further improved technical scheme of the invention, the synthesis of the nano cerium oxide by adopting the microemulsion method specifically comprises the following steps:

dissolving a certain amount of docusate sodium in toluene, adding a certain amount of cerium nitrate, reacting at room temperature for 45-60 min under strong magnetic stirring, and slowly dropwise adding 3mL of H into the reaction system₂O₂Continuously reacting for 1h, standing at room temperature for 8h-72h, and dispersing the synthesized nano cerium oxide in the upper toluene phase; 3mL of H₂O₂The dropping speed of (a) is 100 to 400. mu.L/min.

As a further improved technical solution of the present invention, the preparation of the nano cerium oxide CeAL modified by alendronic acid using a two-phase ligand exchange experiment specifically comprises the following steps:

taking a certain amount of Na₂CO₃Dissolving with Allen phosphoric acid in ultrapure water, adding toluene phase containing nano cerium oxide on the upper layer, and reacting at room temperature for 24-48 h under strong magnetic stirring; collecting the above reaction body fluid, centrifuging at 3000rpm/min-5000rpm/min for 3min-5min, taking the lower layer water phase, transferring into a snake skin dialysis bag with cut-off molecular weight of 10000, dialyzing with ultrapure water for 24h-48h, changing water every 5h-8h, and changing water amount for 3L/time-5L/time; finally obtaining the nanometer cerium oxide CeAL modified by the Allan phosphoric acid with the grain size of 3nm-5 nm.

As a further improved technical scheme of the invention, the docusate sodium, the toluene, the cerium nitrate and the H₂O₂The dosage is 0.3g to 0.5g, 30mL, 1mL to 2mL and 2mL to 4mL respectively;

the volume ratio of the toluene phase containing nano cerium oxide on the upper layer to the ultrapure water on the lower layer is 2-3: 1;

said Na₂CO₃The mass ratio of the alpha-phosphoric acid to the alpha-phosphoric acid is 10-15: 1.

As a further improved technical scheme of the invention, the specific method for adjusting the surface valence of the raw material comprises the following steps:

adding reducing substances or hydrogen peroxide with a certain concentration into the raw materials, stirring with strong magnetic force, reacting at room temperature for 1-8 h, and dialyzing the reaction product in a dialysis bag for 24-48 h.

As a further improved technical scheme of the invention, the reducing substance comprises ascorbic acid, reducing glutathione, SOD enzyme or vitamin E, and the concentration of the reducing substance is 0.005mol/L-1 mol/L; the concentration of the hydrogen peroxide is 0.01mol/L-1 mol/L.

In order to achieve the technical purpose, the invention adopts another technical scheme as follows:

an adding method for adding a cigarette smoke active oxygen scavenger based on nano cerium oxide into a cigarette product comprises a plurality of adding modes, wherein the adding modes are as follows:

(1) adding the CeAL water solution to the hookah;

(2) dripping CeAL mixed solution into the tobacco leaves or the filter tips;

(3) drying the CeAL aqueous solution, and adding CeAL powder to the junction of the filter tip and the tobacco leaves;

(4) drying the CeAL water solution, and adding CeAL powder into the cigarette holder filter.

As a further improved technical scheme of the invention, the Ce element content in the CeAL aqueous solution added into the hookah is 0.01mg/mL-1 mg/mL;

the CeAL mixed solution used for dropping into the tobacco leaves or the filter tips is a mixed solution of a CeAL aqueous solution and ethanol, and the ratio of the CeAL aqueous solution to the ethanol is 1: 1.5-3; the dosage of the CeAL mixed solution dripped into one filter tip is 50-200 mu L, and the concentration is 0.1-1 mg/mL; the dosage of the CeAL mixed solution dripped into one tobacco leaf is 20-100 mu L, and the concentration is 0.2-1 mg/mL;

the drying mode of the CeAL water solution is as follows: vacuum drying at 30-50 deg.c for 16-24 hr or freeze drying at-40 deg.c to-60 deg.c for 16-48 hr; the amount of CeAL powder added to the junction of one filter tip and the tobacco leaf is 5mg-15 mg;

the amount of CeAL powder added to the mouthpiece filter is 10mg-100 mg.

Compared with the existing ROS removing technology of cigarette smoke, the invention has great originality and obvious technical progress:

the invention opens up a new application field for the nano cerium oxide. The Al is adopted to modify the nano cerium oxide, so that the stability and the dispersibility of the nano particles are greatly improved, the nano particles can be fully contacted with smoke, and the oxidation resistance is favorably exerted. CeAL oxidation resistance activity thermostability is strong, receives external environment change influence less, can high-efficiently clear away the multiple ROS that cigarette burning produced, alleviates the harm of smoking to health, has reproducibility simultaneously, can add to can dismantle in the cigarette holder filter, and used repeatedly practices thrift the cost. Through adjusting the surface valence state of CeAL, regulate and control its efficiency of cleaing away to different kinds of ROS, can develop the specificity product that is applicable to different brands cigarette. The ROS removing technology for cigarette smoke does not reduce the taste of the cigarette, and meanwhile, the method is green and economical in reaction, simple in process, low in preparation cost, flexible and easy to control in regulation and control mode, capable of realizing industrial production and good in application prospect in developing low ROS safe and environment-friendly cigarette products.

