CN118026240A

CN118026240A - Cerium oxide microsphere, preparation method and application thereof, and cosmetic

Info

Publication number: CN118026240A
Application number: CN202310697002.XA
Authority: CN
Inventors: 李晓珍
Original assignee: Beijing Weiye Innovation Technology Co ltd
Current assignee: Beijing Weiye Innovation Technology Co ltd
Priority date: 2023-06-13
Filing date: 2023-06-13
Publication date: 2024-05-14

Abstract

The invention provides cerium oxide microspheres, a preparation method, application and cosmetics thereof; wherein the cerium oxide microsphere comprises Ce ³⁺,Ce³⁺ with a mole ratio of 30-38% in the cerium oxide microsphere. Ce ³⁺ and Ce ⁴⁺ in cerium oxide can be reversibly converted, and in the conversion process, along with the generation and elimination of oxygen holes, the characteristic gives the cerium oxide good oxygen storage and release capability, so that the cerium oxide has catalytic performance and oxidation resistance, and when the mole ratio of Ce ³⁺ in the cerium oxide microsphere is up to 30% -38%, the catalytic performance and oxidation resistance of the cerium oxide microsphere can be improved, and further the oxidation resistance effect of the cerium oxide microsphere in cosmetics is improved.

Description

Cerium oxide microsphere, preparation method and application thereof, and cosmetic

Technical Field

The invention relates to the technical field of cosmetics, in particular to cerium oxide microspheres, a preparation method and application thereof, and cosmetics.

Background

Cerium (Ce) is the most abundant element in earth surface rare earth elements, the atomic number of Ce is 57, the outermost electronic structure is [ Xe ]4f ¹5d¹6s², and two oxidation states of +3 and +4 exist. Among all cerium-containing compounds, cerium oxide (CeO ₂) has been widely used in various fields, such as automobile exhaust gas treatment, CO catalytic oxidation, nitrogen oxide reduction, steam reforming reaction, etc., due to its low cost, unique crystal structure and good redox ability.

With the progress of research, cerium oxide is found to be capable of eliminating various active oxidation species, has good biosafety and has important application value in the field of biological antioxidation. . However, in the related art, cerium oxide is rarely applied to cosmetics, so the application and research of cerium oxide in the field of cosmetics are significant.

Disclosure of Invention

Based on this, it is necessary to provide a cerium oxide microsphere, a preparation method, application and cosmetics thereof, so as to research the development and application of cerium oxide in the cosmetic field.

In a first aspect of the present invention, there is provided a cerium oxide microsphere comprising Ce ³⁺, the molar ratio of Ce ³⁺ in the cerium oxide microsphere being 30% to 38%.

In some embodiments, the Ce ³⁺ is present in the cerium oxide microsphere at a molar ratio of 36% -38%.

In some embodiments, the cerium oxide microspheres have an average particle size of 0.2 μm to 3 μm.

In some embodiments, the cerium oxide microspheres have an average particle size of 0.4 μm to 3 μm.

In a second aspect, the present invention provides a method for preparing the cerium oxide microsphere of the first aspect, comprising the steps of:

mixing a cerium-containing precursor with water to prepare a precursor solution;

Adding a pH regulator into the precursor solution to prepare a first solution;

Adding a dispersion medium into the first solution to prepare a second solution; the mass ratio of the cerium-containing precursor in the second solution is 3% -7%;

And heating the second solution for reaction.

In some embodiments, the cerium-containing precursor is present in the second solution at a mass ratio of 5% to 7%.

In some embodiments, the step of preparing the precursor solution comprises at least one of the following conditions:

(1) The cerium-containing precursor comprises one or more of cerium nitrate hexahydrate and cerium nitrate;

(2) The mass volume ratio of the cerium-containing precursor and the water is (20-120) g (10-60) mL.

In some embodiments, the step of preparing the first solution comprises at least one of the following conditions:

(1) The pH regulator comprises one or more of acetic acid, formic acid and propionic acid;

(2) The mass volume ratio of the cerium-containing precursor and the pH regulator is (20-120) g (20-120) mL.

In some embodiments, the step of preparing the second solution comprises at least one of the following conditions:

(1) The dispersion medium comprises ethylene glycol;

(2) The mass volume ratio of the cerium-containing precursor and the dispersion medium is (20-120) g (600-700) mL.

In some embodiments, the heating reaction comprises at least one of the following conditions:

(1) The pressure of the heating reaction is 0.5Mpa-4Mpa;

(2) The temperature of the heating reaction is 160-170 ℃;

(3) The heating reaction time is 140-180 min;

(4) The heating reaction is carried out in a hastelloy pressure-resistant reaction kettle;

Alternatively, the heating reaction is performed in a hastelloy pressure-resistant reaction kettle with a volume of 1L.

In some embodiments, the method of making further comprises the steps of:

and (3) filtering the heated reaction product, washing the suspension after the filtering to be neutral, and drying to prepare the cerium oxide microsphere.

In some embodiments, the filtration treatment is performed with a ceramic filter membrane;

Optionally, the ceramic filter membrane has an average pore size of 100nm to 500nm.

In a third aspect, the present invention provides a cerium oxide microsphere according to the first aspect of the present invention or a cerium oxide microsphere prepared by the preparation method according to the second aspect of the present invention, and its use in preparing cosmetics.

In a fourth aspect, the present invention provides a cosmetic comprising the cerium oxide microsphere of the first aspect of the present invention or the cerium oxide microsphere prepared by the preparation method of the second aspect of the present invention.

In some embodiments, the cosmetic comprises a cosmetic having an anti-aging effect.

The cerium oxide microsphere, the preparation method, the application and the cosmetics thereof, wherein the mass ratio of Ce ³⁺ in the cerium oxide microsphere is 30-38%; ce ³⁺ and Ce ⁴⁺ in cerium oxide can be reversibly converted, and in the conversion process, along with the generation and elimination of oxygen holes, the characteristic endows cerium oxide with good oxygen storage and release capability, so that the cerium oxide has catalytic performance and oxidation resistance, and when the mass ratio of Ce ³⁺ in the cerium oxide microsphere is up to 30% -38%, the catalytic performance and oxidation resistance of the cerium oxide microsphere can be improved; when applied to the field of cosmetics, the anti-oxidation and anti-aging effects are excellent.

Drawings

FIG. 1 is a schematic view showing the appearance of cerium oxide microspheres prepared in examples 1 to 9;

FIG. 2 is an electron scanning electron microscope image of cerium oxide microspheres prepared in example 1, example 4 and example 9;

FIG. 3 is XPS spectroscopy analysis of cerium oxide microspheres prepared in example 1, example 4 and example 9;

FIG. 4 is a graph showing the DPPH radical scavenging effect of example 1, comparative examples 4-8 and VC control;

FIG. 5 is a graph showing the change in DHHP removal rate according to the concentration of cerium oxide microspheres prepared in example 1, example 4 and example 9;

FIG. 6 is a graph showing DPPH effect of the comparative cycle cleaning of cerium oxide microspheres and VC in example 1;

FIG. 7 shows the clearance of the cerium oxide microspheres of example 1 when they were recycled for five DPPH removals;

FIG. 8 is a schematic diagram showing the treatment results of DPPH stock solution with cerium oxide microspheres and VC control in example 1;

FIG. 9 is a graph showing the results of detection of ROS expression in HFF-1 cells based on UVA irradiation of a dispersion of cerium oxide microspheres in example 1;

FIG. 10 is a graph showing the results of the dispersion of cerium oxide microspheres in example 1; wherein, P <0.01 indicates that the difference is very significant compared to NC group;

FIG. 11 is a graph showing the effect of the dispersion of cerium oxide microspheres on the expression of inflammatory factor IL-6 in example 1; wherein, ΔΣ represents extremely significant differences compared to SC (0.1% dmso, lps-) group, P <0.01; delta represents that the difference is very significant compared to BC (LPS-) group, P <0.01; # indicates that the difference was very significant, P <0.01, compared to SC (0.1% dmso, lps+) group; * Indicates that the difference was very significant compared to NC (lps+) group, P <0.01;

FIG. 12 is a graph showing the effect of the dispersion of cerium oxide microspheres on the expression of inflammatory factor IL-1α in example 1; wherein, ΔΣ represents a very significant difference P <0.01 compared to SC 0.1% dmso LPS- -) group; delta represents the very significant difference P <0.01 compared to BC LPS- -) group; # represents a very significant difference P <0.01 compared to SC 0.1% dmso lps+) group; * Indicating significant P <0.05 difference compared to NC lps+) group.

