CN109602914B - Vitamin B2Modified iron-based nanoenzyme and preparation method and application thereof - Google Patents

Vitamin B2Modified iron-based nanoenzyme and preparation method and application thereof Download PDF

Info

Publication number
CN109602914B
CN109602914B CN201910014857.1A CN201910014857A CN109602914B CN 109602914 B CN109602914 B CN 109602914B CN 201910014857 A CN201910014857 A CN 201910014857A CN 109602914 B CN109602914 B CN 109602914B
Authority
CN
China
Prior art keywords
vitamin
iron
nanoenzyme
modified iron
modified
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201910014857.1A
Other languages
Chinese (zh)
Other versions
CN109602914A (en
Inventor
高利增
顾云浩
仇智月
李丹丹
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Yangzhou University
Original Assignee
Yangzhou University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Yangzhou University filed Critical Yangzhou University
Priority to CN201910014857.1A priority Critical patent/CN109602914B/en
Publication of CN109602914A publication Critical patent/CN109602914A/en
Application granted granted Critical
Publication of CN109602914B publication Critical patent/CN109602914B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A61K47/551—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds one of the codrug's components being a vitamin, e.g. niacinamide, vitamin B3, cobalamin, vitamin B12, folate, vitamin A or retinoic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/525—Isoalloxazines, e.g. riboflavins, vitamin B2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/26—Iron; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like

Abstract

The invention provides vitamin B2Modified iron-based nanoenzymes, in particular vitamin B2Modified nano grade ferroferric oxide particle, and vitamin B2A preparation method of modified iron-based nanoenzyme and a composition for promoting the healing of canker sore. The vitamin B provided by the invention2The modified iron-based nanoenzyme has catalase activity and superoxide dismutase activity higher than those of the iron-based nanoenzyme, and simultaneously has vitamin B activity higher than that of vitamin B used alone2And iron-based nanoenzyme, vitamin B provided by the invention2The modified iron-based nanoenzyme shows better protection effect on BALB/3T3 cells and HOK cells in hydrogen peroxide environment, cell level ROS scavenging capacity and capacity of reducing IFN-gamma and IL-6 at ulcer positions, has better treatment effect on oral ulcer, and can be used for preparing medicines for efficiently promoting the healing of oral ulcer.