Drawings

FIG. 1 (a) is a high-resolution transmission electron microscopy analysis chart of CeAL.

In FIG. 1, (b) is a hydrated particle size distribution diagram of CeAL.

FIG. 1 (c) is a diagram showing X-ray photoelectron spectroscopy (XPS) analysis and valence state simulation of CeAL.

FIG. 1 (d) is a thermogravimetric analysis of CeAL.

FIG. 1 (e) is an X-ray diffraction (XRD) analysis chart of CeAL and AL.

FIG. 1 (f) is a Fourier transform infrared (FT-IR) spectrum of CeAL and AL.

Fig. 2 (a) is an XPS analysis chart of Ce3d after adjustment of the CeAL valence state.

FIG. 2 (b) is a Ce3d valence state simulation diagram of low Ce3+ concentration CeAL (Ce3+ L).

FIG. 2 (c) is a Ce3d valence state simulation diagram of CeAL (Ce3+ M) with a middle Ce3+ concentration.

FIG. 2 (d) is a Ce3d valence state simulation diagram of CeAL (Ce3+ H) with high Ce3+ concentration.

In FIG. 3, (a) is CeAL scavenging-O₂ ^-Ultraviolet absorption spectrum of (2).

In FIG. 3, (b) is CeAL scavenging-O₂ ^-Schematic diagram of ultraviolet absorption and clearance.

In FIG. 3, (c) is CeAL scavenging-O₂ ^-Clearance rate and SOD enzyme clearance O₂ ^-The clearance of (c) is compared to the graph.

FIG. 4 (a) is a diagram showing the CeAL-OH eliminating ultraviolet absorption spectrum.

FIG. 4 (b) is a diagram showing the UV absorption and removal rate of CeAL OH.

FIG. 4 (c) is a graph comparing the CeAL OH clearance with the ascorbic acid OH clearance.

In FIG. 5 (a) is CeAL clearance of H₂O₂Dissolved oxygen concentration-time profile of (a).

FIG. 5 (b) shows catalase elimination H₂O₂Dissolved oxygen concentration-time profile of (a).

FIG. 5 (c) shows that CeAL and catalase scavenge H₂O₂Graph comparing dissolved oxygen concentration at 20 min.

In FIG. 6, (a) is O of CeAL and SOD enzyme at different temperatures₂ ^-Comparison of the scavenging activity.

FIG. 6 (b) is a graph comparing the OH scavenging activity of CeAL and ascorbic acid at different temperatures.

FIG. 6 (c) is a graph showing the CeAL clearance of H at different temperatures₂O₂Dissolved oxygen concentration-time profile of (a).

In FIG. 6 (d) is catalase elimination H at different temperatures₂O₂Dissolved oxygen concentration-time profile of (a).

In FIG. 7 (a) is the change in pH vs. CeAL. O₂ ^-Graph of the effect of scavenging activity.

FIG. 7 (b) is a graph showing the effect of pH change on CeAL. OH scavenging activity.

FIG. 7 (c) is the change in pH vs. CeAL H₂O₂Graph of the effect of scavenging activity.

In FIG. 8, (a) is the ratio of the valence states of different surfaces to CeAL. O₂ ^-Graph of the effect of scavenging activity.

FIG. 8 (b) is a graph showing the effect of different surface valence ratios on the OH scavenging activity of CeAL.

FIG. 8 (c) is H of CeAL in the ratio of valence states of different surfaces₂O₂Graph of the effect of scavenging activity.

In FIG. 9, (a) is CeAL vs. O in cigarette smoke₂ ^-Clear uv spectrum.

FIG. 9 (b) is a fluorescence spectrum of CeAL for OH elimination in cigarette smoke.

FIG. 9 (c) shows the CeAL vs. H in cigarette smoke₂O₂Cleared fluorescence spectrum.

In FIG. 9, (d) is CeAL vs. O in cigarette smoke₂ ^-、·OH、H₂O₂The cleaning efficiency map of (1).

FIG. 9 (e) is a plot of the fluorescence spectrum of CeAL for total ROS clearance in cigarette smoke.

FIG. 9 (f) is a graph of the efficiency of CeAL in the clearance of total ROS in cigarette smoke.

FIG. 10 (a) is a graph showing HE staining of mouse lung tissue.

FIG. 10 (b) is a graph showing the concentration of IL-1. beta. in mouse lung tissue lysate.