Detailed Description

In order that the invention may be readily understood, a more complete description of the invention will be rendered by reference to the appended drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

In the invention, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.

In the present invention, the numerical ranges are referred to as continuous, and include the minimum and maximum values of the ranges, and each value between the minimum and maximum values, unless otherwise specified. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range description features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to include any and all subranges subsumed therein.

Only a few numerical ranges are specifically disclosed herein. However, any lower limit may be combined with any upper limit to form a range not explicitly recited; and any lower limit may be combined with any other lower limit to form a range not explicitly recited, and any upper limit may be combined with any other upper limit to form a range not explicitly recited. Furthermore, each separately disclosed point or individual value may itself be combined as a lower limit or upper limit with any other point or individual value or with other lower limit or upper limit to form a range not explicitly recited.

The temperature parameter in the present invention is not particularly limited, and may be a constant temperature treatment or a treatment within a predetermined temperature range. The constant temperature process allows the temperature to fluctuate within the accuracy of the instrument control.

In the description of the invention, the meaning of "plurality" means at least two, for example, two, three, etc., unless specifically defined otherwise.

All embodiments of the invention and alternative embodiments may be combined with each other to form new solutions, unless otherwise specified. All technical features and optional technical features of the invention may be combined with each other to form new technical solutions, unless specified otherwise.

All the steps of the present invention may be performed sequentially or randomly, preferably sequentially, unless otherwise specified.

The invention provides a cerium oxide microsphere, which comprises 30-38% of Ce ³⁺,Ce³⁺ in the cerium oxide microsphere.

The cerium oxide has a fluorite cubic structure, the space group is Fm-3m, and the unit cell parameter a is 0.5411nm. In the crystal structure of cerium oxide, ce ⁴⁺ occupies octahedral voids and O ^2- occupies tetrahedral voids. The low-index crystal planes of cerium oxide are {100}, {110}, and {111}, respectively. Because of the electron transition of O2 p-Ce 4f, cerium oxide has stronger absorption in the ultraviolet region and is light yellow, and can be used as an ultraviolet absorber.

In cerium oxide, ce ³⁺ and Ce ⁴⁺ are able to reversibly switch, with concomitant generation and elimination of oxygen vacancies during this switching process. According toEquation, this process can be described by the following defect response:

Wherein Oo represents O ^2-,Ce_Ce represents Ce ⁴⁺, Is an oxygen vacancy that is electrically neutral, and Ce' _Ce represents Ce ³⁺. When cerium oxide is reduced, oxygen in the tetrahedral voids escapes, and two electrons remaining after oxygen escape are obtained by two adjacent Ce ⁴⁺, and Ce ⁴⁺ is reduced to Ce ³⁺. This property gives CeO2 excellent oxygen storage and release capability, and this defective property is also the basis for its catalysis and oxidation resistance. When the mass ratio of Ce ³⁺ in the cerium oxide microsphere is up to 30% -38%, the catalytic performance and oxidation resistance of the cerium oxide microsphere can be improved; when applied to the field of cosmetics, the anti-oxidation and anti-aging effects are excellent.

The mass ratio of Ce ³⁺ in the cerium oxide microsphere is 30% -38%; for example, it may be, but is not limited to 30％、30.2％、30.5％、30.8％、31％、31.2％、31.5％、31.7％、32％、32.3％、32.5％、32.8％、33％、33.2％、33.5％、33.7％、34％、34.2％、34.5％、34.7％、35％、35.3％、35.5％、35.7％、36％、36.3％、36.5％、36.8％、37％、37.2％、37.5％、37.8％、38％ or a range between any two of the above.

In some embodiments, the mass ratio of Ce ³⁺ in the cerium oxide microspheres is 36% -38%; for example, but not limited to 36％、36.1％、36.2％、36.3％、36.4％、36.5％、36.6％、36.7％、36.8％、36.9％、37％、37％、37.1％、37.2％、37.3％、37.4％、37.5％、37.6％、37.7％、37.8％、37.9％、38％ or a range between any two of the above values.

In some embodiments, the ceria microspheres have an average particle size of 0.2 μm to 3 μm; for example, it may be, but is not limited to 0.2μm、0.3μm、0.4μm、0.5μm、0.6μm、0.7μm、0.8μm、0.9μm、1μm、1.1μm、1.2μm、1.3μm、1.4μm、1.5μm、1.6μm、1.7μm、1.8μm、1.9μm、2μm、2.1μm、2.2μm、2.3μm、2.4μm、2.5μm、2.6μm、2.7μm、2.8μm、2.9μm、3μm or a range between any two of the above. Experiments prove that when the average particle size of the prepared cerium oxide microsphere is in the range, the sedimentation speed of the cerium oxide microsphere from the mother solution and the dispersibility of the cerium oxide microsphere in the aqueous solution can reach the optimal balance.

In some embodiments, the cerium oxide microspheres have an average particle size of 0.4 μm to 3 μm. For example, it may be, but is not limited to 0.4μm、0.5μm、0.6μm、0.7μm、0.8μm、0.9μm、1μm、1.1μm、1.2μm、1.3μm、1.4μm、1.5μm、1.6μm、1.7μm、1.8μm、1.9μm、2μm、2.1μm、2.2μm、2.3μm、2.4μm、2.5μm、2.6μm、2.7μm、2.8μm、2.9μm、3μm or a range between any two of the above.

Mixing a cerium-containing precursor with water to prepare a precursor solution; adding a pH regulator into the precursor solution to prepare a first solution; adding a dispersion medium into the first solution to prepare a second solution; the mass ratio of the cerium-containing precursor in the second solution is 3% -7%; the second solution is heated for reaction.

The preparation method has simple process and mild reaction conditions, and can be used for the amplified production of cerium oxide microspheres. Compared with the conventional cerium oxide preparation process, the concentration of the cerium-containing precursor in the precursor solution can be improved by 1-3 times or more.

In some embodiments, the cerium-containing precursor is present in the second solution in a mass ratio of 5% to 7%; when the mass ratio of the cerium-containing precursor in the second solution is within the above range, the formed cerium oxide is closer to the morphology of the microspheres, the dispersibility is good, and the yield of the cerium oxide is higher. As an example, the mass ratio of the cerium-containing precursor in the second solution may be, but is not limited to, 5%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7% or a range between any two of the above.

In some embodiments, the cerium-containing precursor includes one or more of cerium nitrate hexahydrate and cerium nitrate.

As one embodiment, the mass volume ratio of the cerium-containing precursor and water is (20-120) g (10-60) mL; when the mass-volume ratio of the cerium-containing precursor and water is within the above range, the cerium-containing precursor can be sufficiently dissolved. By way of example, the mass to volume ratio of cerium-containing precursor to water may be, but is not limited to 20g:(10-60)mL、30g:(10-60)mL、40g:(10-60)mL、50g:(10-60)mL、60g:(10-60)mL、70g:(10-60)mL、80g:(10-60)mL、90g:(10-60)mL、100g:(10-60)mL、110g:(10-60)mL or 120g (10-60) mL, etc.