Description

Vitamin B2Modified iron-based nanoenzyme and preparation method and application thereof
Technical Field
The application relates to the technical field of biological nano materials, in particular to vitamin B2Modified iron-based nanoenzyme, and a preparation method and application thereof.
Background
The iron-based nanoenzyme (IONzyme) is a mimic enzyme which has the unique performance of a nano material and also has a catalytic function. At present, experiments show that the iron-based nanoenzyme has Catalase (CAT) activity and superoxide dismutase (SOD) activity at the same time, and has a series of advantages of simple preparation, low cost, stability, easy storage and the like.
Oral ulcer, commonly known as "aphtha", is a common ulcerative injury to the oral mucosa, which is usually found in the inner labial, tongue abdomen, buccal mucosa, vestibular sulcus, soft palate, etc., where the mucosa lacks cutinization layer or has poor keratosis. Tongue ulcer refers to oral ulcer occurring on tongue and tongue abdomen. The pain is severe when the oral ulcer is attacked, the local burning pain is obvious, and serious patients can influence diet and speaking, thereby causing great inconvenience to daily life; can be complicated with halitosis, chronic pharyngitis, constipation, headache, dizziness, nausea, debilitation, dysphoria, fever, lymphadenectasis, etc., and seriously affects normal life and work of people.
Vitamin B2(chemical formula: C)17H20N4O6) Riboflavin, also known as riboflavin, is slightly soluble in water and has certain thermal stability. Is a component of the prosthetic group of internal yellow enzymes, and when lacking, the biological oxidation of the organism is affected, so that the metabolism is obstructed. At the same time, vitamin B2Is also one of the medicines which are commonly used for treating oral ulcer clinically.
At present, researches show that abnormal active oxygen free Radicals (ROS) are generated at the oral ulcer part to cause oxidative stress, which can objectively prevent bacteria on the ulcer surface from breeding to cause infection, but can also cause delayed healing of the ulcer surface. The iron-based nanoenzyme has catalase activity and superoxide dismutase activity, can well remove excessive ROS at ulcer parts, and promotes the healing of ulcer surfaces.
However, in the prior art, vitamin B has not yet appeared2Reports related to modified iron-based nanoenzyme. While the iron-based nano enzyme and vitamin B are mixed2In combination, it can be shown which activities have influence on the activity of the iron-based nanoenzyme, and no relevant conclusion is given in the prior art.
Disclosure of Invention
In order to solve the above problems, in one aspect, the present invention provides a vitamin B2A modified iron-based nanoenzyme, wherein said vitamin B is2Modifying iron-based nanoenzyme into vitamin B2Modified nano-scale ferroferric oxide particles.
Preferably, the vitamin B2The crystal form of the modified iron-based nanoenzyme is a ferroferric oxide crystal form, the particle shape is irregular spherical, the particle size of the particles is 150-300nm, the particle size is in a nanometer range, and the particle size is about 200nm preferably.
In another aspect, the invention also provides vitamin B2The preparation method of the modified iron-based nanoenzyme comprises the step of adding vitamin B in the process of preparing the nano ferroferric oxide particles by utilizing an iron source2A step (2); wherein the iron source and vitamin B2The molar ratio of (A) to (B) is 1: 0.005-30.
Further, the iron source is in combination with vitamin B2Is 1:0.01 to 20, more preferably 1:0.02 to 18, more preferably 1:0.05 to 15, more preferably 1:0.1 to 13, more preferably 1:0.15 to 10, more preferably 1:0.5 to 5, more preferably 1:1 to 4, more preferably 1:2 to 3.
Further, the preparation of the nano ferroferric oxide particles by using the iron source comprises the step of preparing the nano ferroferric oxide particles by using a hydrothermal method or a solvothermal method; preferably, the hydrothermal method or the solvothermal method for preparing the nano ferroferric oxide particles comprises the steps of using an iron source, a water or non-water solvent and an alkaline solution as reactants; more preferably, the preparation method comprises the following steps: mixing iron source, water or non-water solvent, alkaline solution and vitamin B2Mixing to obtain a reaction solution, and preparing the reaction solution into vitamin B by a hydrothermal method or a solvothermal method2Modified iron-based nanoenzymes.
Further, the preparation method comprises the following steps:
s1, dissolving an iron source and a water or non-aqueous solvent in proportion to obtain a reaction solution A;
s2, adding an alkaline solution into the reaction solution A, and stirring to dissolve the alkaline solution to obtain a reaction solution B;
s3, adding vitamin B into the reaction solution B2Stirring to dissolve the mixture to obtain reaction liquid C;
s4, heating the reaction solution C to obtain vitamin B2Modified iron-based nanoenzymes.
Preferably, the step S4 includes a step of heating and drying the reaction solution, wherein the heating temperature is 100-500 ℃, preferably 200-400 ℃; the heating time is 1-48h, preferably 12-24 h.
Optionally, after heating, an alcohol wash is performed.