FIG. 10 (c) is a graph showing the concentration of mouse lung tissue lysate TNF-. alpha.in FIG.

Detailed Description

The present invention will be described in further detail with reference to the following examples and drawings, but the present invention is not limited thereto. On the premise of the conception of the invention, the simple modification of the invention belongs to the protection scope of the invention.

Preparation method example 1: a preparation method of a cigarette smoke active oxygen scavenger based on nano cerium oxide comprises the following steps:

the method is characterized in that nanometer cerium oxide CeAL modified by alendronic acid is used as a raw material, and a cigarette smoke active oxygen scavenger is formed through surface valence state adjustment, and is added into a cigarette product to achieve the purpose of scavenging cigarette smoke active oxygen (ROS).

The preparation method of the raw material CeAL comprises the following steps:

(1) and synthesizing the nano cerium oxide by adopting a micro-emulsion method: 0.4584g of docusate sodium (surfactant) was dissolved in 30mL of toluene, and 1.5mL of cerium nitrate [ Ce (NO)₃)₃.6H₂O](0.1M) and reacted at room temperature for 45min under strong magnetic stirring. 3mL of H was slowly added dropwise to the above reaction system₂O₂(the dropping speed is 200 mu L/min), the reaction is continued for 1h, the mixture is kept standing for 24h at room temperature, and the synthesized nano cerium oxide is dispersed in the upper toluene phase. (2) Preparing nanometer cerium oxide CeAL modified by alendronic acid by adopting a two-phase ligand exchange experiment: collecting 600mg of Na₂CO₃60mg of Alendronic Acid (AL) was dissolved in 15mL of ultrapure water, and 30mL of the above toluene phase containing nano-cerium oxide was added thereto, followed by reaction at room temperature for 24 hours under strong magnetic stirring. Collecting reaction liquid, centrifuging at 3000rpm/min for 3min, transferring the lower water phase into a dialysis bag with snake skin molecular weight cutoff of 10000, dialyzing with ultrapure water for 24h, changing water once every 5h, and changing water volume for 3L/time to remove free cerium salt, AL and other inorganic ions. Finally obtainingAL-modified nano cerium oxide CeAL (15mL, C) with average particle size of 3-5 nm_Ce0.43mg/mL), hydrated particle size 6nm to 8nm, Ce³⁺The content is 46.8%. The high resolution transmission electron microscope (HR-TEM), hydrated particle size analysis (DLS), X-ray photoelectron spectroscopy (XPS) analysis, valence state simulation, thermogravimetric analysis (TGA), X-ray diffraction (XRD) analysis and Fourier transform infrared spectroscopy (FT-IR) analysis diagrams are shown in (a) - (f) of the attached figure 1.

The raw material CeAL is subjected to surface valence state adjustment so as to be suitable for cigarettes of different brands, wherein the specific method for adjusting the raw material CeAL through the surface valence state comprises the following steps:

2mL of CeAL aqueous solution (Ce)³⁺M,C_Ce0.2mg/mL), 0.2mL of ascorbic acid (high Ce) with a concentration of 0.01mol/L was added³⁺Preparation of nano cerium oxide) or 0.2mL of H with concentration of 0.05mol/L₂O₂(high Ce)⁴⁺Preparation of nano cerium oxide), reacting for 4h at room temperature under strong magnetic stirring, taking the reaction product, transferring into a dialysis bag, dialyzing with ultrapure water for 24h to respectively obtain high Ce³⁺Content of CeAL (Ce)³⁺H) And low Ce³⁺Content of CeAL (Ce)³⁺L). After the surface valence state of CeAL is adjusted, the color of the solution is obviously changed (Ce)⁴⁺Yellow), XPS showed Ce³⁺L、Ce³⁺M、Ce³⁺Ce in H³⁺The contents of (a) to (d) in FIG. 2 were 38.1%, 46.8%, and 59.8%, respectively.

Preparation method example 2: a preparation method of a cigarette smoke active oxygen scavenger based on nano cerium oxide comprises the following steps:

The preparation method of the raw material CeAL comprises the following steps:

(1) and synthesizing the nano cerium oxide by adopting a micro-emulsion method: dissolving docusate sodium 0.3g in 30mL toluene, adding cerium nitrate [ Ce (NO)₃)₃.6H₂O](0.1M) and reacted at room temperature for 50min under strong magnetic stirring. To the above-mentioned reactant2mL of H was slowly added dropwise to the system₂O₂(the dropping speed is 100 mu L/min), the reaction is continued for 1h, the mixture is kept standing for 8h at room temperature, and the synthesized nano cerium oxide is dispersed in the upper toluene phase. (2) Preparing nanometer cerium oxide CeAL modified by alendronic acid by adopting a two-phase ligand exchange experiment: 720mg of Na is taken₂CO₃60mg of Alendronic Acid (AL) was dissolved in 12mL of ultrapure water, and 30mL of the above toluene phase containing nano-cerium oxide was added thereto, followed by reaction at room temperature for 36 hours under strong magnetic stirring. Collecting reaction liquid, centrifuging at 4000rpm/min for 4min, transferring the lower-layer water phase into a dialysis bag with the snake skin molecular weight cutoff of 10000, dialyzing with ultrapure water for 36h, changing water once every 6h, and changing water for 4L/time to remove free cerium salt, AL and other inorganic ions. Finally obtaining AL modified nano cerium oxide CeAL (15mL, C) with the average grain diameter of 3nm-5nm_Ce0.38mg/mL), hydrated particle size 5nm to 10nm, Ce³⁺The content is 40.1%. The high-resolution transmission electron microscope (HR-TEM), hydrated particle size analysis (DLS), X-ray photoelectron spectroscopy (XPS), valence state simulation, thermogravimetric analysis (TGA), X-ray diffraction (XRD) analysis and Fourier transform infrared spectroscopy (FT-IR) analysis are shown in the same manner as the analysis graphs (a) to (f) in the attached figure 1.

2mL of CeAL aqueous solution (Ce)³⁺M,C_Ce0.2mg/mL), 0.2mL of ascorbic acid (high Ce) with a concentration of 0.005mol/L was added³⁺Preparation of nano cerium oxide) or 0.2mL of H with concentration of 0.01mol/L₂O₂(high Ce)⁴⁺Preparation of nano cerium oxide), reacting for 1h at room temperature under strong magnetic stirring, taking the reaction product, transferring into a dialysis bag, dialyzing with ultrapure water for 36h to respectively obtain high Ce³⁺Content of CeAL (Ce)³⁺H) And low Ce³⁺Content of CeAL (Ce)³⁺L). After the surface valence state of CeAL is adjusted, the color of the solution is obviously changed (Ce)⁴⁺Yellow), XPS showed Ce³⁺L、Ce³⁺M、Ce³⁺Ce in H³⁺The contents of (A) are 37.9%, 40.1% and 49.4%, respectively.

Preparation method example 3: a preparation method of a cigarette smoke active oxygen scavenger based on nano cerium oxide comprises the following steps:

The preparation method of the raw material CeAL comprises the following steps:

(1) and synthesizing the nano cerium oxide by adopting a micro-emulsion method: dissolving docusate sodium 0.5g in 30mL toluene, adding cerium nitrate [ Ce (NO)₃)₃.6H₂O](0.1M) and reacted at room temperature for 60min under strong magnetic stirring. 4mL of H was slowly added dropwise to the above reaction system₂O₂(the dropping speed is 400 mu L/min), the reaction is continued for 1h, the mixture is kept standing for 72h at room temperature, and the synthesized nano cerium oxide is dispersed in the upper toluene phase. (2) Preparing nanometer cerium oxide CeAL modified by alendronic acid by adopting a two-phase ligand exchange experiment: taking 900mg of Na₂CO₃60mg of Alendronic Acid (AL) was dissolved in 10mL of ultrapure water, and 30mL of the above toluene phase containing nano-cerium oxide was added thereto, followed by reaction at room temperature for 48 hours under strong magnetic stirring. Collecting reaction liquid, centrifuging at 5000rpm/min for 5min, transferring the lower water phase into a dialysis bag with snake skin molecular weight cutoff of 10000, dialyzing with ultrapure water for 48h, changing water once every 8h, and changing water amount for 5L/time to remove free cerium salt, AL and other inorganic ions. Finally obtaining AL modified nano cerium oxide CeAL (10mL, C) with the average grain diameter of 3nm-5nm_Ce0.55mg/mL), hydrated particle size 5nm to 9nm, Ce³⁺The content is 38.4%. The high-resolution transmission electron microscope (HR-TEM), hydrated particle size analysis (DLS), X-ray photoelectron spectroscopy (XPS), valence state simulation, thermogravimetric analysis (TGA), X-ray diffraction (XRD) analysis and Fourier transform infrared spectroscopy (FT-IR) analysis are shown in the same manner as the analysis graphs (a) to (f) in the attached figure 1.

2mL of CeAL aqueous solution (Ce)³⁺M,C_Ce0.2mg/mL), 0.2mL of ascorbic acid (high Ce) was added at a concentration of 1mol/L³⁺Nano oxidationPreparation of cerium) or 0.2mL of H at a concentration of 1mol/L₂O₂(high Ce)⁴⁺Preparation of nano cerium oxide), reacting for 8 hours at room temperature under strong magnetic stirring, taking the reaction product, transferring into a dialysis bag, dialyzing with ultrapure water for 48 hours to respectively obtain high Ce³⁺Content of CeAL (Ce)³⁺H) And low Ce³⁺Content of CeAL (Ce)³⁺L). After the surface valence state of CeAL is adjusted, the color of the solution is obviously changed (Ce)⁴⁺Yellow), XPS showed Ce³⁺L、Ce³⁺M、Ce³⁺Ce in H³⁺The contents of (A) are 34.2%, 38.4% and 51.3%, respectively.