In some embodiments, the pH adjuster includes one or more of acetic acid, formic acid, and propionic acid. Acetic acid not only has the effect of regulating pH, but also can coordinate with Ce (III), and the crystallization rate of CeO ₂ is regulated, so that the acetic acid is a necessary condition for synthesizing cerium oxide microspheres.

In some embodiments, the mass to volume ratio of cerium-containing precursor to pH adjustor is (20-120) g (20-120) mL; when the mass volume ratio of the cerium-containing precursor to the pH regulator is in the above range, the cerium oxide microspheres with different particle sizes and different surface Ce (III) contents can be synthesized under control. By way of example, the mass to volume ratio of cerium-containing precursor and pH adjustor can be, but is not limited to 20g:(20-120)mL、30g:(20-120)mL、40g:(20-120)mL、50g:(20-120)mL、60g:(20-120)mL、70g:(10-120)mL、80g:(20-120)mL、90g:(20-120)mL、100g:(20-120)mL、110g:(20-120)mL or 120g (20-120) mL, and the like.

As a possible embodiment, the dispersion medium comprises ethylene glycol. The glycol has good stability and boiling point reaching 197 ℃, and is used as a dispersion medium of cerium-containing precursor, the dispersion effect is good, and the glycol is easy to separate after the reaction is finished.

In some alternative embodiments, the mass to volume ratio of cerium-containing precursor to dispersion medium is (20-120) g (600-700) mL; when the mass-volume ratio of the cerium-containing precursor and the dispersion medium is within the above range, cerium oxide microspheres with different aggregation degrees can be synthesized, and the products are easy to separate. As an example, the mass to volume ratio of the cerium-containing precursor and the dispersion medium may be, but is not limited to 20g:(600-700)mL、30g:(600-700)mL、40g:(600-700)mL、50g:(600-700)mL、60g:(600-700)mL、70g:(600-700)mL、80g:(600-700)mL、90g:(600-700)mL、100g:(600-700)mL、110g:(600-700)mL or 120g (600-700) mL, etc

In some embodiments, the pressure of the heating reaction is between 0.5Mpa and 4Mpa. As an example, the pressure of the heating reaction may be, but is not limited to, 0.5Mpa, 1Mpa, 1.5Mpa, 2Mpa, 2.5Mpa, 3Mpa, 3.5Mpa, 4Mpa or a range between any two of the above values.

In some embodiments, the temperature of the heating reaction is 160 ℃ to 170 ℃. As an example, the temperature of the heating reaction may be, but is not limited to 160 ℃, 161 ℃, 162 ℃, 163 ℃, 164 ℃, 165 ℃, 166 ℃, 167 ℃, 168 ℃, 169 ℃,170 ℃, or a range between any two of the above.

In some embodiments, the heating reaction is for a period of 140min to 180min. As an example, the time of the heating reaction may be, but is not limited to, 140min, 145min, 150min, 155min, 160min, 165min, 170min, 175min, 180min, or a range between any two of the above.

Compared with the conventional cerium oxide preparation process, the reaction time can be shortened by 20min-60min, and the reaction temperature can be reduced by 10-20 ℃. When the pressure, temperature and time of the heating reaction are controlled within the above ranges, cerium oxide microspheres with good oxidation resistance can be prepared.

In some embodiments, the heating reaction is performed in a hastelloy pressure resistant reaction vessel. Acetic acid is somewhat corrosive, especially at high temperatures. At the temperatures required for the reaction (160 ℃ -170 ℃), conventional 316 stainless steel is corroded by acetic acid, resulting in thinning of the wall material. The Hastelloy pressure-resistant reaction kettle can avoid the problem and ensure the safety in the experimental process in the Hastelloy pressure-resistant reaction kettle. Alternatively, the heating reaction is carried out in a hastelloy pressure-resistant reaction kettle with the volume of 1L; is beneficial to the amplified production of cerium oxide microspheres.

In some embodiments, the method of preparing cerium oxide microspheres further comprises the steps of: and (3) filtering the heated reaction product, washing the filtered filtrate to be neutral, and drying to prepare the cerium oxide microspheres.

As one embodiment, the filtration treatment is performed using a ceramic filter membrane. In some embodiments, the heated reaction product is subjected to a filtration treatment in the form of "cross-flow filtration"; namely: the raw material liquid flows in the membrane tube at a high speed, the permeation liquid containing small molecules is driven by pressure to permeate the membrane outwards along the vertical direction of the membrane layer, and the concentrated liquid containing macromolecular components is intercepted by the membrane, so that the purposes of separating, purifying and concentrating the cerium oxide slurry product are realized. The ceramic filter membrane system has high process integration degree, realizes full-automatic control, can continuously feed, continuously discharge filter residues and filtrate, realizes continuous separation of products, and has clear and transparent filtrate, thereby bringing higher economic benefit.

Optionally, the ceramic filter membrane has an average pore size of 100nm to 500nm; for example, it may be, but is not limited to, 100nm, 150nm, 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm or a range between any two of the above values.

In a third aspect, the present invention provides a use of the cerium oxide microsphere of the first aspect of the present invention or the cerium oxide microsphere prepared by the preparation method of the second aspect of the present invention in the preparation of cosmetics.

The following describes the technical scheme of the present invention in detail with reference to specific examples.

1. Preparation of cerium oxide microspheres

Example 1

20G of cerium nitrate hexahydrate (as a cerium-containing precursor) was weighed, and mixed with 10mL of deionized water to prepare a precursor solution; adding 20mL of acetic acid (as a pH regulator) to the precursor solution to prepare a first solution; adding 600mL of ethylene glycol (serving as a dispersion medium) into the first solution, and uniformly mixing to prepare a second solution; wherein, the mass ratio of the cerium nitrate hexahydrate in the second solution is 3 percent; transferring the second solution into a 1L hastelloy pressure-resistant reaction kettle for heating reaction, wherein the reaction temperature is 165 ℃, the reaction time is 160min, and the reaction pressure is about 0.5 Mpa; filtering and heating the reaction product by adopting a ceramic filter membrane with the average pore diameter of 500nm, washing the filtered filtrate to be neutral by adopting water, and drying at room temperature for 24 hours; and (5) packaging after sterilization to obtain the cerium oxide microspheres.

Examples 2 to 9

The preparation methods of the cerium oxide microspheres in examples 2 to 9 are substantially similar to those of the cerium oxide microspheres in example 1, and the main differences are that: the difference of at least one of the amount of the cerium-containing precursor, the amount of deionized water, the amount of the pH regulator, the amount of the dispersion medium, the mass ratio of cerium nitrate hexahydrate in the second solution, the temperature of the heating reaction and the time of the heating reaction is specifically shown in Table 1.

Comparative examples 1 to 2

The preparation method of the cerium oxide microspheres in comparative examples 1 to 2 is mainly different from the preparation method of the cerium oxide microspheres in example 1 in that: at least one of the amount of the cerium-containing precursor, the amount of deionized water, the amount of the pH adjustor, the amount of the dispersion medium, the mass ratio of cerium nitrate hexahydrate in the second solution, the temperature of the heating reaction and the heating reaction time is different, and the details are shown in Table 1.

Comparative example 3

The difference between the preparation method of the cerium oxide microsphere in comparative example 3 and the preparation method of the cerium oxide microsphere in example 1 is that: the product of the heating reaction was subjected to centrifugal separation at a rotational speed of 10,000rpm for 10 minutes. Details are shown in Table 1.