Further, the iron source is selected from ferrous iron and/or ferric iron, preferably one or more of ferric chloride, ferrous sulfate, ferric nitrate and ferric bromide;
the non-aqueous solvent is one or more of an alcohol solvent, an ether solvent, a ketone solvent, a hydrocarbon solvent and an ester solvent; preferably, the non-aqueous solvent is an alcohol solvent; more preferably, the alcohol solvent is one or more of ethylene glycol, glycerol, ethanol and polyethylene glycol;
the alkaline solution is selected from sodium acetate, sodium citrate, sodium bicarbonate, sodium carbonate, sodium hydroxide, potassium hydroxide, NH4 +Preferably, a solution of sodium acetate, sodium citrate and/or sodium bicarbonate.
The molar ratio of the iron source to the water or non-aqueous solvent is 1: 1-1000; the final concentration of the alkaline solution is 0.01-1 mol/L.
The vitamin B is obtained by hydrothermal method or solvothermal method2Modified iron-based nanoenzymes. The hydrothermal method or the solvothermal method is carried out in a high-pressure container in which fluid participates, and a solvent (water or a solvent) in the sealed container expands to fill the whole container at high temperature, so that high pressure is generated; the dissolved molecules or ions are transported to a low-temperature area of the reaction kettle by utilizing strong convection to form supersaturated solution, and then crystallization is carried out. The nano-particles synthesized by the solvothermal method have the advantages of complete crystal grain development, uniform particle size distribution, good particle dispersibility, controllable particle size and the like.
In another aspect, the present invention also provides the above vitamin B2Application of the modified iron-based nanoenzyme in preparing a medicament for treating and/or preventing ulcer.
Further, the ulcer is a mouth ulcer or a skin ulcer, preferably, a mouth ulcer.
In another aspect, the invention also provides a composition for promoting the healing of canker sores, which comprises the vitamin B2Modified iron-based nanoenzymes.
In another aspect, the present invention also provides the above vitamin B2Use of a modified iron-based nanoenzyme, wherein the use is selected from any one of the following uses I-II:
I. as the use of a catalytic enzyme, preferably, the catalytic enzyme is catalase and/or superoxide dismutase; II. Use for scavenging active oxygen free radicals.
In another aspect, the present invention also provides the above vitamin B2The modified iron-based nanoenzyme is applied to the preparation of drugs or reagents for treating oral ulcer; preferably, the medicament or agent further comprises the following uses: increasing the survival rate of ulcerated oral epithelial keratinocytes (HOK); scavenging intracellular Reactive Oxygen Species (ROS); reduce the content of interferon-gamma (IFN-gamma) and interleukin-6 (IL-6) at the ulcer.
Further, the above-mentioned vitamin B2When the modified iron-based nanoenzyme is applied, the modified iron-based nanoenzyme can be prepared into a liquid preparation, a solid preparation, a semisolid preparation or a gas preparation, preferably, the liquid preparation comprises a solution, an injection, a lotion, a liniment and the like, the solid preparation comprises a powder, a pill, a tablet, a film, a granular preparation and the like, the semisolid preparation comprises an ointment, a gel, a suppository, a paste and the like, and the gas preparation comprises an aerosol, a spray and the like.
Further, the above-mentioned vitamin B2The modified iron-based nanoenzyme can be prepared into a sustained-release preparation, a controlled-release preparation or a nano preparation when being applied.
Further, the above-mentioned vitamin B2The concentration of the modified iron-based nano enzyme in application is not lower than 0.01mg/ml, preferably not lower than 0.05mg/ml, more preferably not lower than 0.1 mg/ml; preferably 0.01-100mg/ml, more preferably 0.05-50mg/ml, more preferably 0.1-20mg/ml, more preferably 0.2-10mg/ml, more preferably 0.25mg/ml, 0.3mg/ml, 0.35mg/ml, 0.4mg/ml, 0.45mg/ml, 0.5mg/ml, 0.55mg/ml, 0.6mg/ml, 0.65mg/ml, 0.7mg/ml, 0.75mg/ml, 0.8mg/ml, 0.85mg/ml, 0.9mg/ml, 0.95mg/ml, 1.0mg/ml, 1.5mg/ml, 2.0mg/ml, 2.5mg/ml, 3.0mg/ml, 3.5mg/ml, 4.0mg/ml, 5.0mg/ml, 6.0mg/ml, 7.0mg/ml, 8mg/ml, 0 mg/ml.
The inventors found that vitamin B is obtained by the above production method2Modified iron-based nanoenzyme having catalase activity and superoxide dismutase activity higher than those of unmodified iron-based nanoenzymeThe activity of the compound also effectively improves the cell survival rate of ulcer HOK cells and BALB/3T3 cells, eliminates excessive ROS in ulcer cells, and reduces the content of interferon-gamma (IFN-gamma) and interleukin-6 (IL-6) in the ulcer cells. Compared with the iron-based nano enzyme or vitamin B used alone2Vitamin B2The modified iron-based nanoenzyme shows more efficient effect of promoting the healing of the oral ulcer, and can be suitable for preparing the medicine for efficiently treating the oral ulcer.
The invention has the following beneficial effects:
1. the vitamin B provided by the invention2The modified iron-based nanoenzyme has the advantages of good dispersibility in water, simple preparation, easy magnetic separation, stable property, good biocompatibility, no harm to human bodies in conventional use, environmental friendliness, ecological friendliness, easy control and low price, can effectively remove excessive ROS at oral ulcer positions by exerting the activity of superoxide dismutase and the activity of catalase, provides a good environment for the regeneration of ulcer surface cells, is beneficial to the healing of oral ulcer, has obvious effect of promoting the healing of the oral ulcer, and is a high-efficiency and low-cost oral ulcer therapeutic agent.
2. The vitamin B provided by the invention2The preparation method of the modified iron-based nanoenzyme can quickly and efficiently prepare the vitamin B2The modified iron-based nanoenzyme is simple to operate, and is non-toxic and pollution-free.
3. The vitamin B provided by the invention2The modified iron-based nanoenzyme has catalase activity and superoxide dismutase activity higher than those of the unmodified iron-based nanoenzyme, and simultaneously is used together with vitamin B2Compared with iron-based nanoenzyme, the compound shows better protective effect on BALB/3T3 cells and HOK cells in hydrogen peroxide environment, cell level ROS scavenging capability and capability of reducing IFN-gamma and IL-6 at ulcer, and has better effect on oral ulcer than vitamin B2And the iron-based nano enzyme, can be used for preparing a composition for efficiently promoting the healing of oral ulcer, a medicament for treating and/or preventing ulcer and the like.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the application and together with the description serve to explain the application and not to limit the application. In the drawings:
FIG. 1 shows vitamin B added in an amount of 0.5g in example 12The prepared vitamin B2Scanning electron microscope image of modified iron-based nanoenzyme;
FIG. 2 shows vitamin B added in an amount of 0.5g in example 12The prepared vitamin B2A transmission electron microscope picture of the modified iron-based nanoenzyme;
FIG. 3 shows vitamin B added in an amount of 0.5g in example 12The prepared vitamin B2Modifying an iron-based nano enzyme element analysis map;
FIG. 4 shows vitamin B added in an amount of 0.5g in example 12The prepared vitamin B2Modifying an X-ray photoelectron spectroscopy analysis map of the iron-based nanoenzyme;
FIG. 5 shows vitamin B added in an amount of 0.5g in example 12The prepared vitamin B2Modifying an X-ray diffraction pattern of the iron-based nanoenzyme;
FIG. 6 shows vitamin B added in an amount of 0.5g2Schematic diagram of modified iron-based nanoenzyme catalase activity;
FIG. 7 shows vitamin B added in an amount of 0.5g2Schematic diagram of the activity of the modified iron-based nanoenzyme superoxide dismutase;
FIG. 8 shows vitamin B added in an amount of 0.5g2The schematic diagram of the protective effect of the modified iron-based nanoenzyme on BALB/3T3 cells and HOK cells in a hydrogen peroxide environment is shown;
FIG. 9 shows vitamin B added in an amount of 0.5g2Schematic diagram of the effect of modified iron-based nanoenzyme on ROS elimination at cellular level;
FIG. 10 shows vitamin B added in an amount of 0.5g2Schematic diagram of the therapeutic effect of the modified iron-based nanoenzyme on oral ulcer;
FIG. 11 shows vitamin B added in an amount of 0.5g2Schematic diagram of the regulation effect of the modified iron-based nanoenzyme on interferon-gamma (IFN-gamma) and interleukin-6 (IL-6) at the oral ulcer.
Detailed Description
In order to more clearly explain the overall concept of the present application, the following detailed description is given by way of example in conjunction with the accompanying drawings. In the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present invention. It will be apparent, however, to one skilled in the art, that the present invention may be practiced without one or more of these specific details. In other instances, well-known features have not been described in order to avoid obscuring the invention.
Example 1 vitamin B2Preparation method of modified iron-based nanoenzyme
Ferric chloride hexahydrate (0.005mol), 1.35g, was dissolved in 40ml of ethylene glycol, stirred with a magnetic stirrer for 30 minutes, and fully dissolved to prepare a ferric chloride solution. 3.6g of anhydrous sodium acetate is added into the ferric chloride solution, and a magnetic stirrer is used for stirring for 30 minutes to fully dissolve the anhydrous sodium acetate, so that a mixed solution is prepared. Adding 0.5g vitamin B into the mixed solution2(0.0013mol), stirring for 30 minutes to ensure that the vitamin B is dissolved2And (4) fully and uniformly mixing to prepare a reaction solution. Pouring all the reaction liquid into a reaction kettle, putting the reaction kettle into an oven, heating the oven to 200 ℃, preserving heat, heating for 12 hours, washing with alcohol for three times, and drying to finally obtain the vitamin B20.3g of modified iron-based nanoenzyme, and the yield is 33.9%.
In the specification, other vitamin B prepared by the method is also provided2Examples of modified iron-based nanoenzymes, the only difference being the addition of vitamin B2The amounts of (A) were varied and were 1.0g (0.0026mol), 1.5g (0.0039mol), 2.5g (0.0065mol), 0.4g (0.0010mol) and 0.2g (0.0005mol), respectively, of the vitamin B obtained in each case2The mass of the modified iron-based nanoenzyme is about 0.3g, and the yield of each example is as follows: 21.6%, 15.9%, 10.4%, 38.2%, 51.2%.