The reducing substance used for the surface valence adjustment in production method example 1, production method example 2, and production method example 3 may be: reduced glutathione, SOD enzyme or vitamin E. The synthesis methods of nano cerium oxide described in the reaction steps of preparation method example 1, preparation method example 2, and preparation method example 3 include a pyrolysis method, a hydrothermal method, or a precipitation method in addition to the microemulsion method.

The various properties of the starting material CeAL were analyzed below.

Analytical example 1: CeAL SOD enzyme mimic activity assay:

through p.O₂ ^-Clearance evaluation of CeAL SOD enzyme mimic activity, the mechanism is riboflavin under light irradiation O₂ ^-，·O₂ ^-The blue product produced by reaction with nitrotetrazolium chloride (NBT) has a maximum absorption peak at 560 nm. Specifically, 200. mu.L of LEDTA (0.1mol/L), 75. mu.L of NBT (2mmol/L) and 2.9mL of PBS (10mmol/L, pH7.8) were mixed to prepare a working solution, which was incubated at 37 ℃ for 5min in the absence of light. 50 μ L riboflavin (0.6mmol/L) and 50 μ L CeAL (C) at different concentrations were taken_Ce0-5. mu.g/mL), adding 100. mu.L of the above working solution, incubating at 37 deg.C in the dark for 5min, illuminating with 27W desk lamp for 2min, and immediately determining OD_560nm。·O₂ ^-Clearance rate [ [ (A)₁-A₂)/(A₁-A₀)]X 100% where A₀OD being NBT_560nmValue, A₁And A₂OD when 50. mu.L of ultrapure water and different concentrations of CeAL were added_560nmThe value is obtained. 0.25. mu.g/mCeAL of L to O₂ ^-The clearance rate reaches 100 percent and is equivalent to the activity of SOD enzyme with 50U/mL. Its ultraviolet absorption spectrum, OD_560nmValue corresponding to₂ ^-Clearance rate, different concentrations of CeAL and SOD enzyme pair O₂ ^-Comparative figures of clearance are shown in fig. 3 (a) - (c).

Analysis example 2: assay for CeAL. OH scavenging Activity:

the mechanism is Fe²⁺And H₂O₂The Fenton reaction produces OH, which reacts with Salicylic Acid (SA) to produce a bluish-violet product with an absorption maximum at 572 nm. Specifically, 20. mu.L of FeSO was taken₄(2mmol/L)、20μL H₂O₂(2mmol/L) to 40. mu.L of different concentrations of CeAL (C)_Ce0-100. mu.g/mL), incubated at room temperature for 3min, 20. mu.L of SA (10mmol/L) was added, and OD was immediately determined_572nm. OH clearance ═ A₁-A₂)/(A₁-A₀)]X 100% where A₀Is OD of SA_572nmValue, A₁And A₂Respectively OD when 40. mu.L of ultrapure water and different concentrations of CeAL were added_572nmThe value is obtained. The CeAL at 20. mu.g/mL reached 100% clearance for OH, which was comparable to the activity of 1. mu.M Ascorbic Acid (AA). Its ultraviolet absorption spectrum, OD_572nmThe values are shown in figure 4 (a) - (c) for OH clearance, comparing the different concentrations of CeAL with ascorbic acid.

Analytical example 3: CeAL catalase mimic activity assay:

by nano cerium oxide and H₂O₂Direct reaction to produce O₂The ability of CeAL to mimic catalase-mimicking activity. 3mL of CeAL (C) at different concentrations were taken_Ce0-100 mug/mL), pumping for 10min with a vacuum pump to remove dissolved oxygen in the liquid, adding 100 μ L of different concentrations of H₂O₂(0-500mmol/L), and immediately measuring the concentration of dissolved oxygen in the reaction for 1-20 min by adopting a dissolved oxygen meter. Concentration of dissolved oxygen in the reaction system according to reaction time, CeAL and H₂O₂The concentration increased, suggesting that CeAL has catalase mimic activity, 1U g/mL CeAL and 50U/mL catalase activity equivalent. It is cleared of H₂O₂Dissolved oxygen concentration-time curve of (1), Catalase scavenging H₂O₂Concentration-time curve of dissolved oxygen, CeAL and catalase-scavenging H₂O₂The 20 th minute dissolved oxygen concentration is shown in the graphs (a) to (c) in FIG. 5.