In comparative example 3, the product of the heating reaction was subjected to centrifugal separation, and the supernatant was still very turbid after the centrifugal separation, and therefore, efficient separation was not achieved, and the separation time was long and the time and labor costs were high.

After the reaction products were heated by filtration with ceramic filter membranes in examples 1-9 and comparative examples 1-2, clear and transparent filter materials were obtained.

The appearance of the cerium oxide microspheres prepared in each of the above examples is shown in fig. 1. In FIG. 1, a-i show the appearance of the cerium oxide microspheres prepared in examples 1-9, respectively. As can be seen from fig. 1, as the mass ratio of cerium nitrate hexahydrate in the second solution increases, the average particle diameter of the cerium oxide microspheres increases.

The average particle diameter of the cerium oxide microspheres prepared in each of the above examples and comparative examples was measured by electron scanning electron microscopy, and the results are shown in Table 1.

An electron scanning electron microscope (sem) image of the cerium oxide microspheres prepared in example 1, example 4 and example 9 is shown in fig. 2. Wherein a-c in FIG. 2 shows a TEM image of the cerium oxide microspheres prepared in example 1; FIG. 2 d-f shows a TEM image of cerium oxide microspheres prepared in example 4; in FIG. 2, g-i shows a TEM image of the cerium oxide microspheres prepared in example 9. As is clear from the results in FIG. 2, when the mass ratio of the cerium-containing precursor in the second solution is 7%, large particle aggregates having an average particle diameter of 3000nm are easily formed.

XPS spectrum analysis was performed on the cerium oxide microspheres prepared in each of the above examples and comparative examples, and the mass ratio of Ce (III) in the cerium oxide microspheres was measured, and the results are shown in Table 1.

The XPS spectrum results of the cerium oxide microspheres prepared in example 1, example 4 and example 9 are shown in fig. 3. Wherein, a in fig. 3 represents an XPS spectrum of the cerium oxide microsphere prepared in example 1; FIG. 3 b shows XPS spectrum of cerium oxide microspheres prepared in example 4; FIG. 3 c shows XPS spectrum of cerium oxide microspheres prepared in example 9; fig. 3 d shows the mass ratio of Ce (III) in the cerium oxide microspheres prepared in example 1, example 4 and example 9.

As can be seen from fig. 3, the cerium (III) oxide microsphere prepared in example 1 has a mass ratio of 30.9%; the cerium (III) oxide microsphere prepared in example 4 has a mass ratio of 36.4%; the cerium (III) oxide microsphere prepared in example 9 has a mass ratio of 37.2%; the cerium oxide microspheres of examples 4 and 9 have 17.8% and 20.3% higher mass ratios of Ce (III), respectively, than example 1.

TABLE 1

Wherein w1 represents the mass ratio of the cerium-containing precursor in the second solution; the Ce (III) content represents the molar ratio of Ce (III) in the cerium oxide microsphere.

As shown in the results in Table 1, when the mass ratio of the cerium-containing precursor in the second solution is 3-7%, the average particle size of the prepared cerium oxide microspheres can reach 200-3000 nm; and the molar ratio of Ce (III) in the cerium oxide microsphere can reach 30-38%.

Comparative example 4 preparation of cerium oxide cubes

10G CH ₃COONa、10ml CH₃ COOH and 2.17g Ce (NO ₃)₃-6H₂ O dissolved and diluted to 85mL with water then the solution was transferred to a 100mL Teflon lined stainless steel autoclave heated to 220℃for 24 hours after cooling, the product was collected, centrifuged (12000 r. P. M.) and washed five times with distilled water and dried to give cerium oxide cubes with individual particle sizes of about 25nm.

Comparative example 5 preparation of cerium oxide polyhedron

Weighing 1.30g (3 mmol) Ce (NO ₃)₃·6H₂ O) in a beaker, adding a proper amount of water for dissolution, placing on a magnetic stirrer for stirring for 10min, then adding 0.24g (6 mmol) NaOH, continuing stirring for 30min, regulating the total volume of the solution to 60mL, then completely transferring the solution into a 100mL polytetrafluoroethylene reaction kettle liner, placing the reaction kettle in a metal shell of the reaction kettle, finally placing the reaction kettle in a 120 ℃ oven for reaction for 24h, repeatedly centrifuging and washing the reaction product with deionized water for 3 times, and drying to obtain the cerium oxide polyhedron.

Comparative example 6 preparation of cerium oxide nanospheres

Ethylene glycol 95% (7.8 mL,0.12 mol) was dispersed in 92mL deionized water. Then 5.16g of Ce (NO ₃)₃·6H₂ O) was added, stirred well, then NH ₃.H₂ O was added to adjust the pH to 9.6, then the mixture was reacted at 50℃until the solution became yellow, centrifuged, washed with water, and dried to obtain cerium oxide nanospheres.

Comparative example 7 preparation of cerium oxide nanorods

1.5G of CeCl ₃·7H₂ O (4 mmol) and 4g of NaOH (100 mol) were dispersed in 60-80mL of deionized water and stirred until homogeneous. Then, the mixture was transferred to a reaction vessel and reacted at 130℃for 18 hours. And (3) centrifugally separating, washing with water, drying, and finally calcining the dried powder product in a muffle furnace (300 ℃ for 4 hours) to prepare CeO ₂ nano-rod powder with rich oxygen defects.

Comparative example 8 preparation of calcium-doped cerium oxide

0.059G CaCl ₂·7H₂O、1.34g CeCl₃·7H₂ O (3.6 mmol), 4g NaOH (100 mol), 0.12g CaCl ₂·7H₂ O, 1.20g CeCl ₃·7H₂ O (3.2 mmol), 4g NaOH (100 mol) were dispersed in 60-80mL deionized water, respectively, and stirred until homogeneous. Then, the mixture was transferred to a reaction vessel and reacted at 130℃for 18 hours. And (3) centrifugally separating, washing with water, drying, and finally calcining the dried powder product in a muffle furnace (300 ℃ for 4 hours) to respectively prepare 10% Ca-doped CeO ₂ nano rod and 20% Ca-doped CeO ₂ nano rod powder.

2. Cerium oxide microsphere Performance test

DPPH radical scavenging efficacy

DPPH (1, 1-disphenyl-2-picryl-hydrazyl), which is chemically named 1, 1-diphenyl-2-trinitrophenylhydrazine, has a molecular formula (C ₆H₅)₂N-NC₆H₂(NO₂)₃) in which an unpaired valence electron is present on one atom of the nitrogen bridge, and the orbital motion of this electron is almost offset by the molecular structure, and is usually taken as an index for evaluating the antioxidant ability, DPPH is a stable organic radical having a maximum absorption peak in the visible region of 517nm, and in ethanol solution, each DPPH molecule generates a stable nitrogen-containing radical in solution, which has a typical purple color, and when it reacts with a radical scavenger (e.g., cerium oxide dispersion) providing 1 electron, a colorless product is generated, which lightens the typical purple color of the solution, and the degree of the discoloration is in a stoichiometric relationship with the number of the paired electrons.

1.1 Instruments and experiments

(1) Analytical balance (d=0.1 mg, available from Sidoris, model BSA2202S-CW, max 220 g);

(2) Vortex mixer (model MX-F);

(3) Multifunctional enzyme labeling instrument (purchased from Tecan), 96 well plate (model number is jet)

(4) Pipette (5 mL, 200. Mu.L, 1000. Mu.L, available from Eppendorf);

(5) DPPH (available from sigma, analytically pure, C ₁₈H₁₂N₅O₆, molecular weight 394.32);

(6) Vc (purchased from bayer di biology ltd., analytically pure);

(7) Absolute ethanol (purchased from national pharmaceutical systems chemical company, inc.).

1.2 Preparation for test

1.2.1 Reagent preparation

(1) 0.2Mmol/L DPPH solution

Weigh 0.0200g DPPH, add absolute ethanol to dissolve and volume to 250mL volumetric flask, shake well.

(2) 200 Μg/mL VC solution (note: on-the-fly)

Weighing 0.0500gVC, adding deionized water for dissolution, and shaking uniformly in a 250mL volumetric flask.

(3) 15 Μg/mL VC solution

200 Mug/mL VC aqueous solution 0.600mL was taken, 7.400mL absolute ethanol was added and mixed well.

(4) 0.5Wt% cerium oxide dispersion

50Mg of cerium oxide sample to be measured is taken and dispersed in 10mL of deionized water, the ultrasonic dispersion is carried out for 10min, and the cerium oxide sample is uniformly shaken, thus obtaining 0.5wt% cerium oxide dispersion.

1.2.2 Test procedure

Setting a blank group C, a negative control group B, a positive control group AVc and a sample group A respectively; wherein, sample group A refers to cerium oxide products with different morphologies prepared in examples 1-9 and comparative examples 4-8, and cerium oxide products with different morphologies were respectively dispersed in water to prepare a dispersion with a concentration of 0.5 wt%.

1.2.3 Results processing

The DPPH radical scavenging rate is calculated as follows: clearance = [ (y+z) -X ]/B × 100%

Wherein: x represents the absorbance value after the sample solution is mixed with DPPH solution; y represents the absorbance value after absolute ethyl alcohol is mixed with DPPH solution; z represents the absorbance after absolute ethanol is mixed with the sample solution.

1.2.3 Experimental procedure

The specific sample addition sequence and the amount are shown in tables 2-1 and 2-2, respectively.

TABLE 2-1 sample tube reagent addition

TABLE 2-2 sample tube reagent addition

In addition, A1 to a10 represent 10 samples of each cerium oxide product were set up for parallel measurement 10 times.

1.2.4 Analysis of results

The DPPH clearance results for the cerium oxide microspheres of examples 1-9 are shown in Table 3.

TABLE 3 Table 3

Group of

Example 1

Example 2

Example 3

Example 4

Example 5

Example 6

Example 7

Example 8

Example 9

Clearance rate of

94.8％

95.6％

94.7％

94.4％

92.1％

92.8％

94.3％

94.5％

95.6％

The DPPH radical scavenging effect of example 1, comparative examples 4-8 and the VC control is shown in FIG. 4. Fig. 4a shows the DPPH stock solution, fig. 4b shows the effect of treating the DPPH stock solution with the cerium oxide microspheres of example 1, and fig. 4 c shows the effect of treating the DPPH stock solution with the positive control group AVc.

As can be seen from the results of FIG. 4, the cerium oxide microspheres prepared in example 1 have the best oxidation resistance. Compared with the positive control group AVc, the average result of three experiments on the DPPH clearance rate of the VC is 62.4 percent, and the average result of three experiments on the DPPH clearance rate of the cerium oxide microsphere in the embodiment 1 is as high as 94.8 percent, which is improved by 29 percent compared with the positive control group AVc. Furthermore, it is apparent from the solution after DPPH reaction that the cerium oxide microspheres exhibit excellent radical scavenging ability. After 30min of reaction, the color of the mixed solution of VC and DPPH is darker than that of the mixed solution of cerium oxide microspheres and DPPH.

The cerium oxide microspheres prepared in example 1, example 4 and example 9 were subjected to DPPH radical scavenging test according to the above procedure, and the results are shown in fig. 5. As can be seen from FIG. 5, the effect concentration of the cerium oxide microspheres for eliminating DPPH in example 9 is as low as 0.05wt%, and the DPPH eliminating rate is more than 90%; the effective concentration is lower than that of the cerium oxide microspheres in examples 1 and 4, and the DPPH clearance is higher than that of the cerium oxide microspheres in examples 1 and 4. The higher the ratio of Ce (III) in the cerium oxide microsphere, the better the DPPH scavenging effect of the cerium oxide microsphere.

The cerium oxide microspheres prepared in example 1 were subjected to DHHP cycle purge experiments to more intuitively illustrate their sustained antioxidant capacity. The specific experimental procedure is as follows:

Primary experiment: 500. Mu.L of the cerium oxide microsphere dispersion with the concentration of 0.5wt% was added to the sample tube, and 500. Mu.L of DPPH solution with the concentration of 0.2mmol/L (consistent with the concentration used in the DPPH removal test) was added and mixed well. After 20min of reaction, separation was carried out for 10min at 10,000rpm using a centrifuge. The supernatant was aspirated into a 96-well plate and absorbance was measured at 517 nm.

DPPH cycle purge experiment: the cerium oxide microsphere precipitate obtained by centrifugal separation is dispersed in 500 mu L of deionized water (neglecting the trace loss in the centrifugal separation process), and 500 mu L of DPPH solution with the concentration of 0.2mmol/L is added for uniform mixing. After 20min of reaction, separation was carried out for 10min at 10,000rpm using a centrifuge. The supernatant was aspirated into a 96-well plate and absorbance was measured at 517 nm. This procedure was repeated 5 times. The results are shown in FIGS. 6-8.

Fig. 8 a shows DPPH stock solution, fig. 8 b shows cerium oxide microspheres circulating once, fig. 8 c shows cerium oxide microspheres circulating twice, fig. 8 d shows cerium oxide microspheres circulating three times, fig. 8 e shows cerium oxide microspheres circulating four times, fig. 8 f shows cerium oxide microspheres circulating five times, and fig. 8h shows VC control.

As can be seen from fig. 6 to 8, the efficiency of DPPH removal can reach 76.6% after the cerium oxide microsphere in example 1 is recycled five times, and still is significantly higher than the DPPH removal rate of VC by 62.4%.

The cerium oxide microspheres used in each of the experiments described below were the cerium oxide microspheres prepared in example 1.

2. Antioxidant in vitro cell model-ROS scavenging experiment

Oxidative stress refers to the process of oxidative damage caused by the fact that when the body is subjected to various stimuli, high-activity molecules such as Reactive Oxygen Species (ROS) of the body are produced excessively beyond the scavenging capacity of an in-vivo antioxidant system, so that the body oxidative system and the antioxidant system are disturbed, and free radicals are accumulated in the body, and the oxidative damage is considered to be one of important factors causing skin aging. Studies have shown that long-wave ultraviolet (UVA 320-400 nm) in sunlight is one of the most dominant environmental factors causing skin aging. UVA can directly damage fibroblasts at the dermis site, producing Reactive Oxygen Species (ROS). The method is based on a human skin fibroblast (HFF-1) model, and the antioxidation effect of the sample is further evaluated by measuring the influence of the sample on the generation of cellular ROS after photodamage.

2.1 Test materials and apparatus

2.1.1 Test materials

Human skin fibroblasts (HFF-1) were purchased from the Chinese academy of sciences stem cell bank. Fetal bovine serum (FBS, BI), DMEM high sugar medium (DMEM, purchased from Gibco), dimethyl sulfoxide (DMSO, purchased from Sigma), phosphate buffer (PBS, purchased from Gibco), vitamin E (VE), vitamin C (VC, purchased from Sigma), thiazole blue (MTT, lab), fluorescent probe DCFH-DA (purchased from Sigma).

2.1.2 Test apparatus

Super clean bench (from tai koilon), inverted microscope (from Leica), carbon dioxide incubator (from Thermo), electric aspirator (from Eppendorf), microplate reader (from Tecan), uv irradiation box (from midoboda), flow cytometer (from beckman).

2.2 Test methods

2.2.1 HFF-1 cell-based toxicity detection

The test adopts MTT method to detect cell activity, and the maximum safe concentration of cell sample is screened. The test set-up negative control (medium), positive control (medium with 10% dmso) and zeroed wells (PBS), sample concentration set-up are shown in table 1,3 replicate wells per concentration set-up. The specific operation steps are as follows:

(1) Inoculating: taking cells in logarithmic growth phase, inoculating the cells to a 96-well plate, and placing the culture plate in a 5% CO ₂ incubator at 37 ℃ for incubation and culture for 18-24h.

(2) Preparing liquid: test substances of different concentrations were prepared according to the concentration settings in table 4.

TABLE 4 sample cytotoxicity concentration setting

(3) Sample feeding: after 18-24h of cell growth, the supernatant was discarded, medium containing different concentrations of the test substance was added, and the plates were placed in a 5% CO ₂ incubator at 37℃for incubation for 18-24h.

(4) And (3) detection: after 18-24h of cell culture, the supernatant was discarded, and the formulated and filtered MTT (0.5 mg/mL) was added, gently mixed, and incubated at 37℃for 4h in the absence of light. After the incubation, the supernatant was discarded, 150. Mu.L of DMSO was added to each well, and the wells were shaken for 20min, and the OD570 nm was read with an ELISA reader.

(5) Relative cell viability calculation formula:

Relative cell viability = (test sample well OD-zeroed well OD)/(negative control OD-zeroed well OD) ×100%.

2.2.2 Detection of ROS expression in HFF-1 cells based on UVA irradiation

(1) Inoculating: cells were inoculated into 6-well plates and incubated at 37℃in a 5% CO ₂ incubator for 18-24h.

(2) Preparing liquid: test and positive controls were formulated according to table 5.

(3) Sample feeding: according to the test group and concentration settings of Table 5, after cell plating in 6 well plates grows for 18-24 hours, group feeding was performed, and 3 duplicate wells were set per group. In group 1, cell culture medium containing 0.1% DMSO was added to both the blank control group and the negative control group, and cell culture medium containing 0.001% vitamin E was added to the positive control group; in group 2, the blank control and the negative control are added with cell culture medium, the positive control is added with cell culture medium containing 0.01% of vitamin C, the sample group is added with cell culture medium containing samples with corresponding concentrations, and the culture is continued for 18-24 hours at 37 ℃ in a 5% CO ₂ incubator.

(4) And (3) irradiation: after 18-24h of incubation, the medium was removed, the washing solution was removed by rinsing twice with 1mL of PBS, 1mL of PBS was added, and the groups to be irradiated were irradiated at a dose of 30J/cm ² UVA, depending on the group setting. After the irradiation dose was reached, the PBS in the wells was removed, washed once with 1mL of pre-warmed PBS, and further incubated with DMEM medium for 2h.

(5) ROS expression detection: after loading the cells with the fluorescent probe DCFH-DA, the cells are collected and detected by a flow cytometer, and the fluorescence intensity value MFI is read.

(6) And (3) data processing: various data obtained in the test were processed and plotted by Excel software. Statistical analysis was performed with SPSS 17.0, and group comparisons were performed using one-way analysis of variance (ANOVA), with significant differences judged when P < 0.05.

TABLE 5 test grouping and concentration setting

2.2.3 Evaluation based on results of detection of ROS expression in HFF-1 cells by UVA irradiation

The ROS expression of the negative control group is obviously increased compared with that of the blank control group, and the ROS expression of the positive control group is obviously reduced compared with that of the negative control group, so that the test system is established. The significant decrease in ROS expression in the sample group compared with the negative control group indicates that the sample has an antioxidant effect.

2.3 Experimental results

The results are shown in Table 6 and FIG. 9.

TABLE 6 ROS expression test results

Wherein, ΔΣ represents extremely significant differences, P <0.01, compared to SC (UVA-, 0.1% dmso) group;

delta represents that the difference is very significant compared to BC (UVA-) group, P <0.01;

# indicates that the difference was very significant, P <0.01, compared to the SC (uva+, 0.1% dmso) group;

* Indicating significant differences compared to NC (uva+) group, P <0.05;

* Indicates that the difference was very significant compared to NC (uva+) group, P <0.01.

As shown in table 6 and fig. 9, the ROS scavenging effect of the cerium oxide microsphere in example 1 was comparable to that of the VE control and VC control, indicating that the cerium oxide microsphere has excellent antioxidant effect. The UVA irradiation (uva+) group caused a significant increase in ROS expression compared to the UVA non-irradiation (UVA-) group; and compared with the SC (UVA+, 0.1% DMSO) group, the PC (VE) group can obviously reduce the ROS expression, which shows that the test system is established. According to the cytotoxicity test result, the cell morphology, the loading concentration of the final selected cerium oxide microsphere dispersion liquid on HFF-1 cells is 0.0313%, 0.0625%, 0.1% and 0.125%. The test results show that compared with NC (UVA+) group, the cerium oxide microsphere dispersion liquid can obviously reduce the expression of ROS when the loading concentration is 0.0313%, 0.0625%, 0.1% and 0.125%. Based on a UVA irradiation human skin fibroblast injury model, the cerium oxide microsphere dispersion liquid can play an antioxidant effect by reducing ROS expression at the concentration of 0.0313%, 0.0625%, 0.1% and 0.125%.

3. Inhibition of matrix metalloproteinase-1 activity (MMP-1)

Skin aging is classified into endogenous aging and exogenous aging. The former is also known as natural aging, and is a natural procedural aging process. The latter is skin aging due to external environmental factors such as ultraviolet light, smoking, blowing, and exposure to chemicals. Human skin fibroblasts are the main cells in the dermis of the skin, are rich in collagen, and their characteristic biological changes play an important role in the skin aging process. Type I collagen (COL-I) is one of the main components of the extracellular matrix of dermis, and is synthesized by dermal fibroblasts into type I procollagen, secreted to the outside of the dermis, and polymerized to form collagen fibers after terminal peptide separation under the action of terminal procollagen peptidase. Once the skin lacks collagen, the collagen fibers crosslink and solidify, and the skin ages. ECM is an intricate network of macromolecules. The main components include collagen, elastin, non-collagen (matrix) glycoprotein and proteoglycan. Matrix metalloproteinase-1 (MMP-1) is capable of degrading ECM, causing skin collagen loss, so over-expression of MMP-1 is one of the important causes of skin aging.

In the test, human skin fibroblasts (HFF-1) are taken as a research object, and after the product is added, the influence of the product on the MMP-1 content in cell supernatant is detected, so that the tightening and anti-wrinkle effects of the product are evaluated. MMPs degrade collagen and elastin in dermis, and are the most important enzymes for causing aging symptoms such as skin shrinkage and fine wrinkles. Collagen and elastin have been considered as an important indicator of aging, and MMP-1 is the most predominant enzyme that degrades type I and type III collagen in the dermis. When MMP-1 is overexpressed, extracellular matrix components are specifically degraded, disrupting the normal structure of collagen and elastin, thereby causing skin aging. The anti-aging efficacy of the test agent can be evaluated by measuring the inhibitory capacity of the test agent on the matrix metalloproteinase activity.

3.1 Test materials and apparatus

3.1.1 Test materials

Human skin fibroblasts (HFF-1) were purchased from the Chinese academy of sciences stem cell bank. Fetal bovine serum (FBS, BI), DMEM high-sugar medium (DMEM, purchased from Gibco), dimethyl sulfoxide (DMSO, purchased from Sigma), phosphate buffer (PBS, purchased from Gibco), tgfβ1 (PEPROTECH), thiazole blue (MTT, lab), matrix metalloproteinase-1 (total) ELISA kit (MMP-1, purchased from bosch).

3.1.2 Test apparatus

Super clean bench (from Thai Colon), inverted microscope (from Leica), carbon dioxide incubator (from Thermo), electric aspirator (from Eppendorf), enzyme-labeled instrument (from Tecan).

3.2 Test methods

3.2.1 HFF-1 cell-based toxicity detection

(2) Preparing liquid: test substances of different concentrations were prepared according to the concentration settings in table 7.

TABLE 7 sample cytotoxicity concentration setting

(5) Relative cell viability calculation formula:

3.2.2 Scratch test detection based on HaCaT cells

(1) Inoculating: cells were first inoculated into 6-well plates with a sign pen from a ruler against two vertical lines at the back of the 6-well plates and incubated overnight at 37℃in a 5% CO ₂ incubator.

(2) Preparing liquid: test and positive controls were formulated according to table 8.

(3) Scoring: after cells in 6-well plates were plated for 18-24h, each well was scored with a 1mL gun head against a ruler, perpendicular to the back cross line, two wells per well, the supernatant discarded, and the scored cells were removed by three washes with PBS.

TABLE 8 test grouping and concentration setting

(4) Dosing and photographing: the prepared samples were each added to 6-well plate corresponding locations for administration and photographed under 5 x mirror for 0h (i.e., immediately). The plates were incubated at 37℃under 5% CO ₂ for a further 18-24h.

(5) Photographing and image analysis: after cell culture for 24 hours, a photograph was taken again under a 5X mirror at the same position as at 0 hours, and then migration area analysis was performed.

(6) Cell mobility calculation: calculated from the cell mobility formula, the calculation formula is as follows: cell mobility (%) = (a ₀-A₂₄)/A₀ ×100)

Wherein A ₀ refers to the blank area when photographing for 0h, and A ₂₄ refers to the blank area of the photographed picture when photographing for 24 h.

(7) And (3) data processing: various data obtained in the test were processed and plotted by Excel software. Statistical analysis was performed with SPSS 17.0, and group comparisons were performed using one-way analysis of variance (ANOVA), with significant differences judged when P < 0.05.

3.2.3 Evaluation of scratch test detection results based on HaCaT cells

The cell mobility of the positive control group is obviously increased compared with that of the negative control group, which indicates that the test system is established. The significant increase in cell mobility of the sample group compared with the negative control group indicates that the sample can promote cell migration to achieve the effect of barrier damage repair.

3.3 Experimental results

The results of MMP-1 expression detection based on natural HFF-1 cells are shown in Table 9 and FIG. 10, and compared with the NC group, the expression of MMP-1 content in the PC (TGF beta 1) group is remarkably reduced, which shows that the test system is established.

TABLE 9 MMP-1 expression assay results

As is clear from the results shown in Table 9 and FIG. 10, the cerium oxide microsphere dispersion in example 1 was able to significantly reduce MMP-1 expression when the concentration of the cerium oxide microsphere dispersion on HFF-1 cells was 0.0313%, 0.0625%, 0.1%, and 0.125% as compared with the NC group. Based on the detection result of MMP-1 expression of natural HFF-1 cells, the cerium oxide microsphere dispersion liquid has certain tightening and anti-wrinkle effects. And the inhibitory effect on MMP was close to PC (TGF-. Beta.1) reference level at the loading concentrations of the cerium oxide microsphere dispersion on HFF-1 cells of 0.0625% and 0.1%.

IL-1 alpha inflammatory factor inhibition

There are three general types of skin inflammation: skin irritation, allergy and sensitization. The skin irritation refers to the process of activating an inflammatory reaction mediated by an innate immune response after skin is contacted with external stimuli, and finally causing skin injury. Macrophages are an important defense line of the immune system of a living body, can cause an innate immune response and an acquired immune response, and can express cell inflammatory factors when being stimulated by the outside, and excessive macrophages can cause the immune disorder of the living body. Lipopolysaccharide (Lipopolysaccharide LPS), one of the components of the cell wall of gram-negative bacteria. When LPS acts on macrophages, inflammatory reactions of the macrophages are induced, so that a large amount of cell inflammatory factors such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and the like are generated.

Skin inflammatory response is also one of the causes of skin aging. Oxidative stress caused by excessive active oxygen can trigger inflammation, and the inflammation is often accompanied with oxidative stress, and the oxidative stress can maintain continuous occurrence of inflammation, so as to form oxidative stress-inflammation-oxidative stress-inflammation vicious circle. ROS in inflammation activate NF- κb signaling pathway, thereby promoting expression of inflammatory factors (TNF- α, IL-6, IL-8, IL-1 α), exacerbating the inflammatory response of the body, while the presence of cerium oxide reduces the production of such pro-inflammatory cytokines, playing an anti-inflammatory role. In addition, cerium oxide can inhibit the formation of gaseous inflammatory mediators NO by inhibiting c-Jun N-terminal kinase (JNKs) to activate the nitric oxide synthase (iNOS) pathway, and can also achieve resistance to inflammatory reactions. The test evaluates the capability of a product to reduce the expression and secretion of cell inflammatory factors based on an LPS-induced mouse macrophage (RAW 264.7) inflammatory model, and further predicts the capability of the product to inhibit further occurrence of inflammatory reaction. The indexes of the test detection are introduced as follows:

Interleukin-6 (IL-6) is identified as a B cell growth factor, can be induced by TNF-alpha and IL-1 beta, has a biological effect similar to IL-1 beta, can stimulate the production of other cytokines, induces organism specific and nonspecific immune responses to occur, can cause fever reaction, plays an important role in the processes of immunoregulation and inflammation reaction, is considered to be one of main endogenous mediators of fever reaction in the processes of inflammation reaction, and once the content of inflammatory factors in skin cells is increased, the skin temperature is higher than normal temperature, and aggravates skin inflammation reaction.

Interleukin-1 alpha (IL-1 alpha), which is produced mainly by monocytes, macrophages, plays a critical role in inflammation and immune response. Interleukin-1 alpha and interleukin-1 beta bind to the same cell surface receptor, causing the same biological response.

4.1 Test materials and apparatus

4.1.1 Test materials

Mouse macrophages (RAW 264.7, purchased from the cell resource center of basic medical institute of the national academy of medicine), fetal bovine serum (FBS BI), DMEM high sugar medium (DMEM, purchased from Gibco), dimethyl sulfoxide (DMSO, purchased from Sigma), phosphate buffer (PBS, purchased from Gibco), thiazole blue (MTT, lab), interleukin 6 enzyme-linked immunosorbent assay kit (Mouse IL 6ELISA kit Boster), mouse interleukin-1 alpha enzyme-linked immunosorbent assay kit (Mouse IL 1 alpha ELISA kit Boster).

4.1.2 Test apparatus

Super clean bench (from Thai Colon), inverted microscope (from Leica), carbon dioxide incubator (from Thermo), electric aspirator (from Eppendorf), cell waste liquid pump (from its Linbell), enzyme-labeled instrument (from Tecan).

4.2 Test methods

4.2.1 RAW264.7 cell-based toxicity detection

The test adopts MTT method to detect cell activity, and the maximum safe concentration of cell sample is screened. The test set-up negative control (medium), positive control (medium with 5% dmso) and zeroed wells (PBS), sample concentration set-up are shown in table 1, 3 replicate wells per concentration set-up. The specific operation steps are as follows:

(1) Inoculating: taking cells in logarithmic growth phase, inoculating the cells to a 96-well plate, and placing the culture plate in a 37-5% CO ₂ incubator for incubation and culture for 18-24h.

(2) Preparing liquid: test substances of different concentrations were prepared according to the concentration settings in table 10.

(4) And (3) detection: after 18-24h of cell culture, the supernatant was discarded, the formulated and filtered MTT (0.5 mg/mL) was added, gently mixed, and incubated at 37℃for 4h in the absence of light. After the incubation, the supernatant was discarded, 150. Mu.L of DMSO was added to each well, and the wells were shaken for 20min, and the absorbance at OD 570nm was read with an ELISA reader.

(5) Relative cell viability calculation formula:

TABLE 10 sample cytotoxicity concentration setting

4.2.2 Detection of RAW264.7 cytokine based on LPS Induction

(1) Inoculating: cells were seeded into 24-well plates and incubated at 37℃in a 5% CO ₂ incubator for 18-24h.

(2) Preparing liquid: test and positive controls were formulated according to table 11.

(3) Sample feeding: according to the test group and concentration settings of Table 11, after cell plating in 24 well plates was grown for 18-24 hours, group feeding was performed with 3 duplicate wells per treatment group. In group 1, the blank and negative control groups were both supplemented with cell culture medium containing 0.1% dmso, and the positive control group was supplemented with cell culture medium containing 0.001% dexamethasone; in group 2, the blank control and the negative control are added with cell culture medium, the sample group is added with cell culture medium containing samples with corresponding concentrations, and the culture is continued for 18-24 hours at 37 ℃ in a 5% CO ₂ incubator.

TABLE 11 test grouping and concentration setting

In table 11, BC represents a blank; NC represents a negative control; SC represents negative control and blank control corresponding to positive substance.

(4) LPS induction: after 18-24h incubation, the plate medium was aspirated, both were washed once with PBS, the blank was added to the cell medium, the negative, sample and positive control were added to the LPS-containing cell medium, and the supernatant was collected after further incubation in a 5% CO ₂ incubator at 37℃for 18-24 h.

(5) Detecting the content of inflammatory factors: cell supernatants from each well were taken and assayed for cellular inflammatory factor content according to ELISA kit protocol.

4.2.3 Evaluation based on LPS-induced RAW264.7 cytokine detection results

The inflammatory factor expression level of the negative control group is obviously increased compared with that of the blank control group, and the inflammatory factor expression level of the positive control group is obviously reduced compared with that of the negative control group, so that the test system is established. Compared with a negative control group, the inflammatory factor expression level of the sample group is obviously reduced, so that the sample has the capability of inhibiting the further occurrence of inflammatory reaction.

4.3 Experimental results

The detection results based on the LPS-induced RAW264.7 cell model are as follows:

The results of the inflammatory factor IL-6 expression test are shown in Table 12 and FIG. 11, and the results of the test show that the LPS-induced (LPS+) group causes a very significant increase in the expression level of IL-6 (P < 0.01) compared with the LPS-uninduced (LPS-) group, indicating that LPS-induced modeling was successful. Compared with SC (0.1% DMSO, LPS+), PC (dexamethasone) dexamethasone can significantly reduce the expression level of IL-6 (P < 0.01) at the sample concentration of 0.001%, which indicates that the test system is established. The dispersion of cerium oxide microspheres in example 1 showed a significant inhibitory effect on IL-6 expression at a concentration of 0.0078% compared to NC (LPS+) group.

TABLE 12 detection of inflammatory factor IL-6 expression

The quantitative limit (minimum concentration of standard curve) of the method was 15.6pg/mL, and the detection limit was 1pg/mL.

The results of the inflammatory factor IL-1α expression test are shown in Table 13 and FIG. 12, and the results of the test show that the expression level of IL-1α is significantly increased (P < 0.01) in the LPS-induced (LPS+) group compared with the LPS-uninduced (LPS-) group, which indicates that LPS-induced modeling is successful.

TABLE 13 detection of inflammatory factor IL-1 alpha expression

Wherein, the quantitative limit (standard curve minimum concentration) of the method is 4.69pg/mL, and the detection limit is 1pg/mL.

As can be seen from the results of FIGS. 11 to 12 and tables 12 to 13, the PC (dexamethasone) group dexamethasone significantly reduced the expression level of IL-1α (P < 0.01) at a sample concentration of 0.001% compared with SC (0.1% DMSO, LPS+), indicating that the test system was established. The dispersion of cerium oxide microspheres in example 1 showed a significant inhibitory effect on the expression of IL-1α at a concentration of 0.0078% compared to NC (LPS+) group.

Compared with the non-induced (LPS-) group of LPS, the induced (LPS+) group of LPS causes the expression of IL-6 and IL-1 alpha to be obviously increased; compared with the SC (0.1% DMSO, LPS+) group, the PC (dexamethasone) group can obviously lower the expression quantity of IL-6 and IL-1 alpha, which shows that the test system is established. According to the cytotoxicity test result, the cell morphology, the loading concentration of the cerium oxide microsphere on the cell in example 1 was selected to be 0.0078%, 0.0156% and 0.0313%. The test results showed that the dispersion of cerium oxide microspheres in example 1 showed significant inhibition of IL-6 and IL-1α expression at a concentration of 0.0078% compared to NC (LPS+) group.

The experimental result shows that when the mass ratio of Ce ³⁺ in the cerium oxide microsphere is up to 30% -38%, the catalytic performance and oxidation resistance of the cerium oxide microsphere can be improved; when applied to the field of cosmetics, the anti-oxidation and anti-aging effects are excellent.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. A cerium oxide microsphere, wherein the cerium oxide microsphere comprises Ce ³⁺, and the molar ratio of Ce ³⁺ in the cerium oxide microsphere is 30% -38%.

2. The cerium oxide microsphere according to claim 1, wherein the molar ratio of Ce ³⁺ in the cerium oxide microsphere is 36% to 38%.

3. The cerium oxide microspheres according to any one of claims 1 to 2, wherein the average particle size of the cerium oxide microspheres is 0.2 μm to 3 μm, optionally 0.4 μm to 3 μm.

4. A method for preparing cerium oxide microspheres according to any one of claims 1 to 3, comprising the steps of:

Adding a pH regulator into the precursor solution to prepare a first solution;

And heating the second solution for reaction.

5. The method for preparing cerium oxide microspheres according to claim 4, wherein the mass ratio of the cerium-containing precursor in the second solution is 5 to 7%.

6. The method of preparing cerium oxide microspheres according to claim 4, wherein the step of preparing the precursor solution comprises at least one of the following conditions:

(2) The mass volume ratio of the cerium-containing precursor to the water is (20-120) g (10-60) mL; and/or

The step of preparing the first solution comprises at least one of the following conditions:

(2) The mass volume ratio of the cerium-containing precursor to the pH regulator is (20-120) g (20-120) mL; and/or

The step of preparing the second solution comprises at least one of the following conditions:

(1) The dispersion medium comprises ethylene glycol;

(2) The mass volume ratio of the cerium-containing precursor and the dispersion medium is (20-120) g (600-700) mL; and/or

The heating reaction includes at least one of the following conditions:

(1) The pressure of the heating reaction is 0.5Mpa-4Mpa;

(2) The temperature of the heating reaction is 160-170 ℃;

(3) The heating reaction time is 140-180 min;

7. The method for preparing cerium oxide microspheres according to any one of claims 4 to 6, further comprising the steps of:

Filtering the heated reaction product, washing the suspension after the filtering treatment to be neutral, and drying to prepare the cerium oxide microspheres;

optionally, the filtering treatment is performed by adopting a ceramic filter membrane;

Further alternatively, the ceramic filter membrane has an average pore size of 100nm to 500nm.

8. Use of the cerium oxide microsphere according to any one of claims 1 to 3 or the cerium oxide microsphere prepared by the preparation method according to any one of claims 4 to 7 in the preparation of cosmetics.

9. Cosmetic comprising the cerium oxide microsphere according to any one of claims 1 to 3 or the cerium oxide microsphere prepared by the preparation method according to any one of claims 4 to 7.

10. The cosmetic according to claim 9, wherein the cosmetic comprises a cosmetic having an anti-aging effect.