Vitamin B in an amount of 0.5g as in example 1 will be added2The obtained vitamin B2The modified iron-based nanoenzyme is taken as an example, and the method is adoptedPreparing the obtained vitamin B2And carrying out structural characterization and biological activity detection on the modified iron-based nanoenzyme. For convenience of description, vitamin B is described below and in the figures2The modified iron-based nanoenzyme can be abbreviated as VB2-IONzyme, said 0.5g VB2The IONzyme, i.e. said example 1, added in an amount of 0.5g vitamin B2The obtained vitamin B2Modifying the iron-based nanoenzyme.
Example 2 vitamin B2Structural characterization of modified iron-based nanoenzymes
Vitamin B2The structural characterization of the modified iron-based nanoenzyme is shown in fig. 1-5, wherein fig. 1-5 respectively show that vitamin B is added in an amount of 0.5g in example 12Obtained VB2-a scanning electron micrograph, a high resolution transmission electron micrograph, an elemental analysis map, an X-ray photoelectron spectroscopy analysis map and an X-ray diffraction map of the lonzyme.
Vitamin B prepared by the above method can be obtained from FIG. 1 and FIG. 22The modified iron-based nanoenzyme is irregular spherical, the particle size of the particles is about 200nm, and the particle size of the particles is in the nano-scale range, which indicates that the obtained vitamin B2The modified iron-based nanoenzyme is in a nanostructure.
As can be seen from FIG. 3, peaks of C element and N element appear in addition to peaks of Fe element and O element, indicating the passage of vitamin B2C element and N element appear on the modified ferroferric oxide nanoenzyme.
As can be seen from FIG. 4, in addition to the peak of Fe2p level orbit and the peak of O1 s level orbit, the peaks of C1 s and N1 s level orbitals appear in the figure, which shows that vitamin B is modified on the ferroferric oxide nanoenzyme2。
From FIG. 5, vitamin B2The crystal form of the modified iron-based nanoenzyme is the same as that of the unmodified iron-based nanoenzyme, and is a ferroferric oxide crystal form.
In conclusion, structural characterization is carried out through a scanning electron microscope, a high-resolution transmission electron microscope, elemental analysis, X-ray photoelectron spectroscopy and X-ray diffraction, and the final product obtained by preparation is confirmed to be vitamin B2Modified iron-based nanoenzymeIt is demonstrated that vitamin B can be prepared using the method provided by the present invention2Modifying the iron-based nanoenzyme.
Example 3 vitamin B2Catalase activity detection of modified iron-based nanoenzyme
Vitamin B2The detection method of catalase activity of the modified iron-based nanoenzyme comprises the following steps:
adding H with different concentrations into a sodium acetate solution system with the pH value of 7.02O2Adding equal concentration vitamin B into the solution2Modifying the iron-based nano enzyme solution or the unmodified iron-based nano enzyme solution with equal concentration, monitoring the dissolved oxygen generated in the system in real time, and fitting to obtain H2O2Concentration-dissolved oxygen generation rate curve. The results are shown in FIG. 6.
As shown in fig. 6, in neutral condition, VB2Both the IONzyme and the IONzyme can decompose hydrogen peroxide, have the activity of catalase, but VB2The decomposition rate at each concentration of the IONzyme is much higher than that of the IONzyme, indicating VB2The IONzyme has a higher hydrogen peroxide activity than the IONzyme, or vitamin B2The modification of the method obviously improves the catalase activity of the iron-based nanoenzyme.
Example 4 vitamin B2Detection of modified iron-based nanoenzyme superoxide dismutase activity
VB2The superoxide dismutase activity of the IONzyme was determined by measuring its absorbance at 560nm and calculating the percentage inhibition. Wherein, the calculation formula of the SOD activity is as follows:
SOD activity (U/mg) ═ inhibition percentage ÷ (1-inhibition percentage) × VAnti-total]÷VSample (A)X sample dilution factor 11.11 x percent inhibition ÷ (1-percent inhibition) × sample dilution factor.
Production of superoxide anion (O) by xanthine and xanthine oxidase reaction systems2-)，O2-The reducible nitro blue tetrazole generates blue formazan, and the blue formazan absorbs at 560 nm; SOD scavenged O2-Thereby inhibiting the formation of formazan; the greater the absorbance at 560nmHigh indicates that the SOD activity is lower, whereas the activity is higher. The results are shown in FIG. 7.
As shown in FIG. 7, VB2-IONzyme purged O2-The amount of (B) is significantly higher than that of IONzyme, indicating VB2The IONzyme can effectively eliminate superoxide anions, and the activity of superoxide dismutase is obviously improved compared with that of unmodified iron-based nanoenzyme.
Example 5 vitamin B2Protection effect of modified iron-based nanoenzyme on BALB/3T3 cells and HOK cells in hydrogen peroxide environment
Detecting VB2An experimental method for the protective effect of the IONzyme on BALB/3T3 cells and HOK cells in a hydrogen peroxide environment specifically comprises the following steps:
(1) BALB/3T3 cells and HOK cells were plated 8000 cells/well in 96-well plates;
(2) removing old culture medium after culturing for 12h, adding VB with different concentrations2、IONzyme、VB2-new medium of lonzyme;