Analysis example 4: thermostability assay of CeAL antioxidant activity:

the experimental method of this procedure was the same as in analysis example 1, analysis example 2, and analysis example 3, except that CeAL or natural antioxidant was incubated at different temperatures for 5min before the experiment. Within the temperature range of 20 ℃ to 90 ℃, there was no significant loss of the SOD enzyme-mimicking activity of CeAL,. OH-scavenging activity, catalase-mimicking activity. The natural antioxidants SOD enzyme, ascorbic acid and catalase are quickly inactivated at the temperature of more than 60 ℃, and the temperature of hot cigarette aerosol of the filter tip can reach 80 ℃ at most. O of CeAL and SOD enzymes at different temperatures₂ ^-Comparison of scavenging Activity, comparison of the OH scavenging Activity of CeAL with ascorbic acid at different temperatures, and CeAL scavenging H at different temperatures₂O₂Concentration of dissolved oxygen versus time curves, Catalase scavenging H at different temperatures₂O₂The dissolved oxygen concentration-time profiles of (a) to (d) in FIG. 6 are shown.

Analysis example 5: effect of pH change on CeAL antioxidant activity:

the experimental method of this process was the same as in analytical examples 1, 2 and 3 except that HCl or NaOH was used to adjust the reaction system to different pH values before the experiment. The pH change (4.5-11.5) has no obvious influence on the SOD enzyme mimicking activity of CeAL; the OH removal activity of the product is slightly reduced under the acidic pH environment; its catalase mimic activity increases with an increase in external pH. O in different pH environments₂ ^-Scavenging ability, OH scavenging ability in different pH environments, H scavenging ability in different pH environments₂O₂Production of O₂The capacity map is shown in fig. 7 (a) - (c).

Analysis example 6: effect of CeAL surface valence state change on its antioxidant activity:

the experimental method of this process was the same as in analysis example 1, analysis example 2 and analysis example 3, except that CeAL (Ce) with different valence ratios was used³⁺L、Ce³⁺M、Ce³⁺H) To carry outAnd (5) carrying out experiments. Ce³⁺The higher the content (Ce)³⁺H) The higher the SOD enzyme-mimicking activity and OH-scavenging activity, the lower the catalase-mimicking activity. The ROS components and the proportions in the cigarette smoke of different brands are different, and products suitable for cigarettes of different brands can be prepared by adjusting the surface valence state of the CeAL. Of different surface valence ratios CeAL O₂ ^-Scavenging capacity, OH scavenging capacity of different surface valence ratios CeAL, scavenging H of different surface valence ratios CeAL₂O₂Production of O₂The capacity map is shown in fig. 8 (a) - (c).

The embodiment also provides an adding method for adding the cigarette smoke active oxygen scavenger based on the nano cerium oxide into the cigarette product, and the adding method of the raw material CeAL is described below.

Addition method example 1: the method for adding the cigarette smoke active oxygen scavenger based on the nano cerium oxide into the cigarette product comprises a plurality of adding modes, which are respectively as follows:

1. adding the CeAL aqueous solution into the hookah, wherein the Ce element content in the CeAL aqueous solution added into the hookah is 0.1 mg/mL.

2. Dripping CeAL mixed liquid into the tobacco leaves or the filter tip; the CeAL mixed solution used for dropping into the tobacco leaves or the filter tips is a mixed solution of a CeAL aqueous solution and ethanol, and the ratio of the CeAL aqueous solution to the ethanol is 1: 2; the dosage of the CeAL mixed solution dripped into one filter tip is 100 mu L, and the concentration is 0.5 mg/mL; the dosage of the CeAL mixed solution dripped into one tobacco leaf is 60 mu L, and the concentration is 0.6 mg/mL.

3. Drying the CeAL water solution, and adding CeAL powder to the junction of the filter tip and the tobacco leaves; the drying mode of the CeAL water solution is as follows: vacuum drying at 40 deg.C for 20 hr, or freeze drying at-50 deg.C for 32 hr; the amount of CeAL powder added to the junction of a filter tip and tobacco leaf was 10 mg.

4. Drying CeAL water solution, adding CeAL powder into the cigarette holder filter, and reusing, wherein the amount of CeAL powder added into the cigarette holder filter is 60 mg.

The CeAL water solution is formed by adjusting the surface valence state of the raw material, and when the cigarette is burnt, the product can remove ROS in tobacco smoke, reduce the harm of smoking to health, and has the effects of softening smoke and removing offensive odor.

Addition method example 2: the method for adding the cigarette smoke active oxygen scavenger based on the nano cerium oxide into the cigarette product comprises a plurality of adding modes, which are respectively as follows:

1. adding the CeAL aqueous solution into the hookah, wherein the Ce element content in the CeAL aqueous solution added into the hookah is 1 mg/mL.