(3) after acting for 15min, adding hydrogen peroxide with the final concentration of 200 mu M, and incubating for 24 h;
(4) washing off the culture medium, washing with PBS, and adding MTT solution with final concentration of 0.5mg/mL for acting for 4 h;
(5) after adding 100. mu.L of DMSO and sufficiently dissolving, the absorbance value was measured at a wavelength of 490nm, and the cell viability was calculated from the absorbance value.
The results obtained using the above method are shown in FIG. 8.
As shown in FIG. 8, VB concentrations of 12.5. mu.g/ml and 25. mu.g/ml were found compared to the blank (200. mu.M hydrogen peroxide only)2The survival rate of HOK cells of the IONzyme is improved under the hydrogen peroxide environment, and the concentration is 50 mu g/ml and 100 mu g/ml VB2The survival rate of HOK cells and BALB/3T3 cells of the IONzyme in a hydrogen peroxide environment is obviously improved. At the same time as compared with VB used alone2And IONzyme, VB2The survival rates of BALB/3T3 cells and HOK cells in hydrogen peroxide environment can be remarkably improved by the IONzyme, which shows that VB2The IONzyme has better VB performance on BALB/3T3 cells and HOK cells in hydrogen peroxide environment2And the protective effect of the IONzyme.
Example 6 vitamin B2Detection of ROS scavenging ability of modified iron-based nanoenzyme at cellular level
Detecting VB2-a method for ROS scavenging of an lonzyme at cellular level, comprising the steps of:
(1) BALB/3T3 cells and HOK cells were plated 8000 cells/well in 96-well plates;
(2) removing old culture medium after culturing for 12h, adding VB with different concentrations2、IONzyme、VB2-new medium of lonzyme;
(3) after acting for 15min, adding hydrogen peroxide with the final concentration of 200 mu M, and incubating for 24 h;
(4) washing off the culture medium, washing with PBS, adding a DCFH-DA probe, and incubating for 30 min;
(5) PBS was washed, fresh PBS was added, and the fluorescence intensity was measured at an excitation wavelength of 488nm and an emission wavelength of 525 nm.
The results obtained using the above method are shown in FIG. 9.
As shown in fig. 9, vs compared to VB used alone2And IONzyme, VB2Significant reduction of ROS levels in HOK cells and BALB/3T3 cells in the IONzyme treated oxygenated water environment, indicating VB2The IONzyme can effectively eliminate ROS in HOK cells and BALB/3T3 cells in hydrogen peroxide environment, and the elimination effect is better than that of VB used alone2And an lonzyme.
Example 7 vitamin B2Evaluation of therapeutic effect of modified iron-based nanoenzyme on oral ulcer
VB2The method for detecting the treatment effect of the IONzyme on the oral ulcer comprises the step of establishing an oral ulcer model, and specifically comprises the following steps:
(1) dipping filter paper with glacial acetic acid and diameter of about 4mm on the tongue of the mouse for 30s, and cleaning with normal saline;
(2) after 24h, the ulcer surface is formed, normal saline is given to the ulcer surface of the control group, and 2mg/ml VB after sterilization is given to the ulcer surface of the drug group210mg/ml IONzyme and 10mg/ml VB2-lonzyme, administered for 6 days;
(3) observing and photographing the ulcer surface every two days during the administration period;
(4) after the sixth application, the mice were sacrificed and the tongue of the ulcer surface of the mice was cut off for H & E staining and ELISA for the IL-6 and IFN-y content of the tongue.
The results obtained using the above method are shown in FIGS. 10-11.
As shown in FIG. 10, VB and the control group2VB in the treated group compared to the IONzyme treated group2The regeneration speed of tongue epithelial tissues of mice treated by the IONzyme is obviously accelerated, inflammatory cells infiltrate less and the recovery is good.
As shown in FIG. 11, VB and the control group2VB in the treated group compared to the IONzyme treated group2Significant reduction in IFN-. gamma.and IL-6 levels in tongue ulcers in mice after IONzyme treatment, further indicating via VB2The tongue ulcers recovered better in mice after treatment with lonzyme, whereas the remaining treatment groups did not.
In conclusion, the preparation method provided by the invention can be used for obtaining the vitamin B with the particle size of nano-scale2A modified iron-based nanoenzyme; the vitamin B provided by the invention2The modified iron-based nanoenzyme has catalase activity and superoxide dismutase activity higher than those of the unmodified iron-based nanoenzyme, and simultaneously is used together with vitamin B2Compared with iron-based nanoenzyme, vitamin B2The modified iron-based nanoenzyme shows better protection effect on BALB/3T3 cells and HOK cells in hydrogen peroxide environment, ROS scavenging capacity at the cellular level and capacity of reducing IFN-gamma and IL-6 at ulcer, and indicates that vitamin B2The modified iron-based nanoenzyme has better effect on oral ulcer than vitamin B2And the iron-based nano enzyme, and can be used for preparing a composition for efficiently promoting the healing of the oral ulcer and a medicament for treating the oral ulcer.
The above description is only an example of the present application and is not intended to limit the present application. Various modifications and changes may occur to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims (15)