2. Dripping CeAL mixed liquid into the tobacco leaves or the filter tip; the CeAL mixed solution used for dropping into the tobacco leaves or the filter tips is a mixed solution of a CeAL aqueous solution and ethanol, and the ratio of the CeAL aqueous solution to the ethanol is 1: 3; the dosage of the CeAL mixed solution dripped into one filter tip is 200 mu L, and the concentration is 1 mg/mL; the dosage of the CeAL mixed solution dripped into one tobacco leaf is 100 mu L, and the concentration is 1 mg/mL.

3. Drying the CeAL water solution, and adding CeAL powder to the junction of the filter tip and the tobacco leaves; the drying mode of the CeAL water solution is as follows: vacuum drying at 50 deg.C for 24 hr, or freeze drying at-60 deg.C for 48 hr; the amount of CeAL powder added to the junction of a filter tip and tobacco leaf was 15 mg.

4. The aqueous solution of CeAL was dried and CeAL powder was added to the mouthpiece filter in an amount of 100 mg.

Addition method example 3: the method for adding the cigarette smoke active oxygen scavenger based on the nano cerium oxide into the cigarette product comprises a plurality of adding modes, which are respectively as follows:

1. adding the CeAL aqueous solution into the hookah, wherein the Ce element content in the CeAL aqueous solution added into the hookah is 0.01 mg/mL.

2. Dripping CeAL mixed liquid into the tobacco leaves or the filter tip; the CeAL mixed solution used for dropping into the tobacco leaves or the filter tips is a mixed solution of a CeAL aqueous solution and ethanol, and the ratio of the CeAL aqueous solution to the ethanol is 1: 1.5; the dosage of the CeAL mixed solution dripped into one filter tip is 50 mu L, and the concentration is 0.1 mg/mL; the dosage of the CeAL mixed solution dripped into one tobacco leaf is 20 mu L, and the concentration is 0.2 mg/mL.

3. Drying the CeAL water solution, and adding CeAL powder to the junction of the filter tip and the tobacco leaves; the drying mode of the CeAL water solution is as follows: vacuum drying at 30 deg.C for 16h, or freeze drying at-40 deg.C for 16 h; the amount of CeAL powder added to the junction of a filter tip and tobacco leaf is 5 mg.

4. The aqueous solution of CeAL was dried and CeAL powder was added to the mouthpiece filter in an amount of 10 mg.

Addition method example 4: the method for adding the cigarette smoke active oxygen scavenger based on the nano cerium oxide into the cigarette product comprises a plurality of adding modes, which are respectively as follows:

1. mixing the CeAL water solution with ethanol according to the volume ratio of 1:2 to ensure that the CeAL concentration in the CeAL mixed solution is 0.2 mg/mL; dripping 200 μ L of CeAL mixture solution into 1 cigarette (brand: Su cigarette) filter tip (simulated cigarette filter tip bead blasting);

2. taking 15mL of CeAL aqueous solution (C)_Ce0.2mg/mL) was placed in a vacuum freeze dryer and dried at-60 ℃ for 24h to give pale yellow CeAL powder; 5mg of CeAL powder is added to the junction of the filter tip (namely the filter tip) and the tobacco leaf.

Cigarette smoke ROS was collected using a vacuum pump and a commercially available hookah containing 2mL of different solutions. Wherein O₂ ^-The solution used for the measurement was PBS (pH7.8) containing 0.1mol/L EDTA and 2mmol/L NBT, and the OD of the solution was measured_560nmThe value is obtained. 0.2mol/L solution of terephthalic acid for OH determination(PTA, ph7.4) and the detection method is to measure the fluorescence intensity of the solution (Ex/Em: 320/405 nm). H₂O₂The solution used for the measurement was a sodium acetate solution (ph4.5) containing 0.1 mmol/L10-acetyl-3, 7-dihydroxybenzoxazine (ADHP) and 0.4U/mL horseradish peroxidase (HRP), and the fluorescence intensity of the solution was measured (Ex/Em ═ 540/585 nm). The total ROS was measured using PBS solution (ph7.4) containing 0.02mmol/L2, 7-Dichlorofluorescein (DCFH), and the fluorescence intensity of the solution was measured (Ex/Em ═ 480/525 nm). The solution used for safety analysis is PBS (pH7.4), and the detection method comprises the steps of adding 10 times volume of concentrated sulfuric acid, digesting for 3 hours at 70 ℃, and then measuring the concentration of cerium element in the solution by adopting ICP-MS. The results show that the method of dripping the CeAL mixed liquid is adopted to simulate the bead blasting of the cigarette filter tip, which is to the smoke gas O₂ ^-、·OH、H₂O₂And total ROS clearance of 93.5%, 20.1%, 38.9% and 66.2% respectively; adding CeAL powder at the junction of the filter tip and the tobacco leaf, and neutralizing the smoke O₂ ^-、·OH、H₂O₂And total ROS clearance of 94.4%, 57.7%, 67.4% and 82.8%, respectively. The concentrations of cerium in the cigarette without adding CeAL, the cigarette with the CeAL mixed solution added dropwise and the cigarette smoke collecting liquid with the powder are respectively 8.75mg/L, 9.12mg/L and 6.47mg/L, which indicates that the method has high application safety. The graphs of the ultraviolet absorption spectrum or fluorescence spectrum of the reaction solution and the scavenging efficiency of different types of ROS in cigarette smoke by CeAL after NBT, PTA, ADHP-HRP and DCFH react with corresponding ROS are shown in (a) - (f) in the attached figure 9.