1. Vitamin B2Modified iron-based nanoenzyme, characterized in that said vitamin B is2Modifying iron-based nanoenzyme into vitamin B2Modified nano-sized ferroferric oxide particles;
the vitamin B2The preparation method of the modified iron-based nanoenzyme comprises the step of adding vitamin B in the process of preparing the nano ferroferric oxide particles by utilizing an iron source2A step (2); the iron source and vitamin B2Is 1: 0.26.
2. Vitamin B in accordance with claim 12The modified iron-based nanoenzyme is characterized in that the step of preparing the nano ferroferric oxide particles by using an iron source comprises the step of preparing the nano ferroferric oxide particles by using a hydrothermal method or a solvothermal method.
3. Vitamin B in accordance with claim 22The modified iron-based nanoenzyme is characterized in that the preparation of the nano ferroferric oxide particles by using a hydrothermal method or a solvothermal method comprises the step of using an iron source, a water or non-water solvent and an alkaline solution as reactants.
4. Vitamin B in accordance with claim 32The modified iron-based nanoenzyme is characterized in that the preparation method comprises the following steps: mixing iron source, water or non-water solvent, alkaline solution and vitamin B2Mixing to obtain a reaction solution, and preparing the reaction solution into vitamin B by a hydrothermal method or a solvothermal method2Modified iron-based nanoenzymes.
5. Vitamin B in accordance with claim 3 or 42The modified iron-based nanoenzyme is characterized in that the preparation method comprises the following steps:
s1, dissolving an iron source and a water or non-aqueous solvent in proportion to obtain a reaction solution A;
s2, adding an alkaline solution into the reaction solution A, and stirring to dissolve the alkaline solution to obtain a reaction solution B;
s3, adding vitamin B into the reaction solution B2Stirring to dissolve the mixture to obtain reaction liquid C;
s4, heating the reaction solution C to obtain vitamin B2Modified iron-based nanoenzymes.
6. Vitamin B in accordance with claim 52Modified iron-based nanoenzymes, characterized in that the iron source is selected from ferrous and/or ferric iron.
7. Vitamin B in accordance with claim 62The modified iron-based nanoenzyme is characterized in that the iron source is selected from one or more of ferric chloride, ferrous sulfate, ferric nitrate and ferric bromide.
8. Vitamin B in accordance with claim 52The modified iron-based nanoenzyme is characterized in that the non-aqueous solvent is one or more of an alcohol solvent, an ether solvent, a ketone solvent, a hydrocarbon solvent and an ester solvent.
9. Vitamin B in accordance with claim 82The modified iron-based nanoenzyme is characterized in that the nonaqueous solvent is an alcohol solvent.
10. Vitamin B in accordance with claim 92The modified iron-based nanoenzyme is characterized in that the alcohol solvent is one or more of ethylene glycol, glycerol, ethanol and polyethylene glycol.
11. Vitamin B in accordance with claim 52The modified iron-based nano enzyme is characterized in that the alkaline solution is selected from sodium acetate, sodium citrate, sodium bicarbonate, sodium carbonate, sodium hydroxide, potassium hydroxide and NH4 +A solution of one or more of (a).
12. Vitamin B in accordance with claim 112Modified iron-based nanoRice enzyme, characterized in that the alkaline solution is selected from solutions of sodium acetate, sodium citrate and/or sodium bicarbonate.
13. Vitamin B as claimed in any of claims 1 to 122Application of modified iron-based nanoenzyme in preparation of medicine for treating and/or preventing ulcer.
14. The use of claim 13, wherein the ulcer is a mouth ulcer or a skin ulcer.
15. A composition for promoting healing of canker sores comprising the vitamin B of any one of claims 1-122Modified iron-based nanoenzymes.
CN201910014857.1A 2019-01-08 2019-01-08 Vitamin B2Modified iron-based nanoenzyme and preparation method and application thereof Active CN109602914B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910014857.1A CN109602914B (en) 2019-01-08 2019-01-08 Vitamin B2Modified iron-based nanoenzyme and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910014857.1A CN109602914B (en) 2019-01-08 2019-01-08 Vitamin B2Modified iron-based nanoenzyme and preparation method and application thereof