Application example 1: use of a CeAL-based cigarette smoke ROS scavenger:

the filter plug dropping CeAL mixed solution and the cigarette (brand: Su cigarette) loaded with the powder were prepared by the method of addition example 4. A self-made mouse smoking device is characterized in that male Balb/c mice with the age of 6 weeks are placed in a glass container (5 mice/group), a one-way air pump is connected, and smoke of burning cigarettes enters the container through a filter tip due to negative pressure caused by air suction. The air extraction flow is 0.2L/min, the mouse exposure mode is 10 min/cigarette, 10 cigarettes/day, every two cigarettes are exposed at intervals, the mice are placed in fresh air for rest for 30min, and the mice are continuously exposed for 3 days. Taking the lung of the mouse on the 4 th day, fixing one part of the lung by 10% neutral formalin, embedding the part of the lung by paraffin, slicing the lung, and observing the lung by HE staining; another portion was weighed, homogenized, supernatant was taken, and the tissue homogenate was assayed for IL-1. beta. and TNF-. alpha.concentration by ELISA. When a mouse is exposed to cigarette smoke without CeAL, lung tissues of the mouse show severe congestion, a large number of inflammatory cells infiltrate, alveolar walls are obviously fused, thinned and broken, and the concentrations of IL-1 beta and TNF-alpha in lung tissue lysate are obviously increased; the situation is obviously improved when the mouse is exposed to the mouse added with the CeAL cigarette smoke, and the effect of loading the CeAL powder is stronger than that of the dropping mode of the CeAL mixed liquid. The HE staining of lung tissue sections of mice, and the concentration profiles of IL-1. beta. and TNF-. alpha.in the tissue lysates are shown in (a) - (c) of FIG. 10.

The scope of the present invention includes, but is not limited to, the above embodiments, and the present invention is defined by the appended claims, and any alterations, modifications, and improvements that may occur to those skilled in the art are all within the scope of the present invention.

Claims

1. A preparation method of a cigarette smoke active oxygen scavenger based on nano cerium oxide is characterized by comprising the following steps: the method comprises the following steps:

2. The method of preparing a nano ceria-based cigarette smoke active oxygen scavenger according to claim 1, wherein: the synthesis method of the raw materials comprises the following steps:

synthesizing nano cerium oxide by a microemulsion method;

3. The method of preparing a nano ceria-based cigarette smoke active oxygen scavenger according to claim 2, wherein: the method for synthesizing the nano cerium oxide by adopting the microemulsion method specifically comprises the following steps:

4. The method of claim 3 for preparing a nano ceria-based cigarette smoke active oxygen scavenger, wherein: the method for preparing the nano cerium oxide CeAL modified by the alendronic acid by adopting a two-phase ligand exchange experiment specifically comprises the following steps:

5. The method of preparing a nano ceria-based cigarette smoke active oxygen scavenger according to claim 1, wherein:

the docusate sodium, the toluene, the cerium nitrate and the H₂O₂The dosage is 0.3g to 0.5g, 30mL, 1mL to 2mL and 2mL to 4mL respectively;

6. The method of preparing a nano ceria-based cigarette smoke active oxygen scavenger according to claim 1, wherein:

the specific method for adjusting the surface valence of the raw material comprises the following steps:

7. The method of claim 6 for preparing a nano ceria-based cigarette smoke active oxygen scavenger, wherein:

the reducing substance comprises ascorbic acid, reducing glutathione, SOD enzyme or vitamin E, and the concentration of the reducing substance is 0.005mol/L-1 mol/L; the concentration of the hydrogen peroxide is 0.01mol/L-1 mol/L.

8. An adding method for adding a cigarette smoke active oxygen scavenger based on nano cerium oxide into a cigarette product is characterized in that: the adding method comprises a plurality of adding modes, wherein the plurality of adding modes are respectively as follows:

(1) adding the CeAL water solution to the hookah;

(2) dripping CeAL mixed solution into the tobacco leaves or the filter tips;

9. The method of adding a nano-ceria based cigarette smoke active oxygen scavenger to a smoking article according to claim 8, wherein:

the Ce element content in the CeAL aqueous solution added into the water pipe is 0.01mg/mL-1 mg/mL;

the amount of CeAL powder added to the mouthpiece filter is 10mg-100 mg.