Publications (2)

Publication Number Publication Date
CN109602914A CN109602914A (en) 2019-04-12
CN109602914B true CN109602914B (en) 2022-05-17

Family

ID=66015630

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910014857.1A Active CN109602914B (en) 2019-01-08 2019-01-08 Vitamin B2Modified iron-based nanoenzyme and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN109602914B (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110935020B (en) * 2019-11-19 2021-12-31 同济大学 Multifunctional antioxidant magnetic nano composite material and preparation method thereof
CN113265021A (en) * 2021-05-31 2021-08-17 青岛科技大学 Preparation method and application of iron-based nano enzyme hydrogel
CN114917191B (en) * 2022-04-08 2023-07-28 南通大学 Preparation method and application of vitamin B2-based acousto-optic (photo) sensitive nanoparticle VFNS

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105101957A (en) * 2013-03-06 2015-11-25 光电子纳米技术研究所 Novel formula of iron based nanocomposites for rapid and efficient treatment of iron deficiency anemia

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105101957A (en) * 2013-03-06 2015-11-25 光电子纳米技术研究所 Novel formula of iron based nanocomposites for rapid and efficient treatment of iron deficiency anemia

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
V-c-Functionalized Fe3O4 Nanocomposites as Peroxidase-like Mimetics for H2O2 and Glucose Sensing;Ren, HJ, et al.;《Chemical Research in Chinese Universities》;20180316;第34卷(第2期);第260-268页 *
维生素修饰四氧化三铁纳米酶的抗氧化作用及其机制研究;顾云浩;《中国硕士学位论文全文数据库 电子期刊》;医药卫生科技辑;20200229;全文 *

Also Published As

Publication number Publication date
CN109602914A (en) 2019-04-12

Similar Documents

Publication Publication Date Title
CN109602914B (en) Vitamin B2Modified iron-based nanoenzyme and preparation method and application thereof
Wang et al. Insights into rapid photodynamic inactivation mechanism of Staphylococcus aureus via rational design of multifunctional nitrogen-rich carbon-coated bismuth/cobalt nanoparticles
TW436288B (en) Pharmaceutical composition of lanthanum carbonate hydrates for hyperphosphataemia treatment
CN101340979B (en) Method for purifying montmorillonite, purified montmorillonite and combination thereof
JP6916184B2 (en) Formulation manufacturing method
Wu et al. Self-driven electron transfer biomimetic enzymatic catalysis of bismuth-doped PCN-222 MOF for rapid therapy of bacteria-infected wounds
CN110585237B (en) Nano diagnosis and treatment agent and preparation method and application thereof
JP2002541218A5 (en)
WO2008005705A2 (en) Metal-containing formulations and methods of use
Rastinfard et al. Aqueous decomposition behavior of solid peroxides: Effect of pH and buffer composition on oxygen and hydrogen peroxide formation
CN110680926B (en) Nano diagnosis and treatment agent and preparation method and application thereof
Miao et al. High special surface area and “warm light” responsive ZnO: Synthesis mechanism, application and optimization
WO2019242044A1 (en) Use of composite nanoparticle of carbon and copper
CN113648414B (en) Metal ion coordinated carbon dot/titanium dioxide heterojunction and preparation method and application thereof
CN111972435A (en) Preparation method of nano-silver-graphene oxide composite material
JP3130262B2 (en) Bath additive
CN113274505A (en) CoMn-Fe-Talc-based nanocomposite and preparation method and application thereof
JPS61130231A (en) Gelatinous antibiotic composition for local use
US8865227B2 (en) Metal carbonate particles and methods of making thereof
US20110070159A1 (en) Compositions comprising red microalgae polysaccharides and metals
KR20140143195A (en) Benzene polycarboxylic acid compounds and their use as drug
CN109453265B (en) Nano zinc green tea sustained release preparation wrapped by poly n-butyl cyanoacrylate and application thereof
JP3714050B2 (en) Solid baking soda dialysis agent and method for producing the same
Liao et al. Stimuli-responsive graphdiyne-silver nanozymes for catalytic ion therapy of dental caries through targeted biofilms removal and remineralization
CN116139167B (en) Thorn-like TiO 2 Ir cluster-loaded enzyme-like material and preparation and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant