Cerium oxide microspheres and preparation method, application and cosmetics thereof

Abstract

The present invention provides a cerium oxide microsphere and a preparation method, application and cosmetics thereof; wherein the cerium oxide microsphere comprises Ce 3+ , Ce 3+ The molar proportion of Ce in the cerium oxide microspheres is 30%-38%. 3+ and Ce 4+ It can be converted reversibly, and in this conversion process, oxygen vacancies are generated and eliminated. This characteristic gives cerium oxide good oxygen storage and release capabilities, making it have catalytic and antioxidant properties. 3+ When the molar proportion in cerium oxide microspheres is as high as 30%-38%, the catalytic performance and antioxidant properties of cerium oxide microspheres can be improved, thereby improving its antioxidant effect in cosmetics.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and in particular to cerium oxide microspheres, a preparation method thereof, an application thereof and cosmetics. Background Art

[0002] Cerium (Ce) is the most abundant rare earth element on the earth. The atomic number of Ce is 57, and the outermost electron structure is [Xe]4f 1 5d 1 6s 2 , there are two oxidation states +3 and +4. Among all cerium-containing compounds, cerium dioxide (CeO2) is widely used in many fields due to its low cost, unique crystal structure and good redox ability, such as automobile exhaust treatment, CO catalytic oxidation, nitrogen oxide reduction, water steam reforming reaction, etc.

[0003] With the progress of research, people have found that cerium oxide can eliminate a variety of active oxidative species and has good biosafety, and has important application value in the field of biological antioxidants. However, in related technologies, the application of cerium oxide in cosmetics is relatively small, so the development and application research of cerium oxide in the field of cosmetics is of great significance. Summary of the invention

[0004] Based on this, it is necessary to provide a cerium oxide microsphere and a preparation method, application and cosmetics thereof, so as to study the development and application of cerium oxide in the field of cosmetics.

[0005] The first aspect of the present invention provides a cerium oxide microsphere, wherein the cerium oxide microsphere comprises Ce 3+ , the Ce 3+ The molar proportion in the cerium oxide microspheres is 30%-38%.

[0006] In some embodiments, the Ce 3+ The molar proportion in the cerium oxide microspheres is 36%-38%.

[0007] In some embodiments, the average particle size of the cerium oxide microspheres is 0.2 μm-3 μm.

[0008] In some embodiments, the average particle size of the cerium oxide microspheres is 0.4 μm-3 μm.

[0009] The second aspect of the present invention provides a method for preparing the cerium oxide microspheres of the first aspect, comprising the following steps:

[0010] mixing a cerium-containing precursor with water to prepare a precursor solution;

[0011] Adding a pH adjuster to the precursor solution to prepare a first solution;

[0012] Adding a dispersion medium to the first solution to prepare a second solution; the mass proportion of the cerium-containing precursor in the second solution is 3%-7%;

[0013] The second solution is heated for reaction.

[0014] In some embodiments, the cerium-containing precursor accounts for 5%-7% by weight in the second solution.

[0015] In some embodiments, the step of preparing the precursor solution includes at least one of the following conditions:

[0016] (1) The cerium-containing precursor includes one or more of cerium nitrate hexahydrate and cerium nitrate;

[0017] (2) The mass volume ratio of the cerium-containing precursor and the water is (20-120) g:(10-60) mL.

[0018] In some embodiments, the step of preparing the first solution comprises at least one of the following conditions:

[0019] (1) The pH adjuster includes one or more of acetic acid, formic acid and propionic acid;

[0020] (2) The mass volume ratio of the cerium-containing precursor and the pH adjuster is (20-120) g: (20-120) mL.

[0021] In some embodiments, the step of preparing the second solution comprises at least one of the following conditions:

[0022] (1) The dispersion medium includes ethylene glycol;

[0023] (2) The mass volume ratio of the cerium-containing precursor to the dispersion medium is (20-120) g: (600-700) mL.

[0024] In some embodiments, the heating reaction comprises at least one of the following conditions:

[0025] (1) The pressure of the heating reaction is 0.5Mpa-4Mpa;

[0026] (2) The temperature of the heating reaction is 160° C.-170° C.;

[0027] (3) The heating reaction time is 140 min-180 min;

[0028] (4) The heating reaction is carried out in a Hastelloy pressure-resistant reactor;

[0029] Optionally, the heating reaction is carried out in a Hastelloy pressure-resistant reactor with a volume of 1 L.

[0030] In some embodiments, the preparation method further comprises the following steps:

[0031] The heated reaction product is filtered, and the filtered suspension is washed to neutrality and dried to prepare the cerium oxide microspheres.

[0032] In some embodiments, the filtration process is performed using a ceramic filter membrane;

[0033] Optionally, the average pore size of the ceramic filter membrane is 100nm-500nm.

[0034] The third aspect of the present invention provides a use of the cerium oxide microspheres according to the first aspect of the present invention or the cerium oxide microspheres prepared by the preparation method of the second aspect of the present invention in the preparation of cosmetics.

[0035] The fourth aspect of the present invention provides a cosmetic comprising the cerium oxide microspheres according to the first aspect of the present invention or the cerium oxide microspheres prepared by the preparation method according to the second aspect of the present invention.

[0036] In some embodiments, the cosmetic comprises a cosmetic having an anti-aging effect.

[0037] The cerium oxide microspheres, preparation method, application and cosmetics thereof, wherein Ce 3+ The mass proportion of Ce in cerium oxide microspheres is 30%-38%; 3+ and Ce 4+ It can be converted reversibly, and in this conversion process, oxygen vacancies are generated and eliminated. This property gives cerium oxide good oxygen storage and release capabilities, making it have catalytic and antioxidant properties. 3+ When the mass proportion in cerium oxide microspheres is as high as 30%-38%, the catalytic performance and antioxidant performance of cerium oxide microspheres can be improved; when it is applied in the field of cosmetics, it has excellent antioxidant and anti-aging effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Schematic diagram of the appearance of cerium oxide microspheres prepared in Example 1-9;

[0039] Figure 2 The electron scanning electron micrographs of the cerium oxide microspheres prepared in Example 1, Example 4 and Example 9;

[0040] Figure 3 XPS spectrum analysis of the cerium oxide microspheres prepared in Example 1, Example 4 and Example 9;

[0041] Figure 4 It is a schematic diagram of the DPPH free radical scavenging effect of Example 1, Comparative Examples 4-8 and VC control;

[0042] Figure 5 Schematic diagram showing the variation of DHHP removal rate with the concentration of cerium oxide microspheres prepared in Example 1, Example 4 and Example 9;

[0043] Figure 6 This is a schematic diagram of the DPPH removal effect of cerium oxide microspheres and VC control cycles in Example 1;

[0044] Figure 7 The scavenging rate of DPPH when the cerium oxide microspheres are used for five cycles in Example 1;

[0045] Figure 8 This is a schematic diagram of the results of treating DPPH stock solution with cerium oxide microspheres and VC control in Example 1;

[0046] Fig. 9 The results of the ROS expression test of HFF-1 cells irradiated by UVA based on the dispersion of cerium oxide microspheres in Example 1;

[0047] Fig.10 The figure is a schematic diagram showing the results of the dispersion of cerium oxide microspheres in Example 1; wherein ** indicates that the difference is extremely significant compared with the NC group, P<0.01;

[0048] Fig.11 Schematic diagram of the effect of the dispersion of cerium oxide microspheres on the expression of inflammatory factor IL-6 in Example 1; wherein ▲▲ indicates that compared with the SC (0.1% DMSO, LPS-) group, the difference is extremely significant, P<0.01; △△ indicates that compared with the BC (LPS-) group, the difference is extremely significant, P<0.01; ## indicates that compared with the SC (0.1% DMSO, LPS+) group, the difference is extremely significant, P<0.01; ** indicates that compared with the NC (LPS+) group, the difference is extremely significant, P<0.01;

[0049] Fig.12 Schematic diagram of the effect of the dispersion of cerium oxide microspheres in Example 1 on the expression of the inflammatory factor IL-1α; wherein, ▲▲ indicates that compared with the SC 0.1% DMSO LPS-) group, the difference is extremely significant P<0.01; △△ indicates that compared with the BC LPS-) group, the difference is extremely significant P<0.01; ## indicates that compared with the SC 0.1% DMSO LPS+) group, the difference is extremely significant P<0.01; * indicates that compared with the NC LPS+) group, the difference is significant P<0.05. DETAILED DESCRIPTION

[0050] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0052] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0053] In the present invention, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values ​​of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0054] Only some numerical ranges are specifically disclosed herein. However, any lower limit can be combined with any upper limit to form an unspecified range; and any lower limit can be combined with other lower limits to form an unspecified range, and likewise any upper limit can be combined with any other upper limit to form an unspecified range. In addition, each separately disclosed point or single value can itself be combined as a lower limit or upper limit with any other point or single value or with other lower limits or upper limits to form an unspecified range.

[0055] The temperature parameters in the present invention, if not specifically limited, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument.

[0056] In the description of the invention, “plurality” means at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0057] If there is no special description, all embodiments and optional embodiments of the present invention can be combined with each other to form a new technical solution. If there is no special description, all technical features and optional technical features of the present invention can be combined with each other to form a new technical solution.

[0058] If not specifically stated, all steps of the present invention may be performed sequentially or randomly, but are preferably performed sequentially.

[0059] The invention provides a cerium oxide microsphere, the cerium oxide microsphere comprises Ce 3+ , Ce 3+ The molar proportion in the cerium oxide microspheres is 30%-38%.

[0060] Cerium oxide has a fluorite cubic structure, a space group of Fm-3m, and a unit cell parameter a of 0.5411nm. In the crystal structure of cerium oxide, Ce 4+ Occupies the octahedral void, O 2- Occupies tetrahedral voids. The low-index crystal faces of cerium oxide are {100}, {110}, and {111}. Due to the electron transition of O 2p-Ce 4f, cerium oxide has strong absorption in the ultraviolet region and is light yellow, and can be used as an ultraviolet absorber.

[0061] In cerium oxide, Ce 3+ and Ce 4+ It can be reversibly converted, with the creation and elimination of oxygen vacancies during this conversion process. Equation, this process can be described by the following defect reaction:

[0062]

[0063] Among them, Oo represents O 2- , Ce Ce Representative Ce 4+ , is an electrically neutral oxygen vacancy, while Ce' Ce Representative Ce 3+ When cerium oxide is reduced, the oxygen in the tetrahedral gap will escape, and the remaining two electrons will be taken by the two adjacent Ce 4+ Obtain, and then Ce 4+ Reduced to Ce 3+ This characteristic endows CeO2 with excellent oxygen storage and release capabilities, and this defect property is also the basis of its catalytic and anti-oxidation properties. 3+ When the mass proportion in cerium oxide microspheres is as high as 30%-38%, the catalytic performance and antioxidant performance of cerium oxide microspheres can be improved; when it is applied in the field of cosmetics, it has excellent antioxidant and anti-aging effects.

[0064] Ce 3+The mass proportion in the cerium oxide microspheres is 30%-38%; for example, it can be but 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 the range between any two of the above values.

[0065] In some embodiments, Ce 3+ The mass proportion in the cerium oxide microspheres is 36%-38%; for example, it can be 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 the range between any two of the above values.

[0066] In some embodiments, the average particle size of the cerium oxide microspheres is 0.2 μm-3 μm; for example, it can be but not limited to 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 1.1 μm, 1.2 μm, 1.3 μm, 1.4 μm, 1.5 μm, 1.6 μm, 1.7 μm, 1.8 μm, 1.9 μm, 2 μm, 2.1 μm, 2.2 μm, 2.3 μm, 2.4 μm, 2.5 μm, 2.6 μm, 2.7 μm, 2.8 μm, 2.9 μm, 3 μm or the range between any two of the above values. It has been experimentally verified that when the average particle size of the prepared cerium oxide microspheres is within this range, the sedimentation rate from the mother solution and its dispersibility in the aqueous solution can achieve the best balance.

[0067] In some embodiments, the average particle size of the cerium oxide microspheres is 0.4 μm-3 μm. For example, it can be, but is not limited to, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 1.1 μm, 1.2 μm, 1.3 μm, 1.4 μm, 1.5 μm, 1.6 μm, 1.7 μm, 1.8 μm, 1.9 μm, 2 μm, 2.1 μm, 2.2 μm, 2.3 μm, 2.4 μm, 2.5 μm, 2.6 μm, 2.7 μm, 2.8 μm, 2.9 μm, 3 μm, or a range between any two of the above values.

[0068] The second aspect of the present invention provides a method for preparing the cerium oxide microspheres of the first aspect, comprising the following steps:

[0069] A cerium-containing precursor is mixed with water to prepare a precursor solution; a pH regulator is added to the precursor solution to prepare a first solution; a dispersion medium is added to the first solution to prepare a second solution; the mass proportion of the cerium-containing precursor in the second solution is 3%-7%; and the second solution is heated to react.

[0070] The preparation method has a simple process and mild reaction conditions and can be used for the large-scale production of cerium oxide microspheres. Compared with the conventional cerium oxide preparation process, the concentration of the cerium precursor in the precursor solution can be increased by 1-3 times or more.

[0071] In some embodiments, the mass proportion of the cerium-containing precursor in the second solution is 5%-7%; when the mass proportion of the cerium-containing precursor in the second solution is within the above range, the formed cerium oxide is closer to the micron sphere morphology, has good dispersibility, and has a high yield of cerium oxide. As an example, the mass proportion of the cerium-containing precursor in the second solution can 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 values.

[0072] In some embodiments, the cerium-containing precursor includes one or more of cerium nitrate hexahydrate and cerium nitrate.

[0073] As an 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 fully dissolved. As an example, the mass volume ratio of the cerium-containing precursor and water can be, but is not limited to, 20 g: (10-60) mL, 30 g: (10-60) mL, 40 g: (10-60) mL, 50 g: (10-60) mL, 60 g: (10-60) mL, 70 g: (10-60) mL, 80 g: (10-60) mL, 90 g: (10-60) mL, 100 g: (10-60) mL, 110 g: (10-60) mL or 120 g: (10-60) mL, etc.

[0074] In some embodiments, the pH regulator 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) to regulate the crystallization rate of CeO2, which is a necessary condition for synthesizing cerium oxide microspheres.

[0075] In some embodiments, the mass volume ratio of the cerium-containing precursor and the pH adjuster is (20-120) g: (20-120) mL; when the mass volume ratio of the cerium-containing precursor and the pH adjuster is within the above range, cerium oxide microspheres with different particle sizes and different surface Ce (III) contents can be controlled to be synthesized. As an example, the mass volume ratio of the cerium-containing precursor and the pH adjuster 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, etc.

[0076] As a possible implementation, the dispersion medium includes ethylene glycol. Ethylene glycol has good stability and a boiling point of 197° C. As a dispersion medium for the cerium-containing precursor, it has a good dispersion effect and is easy to separate after the reaction is completed.

[0077] In some optional embodiments, the mass volume ratio of the cerium-containing precursor and the 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 product is easy to separate. As an example, the mass volume ratio of the cerium-containing precursor to the dispersion medium can be, but is not limited to, 20 g: (600-700) mL, 30 g: (600-700) mL, 40 g: (600-700) mL, 50 g: (600-700) mL, 60 g: (600-700) mL, 70 g: (600-700) mL, 80 g: (600-700) mL, 90 g: (600-700) mL, 100 g: (600-700) mL, 110 g: (600-700) mL or 120 g: (600-700) mL, etc.

[0078] In some embodiments, the pressure of the heating reaction is 0.5 MPa-4 MPa. As an example, the pressure of the heating reaction can be, but is not limited to, 0.5 MPa, 1 MPa, 1.5 MPa, 2 MPa, 2.5 MPa, 3 MPa, 3.5 MPa, 4 MPa, or a range between any two of the above values.

[0079] In some embodiments, the temperature of the heating reaction is 160° C.-170° C. As an example, the temperature of the heating reaction can be, but is not limited to, 160° C., 161° C., 162° C., 163° C., 164° C., 165° C., 166° C., 167° C., 168° C., 169° C., 170° C., or a range between any two of the above values.

[0080] In some embodiments, the heating reaction time is 140 min-180 min. As an example, the heating reaction time can be but is not limited to 140 min, 145 min, 150 min, 155 min, 160 min, 165 min, 170 min, 175 min, 180 min or a range between any two of the above values.

[0081] Compared with the conventional cerium oxide preparation process, the reaction time can be shortened by 20-60 minutes, and the reaction temperature can be reduced by 10-20° C. When the pressure, temperature and time of the heating reaction are controlled within the above ranges, cerium oxide microspheres with good antioxidant properties can be prepared.

[0082] In some embodiments, the heating reaction is carried out in a Hastelloy pressure-resistant reactor. Acetic acid is corrosive to a certain extent, especially under high temperature conditions. At the temperature required for the reaction (160°C-170°C), traditional 316 stainless steel will be corroded by acetic acid, causing the wall material to become thinner. The use of a Hastelloy pressure-resistant reactor can avoid this problem, and the safety of the experiment is ensured in a Hastelloy pressure-resistant reactor. Optionally, the heating reaction is carried out in a Hastelloy pressure-resistant reactor with a volume of 1L; this is conducive to the scaled-up production of cerium oxide microspheres.

[0083] In some embodiments, the method for preparing cerium oxide microspheres further comprises the following steps: filtering the heated reaction product, washing the filtered filtrate to neutrality, and drying to prepare cerium oxide microspheres.

[0084] As an embodiment, the filtration treatment is carried out using a ceramic filter membrane. In some embodiments, the heating reaction product is filtered in the form of "cross-flow filtration"; that is, the raw liquid flows at a high speed in the membrane tube, and the permeate containing small molecules passes through the membrane in the vertical direction of the membrane layer under pressure, and the concentrated liquid containing large molecular components is retained by the membrane, thereby achieving the purpose of separation, purification, and concentration of the product cerium oxide slurry. The ceramic filter membrane system has a high degree of process integration and realizes full automatic control. It can continuously feed, continuously discharge filter residue and filtrate, realize continuous separation of products, and the obtained filtrate is clear and transparent, which will bring higher economic benefits.

[0085] Optionally, the average pore size of the ceramic filter membrane is 100nm-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.

[0086] The third aspect of the present invention provides a use of the cerium oxide microspheres according to the first aspect of the present invention or the cerium oxide microspheres prepared by the preparation method of the second aspect of the present invention in the preparation of cosmetics.

[0087] The fourth aspect of the present invention provides a cosmetic comprising the cerium oxide microspheres according to the first aspect of the present invention or the cerium oxide microspheres prepared by the preparation method of the second aspect of the present invention.

[0088] In some embodiments, the cosmetic comprises a cosmetic having an anti-aging effect.

[0089] The technical solution of the present invention is described in detail below in conjunction with specific embodiments.

[0090] 1. Preparation of cerium oxide microspheres

[0091] Example 1

[0092] Weigh 20g of cerium nitrate hexahydrate (as a cerium-containing precursor), mix it with 10mL of deionized water to prepare a precursor solution; add 20mL of acetic acid (as a pH adjuster) to the precursor solution to prepare a first solution; add 600mL of ethylene glycol (as a dispersion medium) to the first solution and mix evenly to prepare a second solution; wherein the mass proportion of cerium nitrate hexahydrate in the second solution is 3%; transfer the second solution to a 1L Hastelloy pressure-resistant reactor for heating reaction, the reaction temperature is 165°C, the reaction time is 160min, and the reaction pressure is about 0.5Mpa; use a ceramic filter membrane with an average pore size of 500nm to filter the product of the heating reaction, wash the filtered filtrate with water to neutrality, and dry it at room temperature for 24h; sterilize and package it to obtain cerium oxide microspheres.

[0093] Embodiment 2-9

[0094] The preparation method of cerium oxide microspheres in Examples 2-9 is basically similar to the preparation method of cerium oxide microspheres in Example 1, with the main difference being that at least one of the amount of cerium-containing precursor, the amount of deionized water, the amount of pH adjuster, the amount of dispersion medium, the mass proportion of cerium nitrate hexahydrate in the second solution, the temperature of the heating reaction, and the time of the heating reaction is different, as shown in Table 1 for details.

[0095] Comparative Example 1-2

[0096] The difference between the preparation method of cerium oxide microspheres in Comparative Example 1-2 and the preparation method of cerium oxide microspheres in Example 1 is mainly that: at least one of the amount of cerium-containing precursor, the amount of deionized water, the amount of pH adjuster, the amount of dispersion medium, the mass proportion of cerium nitrate hexahydrate in the second solution, the temperature of the heating reaction and the heating reaction time is different, see Table 1 for details.

[0097] Comparative Example 3

[0098] The difference between the preparation method of cerium oxide microspheres in Comparative Example 3 and the preparation method of cerium oxide microspheres in Example 1 is that the product of the heating reaction is centrifuged at a speed of 10,000 rpm for 10 minutes. See Table 1 for details.

[0099] It should be noted that, when the product of the heating reaction is centrifuged in Comparative Example 3, the supernatant is still turbid after centrifugation, and efficient separation cannot be achieved. In addition, the separation time is long, and the time and labor costs are high.

[0100] In Example 1-9 and Comparative Example 1-2, after filtering the product of the heating reaction using a ceramic filter membrane, a clear and transparent filtrate can be obtained.

[0101] The appearance of the cerium oxide microspheres prepared in the above embodiments is as follows: Figure 1 shown. Figure 1 Wherein ai respectively represent the appearance of the cerium oxide microspheres prepared in Examples 1-9. Figure 1 It can be seen that as the mass proportion of cerium nitrate hexahydrate in the second solution increases, the average particle size of the cerium oxide microspheres increases.

[0102] The cerium oxide microspheres prepared in the above-mentioned embodiments and comparative examples were observed by electron scanning electron microscopy, and the average particle size of the cerium oxide microspheres was measured. The results are shown in Table 1.

[0103] The electron scanning electron microscopy images of the cerium oxide microspheres prepared in Example 1, Example 4 and Example 9 are as follows: Figure 2 As shown. Among them, Figure 2 ac represent TEM images of cerium oxide microspheres prepared in Example 1; Figure 2 df in the figure represents the TEM image of the cerium oxide microspheres prepared in Example 4; Figure 2 gi represents the TEM image of the cerium oxide microspheres prepared in Example 9. Figure 2 It can be seen from the results that when the mass proportion of the cerium-containing precursor in the second solution is 7%, large particle aggregates with an average particle size of 3000 nm are easily formed.

[0104] XPS energy spectrum analysis was performed on the cerium oxide microspheres prepared in the above embodiments and comparative examples to determine the mass percentage of Ce(III) in the cerium oxide microspheres. The results are shown in Table 1.

[0105] The XPS spectra of the cerium oxide microspheres prepared in Example 1, Example 4 and Example 9 are as follows: Figure 3 As shown. Among them, Figure 3 a represents the XPS spectrum of the cerium oxide microspheres prepared in Example 1; Figure 3 b represents the XPS spectrum of the cerium oxide microspheres prepared in Example 4; Figure 3 c represents the XPS spectrum of the cerium oxide microspheres prepared in Example 9; Figure 3 Wherein d represents the mass proportion of Ce(III) in the cerium oxide microspheres prepared in Example 1, Example 4 and Example 9.

[0106] Depend on Figure 3 It can be seen that the mass proportion of Ce(III) in the cerium oxide microspheres prepared in Example 1 is 30.9%; the mass proportion of Ce(III) in the cerium oxide microspheres prepared in Example 4 is 36.4%; the mass proportion of Ce(III) in the cerium oxide microspheres prepared in Example 9 is 37.2%; compared with Example 1, the mass proportions of Ce(III) in the cerium oxide microspheres in Examples 4 and 9 increased by 17.8% and 20.3%, respectively.

[0107] Table 1

[0108]

[0109] Wherein, w1 represents the mass proportion of the cerium-containing precursor in the second solution; Ce(III) content represents the molar proportion of Ce(III) in the cerium oxide microspheres.

[0110] From the results in Table 1, it can be seen that when the mass proportion of the cerium-containing precursor in the second solution is 3-7%, the average particle size of the prepared cerium oxide microspheres can reach 200nm-3000nm; and the molar proportion of Ce(III) in the cerium oxide microspheres can reach 30%-38%.

[0111] Comparative Example 4. Preparation of Cerium Oxide Cubes

[0112] 10g CH3COONa, 10ml CH3COOH and 2.17g Ce(NO3)3-6H2O were dissolved and diluted to 85mL with water. The solution was then transferred to a 100ml Teflon-lined stainless steel autoclave and heated to 220°C for 24 hours. After cooling, the product was collected, centrifuged (12000r.pm), washed five times with distilled water, and dried to obtain cerium oxide cubes. The size of a single particle was about 25nm.

[0113] Comparative Example 5. Preparation of cerium oxide polyhedrons

[0114] Weigh 1.30g (3mmol) Ce (NO3) 3·6H2O in a beaker, add appropriate amount of water to dissolve, place on a magnetic stirrer and stir for 10min, then add 0.24g (6mmol) NaOH and continue stirring for 30min. Adjust the total volume of the solution to 60mL, then transfer the solution completely to a 100mL polytetrafluoroethylene reactor liner and place it in the metal shell of the reactor. Finally, place the reactor in a 120℃ oven for 24h. The reaction product was repeatedly centrifuged and washed 3 times with deionized water, and dried to obtain cerium oxide polyhedrons. The morphology of the prepared product is quasi-spherical, and the size is between 5nm-10nm.

[0115] Comparative Example 6. Preparation of cerium oxide nanospheres

[0116] Disperse 95% ethylene glycol (7.8 mL, 0.12 mol) in 92 mL deionized water. Then add 5.16 g of Ce(NO3)3·6H2O, stir thoroughly, add NH3.H2O to adjust the pH to 9.6, and then react at 50°C until the solution turns yellow. Centrifuge, wash with water, and dry to obtain cerium oxide nanospheres.

[0117] Comparative Example 7. Preparation of cerium oxide nanorods

[0118] 1.5 g of CeCl3·7H2O (4 mmol) and 4 g of NaOH (100 mol) were dispersed in 60-80 mL of deionized water and stirred until uniform. Then, the mixture was transferred to a reactor and reacted at 130°C for 18 h. After centrifugation, the mixture was washed with water and dried. Finally, the dried powder product was calcined in a muffle furnace (300°C, 4 h) to obtain CeO2 nanorod powder with abundant oxygen defects.

[0119] Comparative Example 8. Preparation of Calcium-doped Cerium Oxide

[0120] 0.059g CaCl2·7H2O, 1.34g CeCl3·7H2O (3.6mmol), 4g NaOH (100mol) and 0.12g CaCl2·7H2O, 1.20g CeCl3·7H2O (3.2mmol), 4g NaOH (100mol) were dispersed in 60-80mL deionized water and stirred until uniform. Then, they were transferred to a reactor and reacted at 130°C for 18h. After centrifugation, washing and drying, the dried powder product was calcined in a muffle furnace (300°C, 4h) to prepare 10% Ca-doped CeO2 nanorods and 20% Ca-doped CeO2 nanorods powders, respectively.

[0121] 2. Cerium Oxide Microspheres Performance Test

[0122] 1.DPPH free radical scavenging effect

[0123] It should be noted that DPPH (1,1-disphenyl-2-picryl-hydrazyl), chemically named 1,1-diphenyl-2-trinitrophenylhydrazine, has a molecular formula of (C6H5)2N-NC6H2(NO2)3. In this compound, there is an unpaired valence electron on one atom of the nitrogen bridge, and the orbital motion of this electron is almost offset by the molecular structure. The capture of DPPH free radicals is usually used as an indicator for assessing antioxidant capacity. DPPH is a stable organic free radical with a maximum absorption peak of 517nm in the visible light region. In ethanol solution, each DPPH molecule can generate a stable nitrogen-containing free radical in the solution with a typical purple color. When it reacts with a free radical scavenger that provides 1 electron (for example, cerium oxide dispersion), a colorless product is generated, which makes the typical purple color of the solution lighter. The degree of fading is stoichiometrically related to the number of paired electrons. Therefore, spectrophotometry can be used for quantitative analysis to detect the free radical scavenging situation, so as to evaluate the free radical scavenging ability of the sample; the DPPH scavenging reaction process of cerium oxide dispersion is as follows:

[0124]

[0125] 1.1 Instruments and Experiments

[0126] (1) Analytical balance (d = 0.1 mg, purchased from Sartorius, model: BSA2202S-CW, Max 220 g);

[0127] (2) Vortex mixer (model MX-F);

[0128] (3) Multifunctional microplate reader (purchased from Tecan), 96-well plate (model: jet)

[0129] (4) Pipette (5 mL, 200 μL, 1000 μL, purchased from Eppendorf);

[0130] (5) DPPH (purchased from Sigma, analytical grade, C 18 H 12 N5O6, molecular weight 394.32);

[0131] (6) Vitamin C (purchased from Bio-Di Biotechnology Co., Ltd., analytical grade);

[0132] (7) Anhydrous ethanol (purchased from Sinopharm Chemical Reagent Co., Ltd., analytical grade).

[0133] 1.2 Experimental preparation

[0134] 1.2.1 Reagent preparation

[0135] (1) 0.2mmol / L DPPH solution

[0136] Weigh 0.0200 g of DPPH, add anhydrous ethanol to dissolve and dilute to volume in a 250 mL volumetric flask, and shake well.

[0137] (2) 200 μg / mL VC solution (Note: Prepare immediately before use)

[0138] Weigh 0.0500g VC, add deionized water to dissolve and dilute to volume in a 250mL volumetric flask, and shake well.

[0139] (3) 15 μg / mL VC solution

[0140] Take 0.600 mL of 200 μg / mL VC aqueous solution, add 7.400 mL of anhydrous ethanol, and mix well.

[0141] (4) 0.5 wt% cerium oxide dispersion

[0142] Take 50 mg of the cerium oxide sample to be tested, disperse it in 10 mL of deionized water, ultrasonically disperse it for 10 minutes, and shake it well to obtain a 0.5 wt% cerium oxide dispersion.

[0143] 1.2.2 Test operation

[0144] A blank group C, a negative control group B, a positive control group AVc and a sample group A were set up respectively; wherein, the sample group A refers to the cerium oxide products of different morphologies prepared in Examples 1-9 and Comparative Examples 4-8, and the cerium oxide products of different morphologies were dispersed in water respectively to prepare a dispersion with a concentration of 0.5wt%.

[0145] 1.2.3 Result processing

[0146] The calculation formula of DPPH free radical scavenging rate is: scavenging rate = [(Y+Z)-X] / B*100%

[0147] Wherein: X represents the absorbance value after the sample solution is mixed with the DPPH solution; Y represents the absorbance value after the anhydrous ethanol is mixed with the DPPH solution; Z represents the absorbance value after the anhydrous ethanol is mixed with the sample solution.

[0148] 1.2.3 Experimental operation process

[0149] The specific sample addition order and dosage are shown in Table 2-1 and Table 2-2 respectively.

[0150] Table 2-1. Reagent addition in sample tubes

[0151]

[0152] Table 2-2. Reagent addition in sample tubes

[0153]

[0154] It should be noted that A1-A10 means that 10 samples of each cerium oxide product were set to be measured 10 times in parallel.

[0155] 1.2.4 Result Analysis

[0156] The DPPH removal results of cerium oxide microspheres in Examples 1-9 are shown in Table 3.

[0157] Table 3

[0158] Group Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Example 8 Example 9 Clearance 94.8％ 95.6％ 94.7％ 94.4％ 92.1％ 92.8％ 94.3％ 94.5％ 95.6％

[0159] The DPPH free radical scavenging effects of Example 1, Comparative Examples 4-8 and VC control are as follows: Figure 4 shown. Figure 4 a in the middle represents DPPH stock solution, Figure 4 b shows the effect of treating DPPH stock solution with cerium oxide microspheres in Example 1. Figure 4 The middle c shows the effect of using the positive control group AVc to treat the DPPH stock solution.

[0160] Depend on Figure 4 The results show that the cerium oxide microspheres prepared in Example 1 have the best antioxidant capacity. Compared with the positive control group AVc, the average result of the DPPH scavenging rate of VC in three experiments was 62.4%, and the average result of the DPPH scavenging rate of the cerium oxide microspheres in Example 1 in three experiments was as high as 94.8%, which was 29% higher than the positive control group AVc. In addition, it can be clearly seen from the solution after the DPPH reaction that the cerium oxide microspheres exhibit excellent free radical scavenging ability. After 30 minutes of reaction, the color of the mixed solution of VC and DPPH is darker than the color of the mixed solution of cerium oxide microspheres and DPPH.

[0161] The cerium oxide microspheres prepared in Example 1, Example 4 and Example 9 were subjected to DPPH free radical scavenging test according to the above process. The results are as follows: Figure 5 As shown. Figure 5 It can be seen that the effective concentration of cerium oxide microspheres in Example 9 for scavenging DPPH is as low as 0.05wt%, and the DPPH scavenging rate is >90%; its effective concentration is lower than that of cerium oxide microspheres in Examples 1 and 4, and the DPPH scavenging rate is higher than that of cerium oxide microspheres in Examples 1 and 4. This shows that the higher the proportion of Ce(III) in cerium oxide microspheres, the better the DPPH scavenging effect of cerium oxide microspheres.

[0162] The cerium oxide microspheres prepared in Example 1 were subjected to a DHHP cycle removal experiment to more intuitively illustrate their sustained antioxidant capacity. The specific experimental process is as follows:

[0163] Initial experiment: Take 500 μL of 0.5wt% cerium oxide microsphere dispersion and add it to the sample tube, add 500 μL of 0.2mmol / L DPPH solution (consistent with the concentration used in the DPPH scavenging experiment) and mix well. After reacting for 20 minutes, use a centrifuge to separate at 10,000 rpm for 10 minutes. Pipette out the supernatant and add it to a 96-well plate, and measure the absorbance at 517nm.

[0164] DPPH cycle removal experiment: Disperse the cerium oxide microsphere precipitate obtained by centrifugation in 500 μL deionized water (ignore the slight loss during centrifugation), add 500 μL 0.2 mmol / L DPPH solution and mix well. After reacting for 20 minutes, use a centrifuge to separate at 10,000 rpm for 10 minutes. Pipette out the supernatant and add it to a 96-well plate, and measure the absorbance at 517 nm. Repeat this process 5 times. The results are as follows: Figure 6-8 shown.

[0165] Figure 8 a in the middle represents DPPH stock solution, Figure 8 b in the figure indicates that the cerium oxide microspheres are cycled once. Figure 8 c in the figure indicates that the cerium oxide microspheres are cycled twice. Figure 8 The d in the figure indicates that the cerium oxide microspheres are cycled three times. Figure 8 e in the figure indicates that the cerium oxide microspheres are cycled four times. Figure 8 f means that the cerium oxide microspheres are cycled five times. Figure 8 h in the middle indicates VC control.

[0166] Depend on Figure 6-8 It can be seen that after the cerium oxide microspheres in Example 1 are recycled for five times, the efficiency of removing DPPH can reach 76.6%, which is still significantly higher than the DPPH removal rate of VC of 62.4%.

[0167] It should be noted that the cerium oxide microspheres used in the following experiments are all the cerium oxide microspheres prepared in Example 1.

[0168] 2. Antioxidant in vitro cell model - ROS scavenging experiment

[0169] Oxidative stress refers to the process in which the body's highly active molecules such as reactive oxygen species (ROS) are produced excessively when the body is subjected to various stimuli, exceeding the clearance capacity of the body's antioxidant system, causing disorders in the body's oxidative and antioxidant systems, leading to the accumulation of free radicals in the body and causing oxidative damage. It is considered to be one of the important factors leading to skin aging. Studies have shown that long-wave ultraviolet rays (UVA 320-400nm) in sunlight are one of the most important environmental factors causing skin aging. UVA can directly damage fibroblasts in the dermis and produce reactive oxygen species (ROS). This method is based on the human skin fibroblast (HFF-1) model, and evaluates the antioxidant effect of the sample by measuring the effect of the sample on the production of cellular ROS after light damage.

[0170] 2.1 Test materials and equipment

[0171] 2.1.1 Test materials

[0172] Human skin fibroblasts (HFF-1) were purchased from the Stem Cell Bank of the Chinese Academy of Sciences. Fetal bovine serum (FBS, BI), DMEM high glucose medium (DMEM, purchased from Gibco), dimethyl sulfoxide (DMSO, purchased from Sigma), phosphate buffered saline (PBS, purchased from Gibco), vitamin E (VE), vitamin C (VC, purchased from Sigma), thiazolyl blue (MTT, Lab), and fluorescent probe DCFH-DA (purchased from Sigma).

[0173] 2.1.2 Test equipment

[0174] Clean bench (purchased from Yatai Cologne), inverted microscope (purchased from Leica), carbon dioxide incubator (purchased from Thermo), electric suction assister (purchased from Eppendorf), microplate reader (purchased from Tecan), ultraviolet irradiation box (purchased from Zhongkeboda), flow cytometer (purchased from Beckman).

[0175] 2.2 Test methods

[0176] 2.2.1 Toxicity assay based on HFF-1 cells

[0177] This experiment uses the MTT method to detect cell activity and screen the maximum safe concentration of cell samples. The experiment sets up negative control (culture medium), positive control (culture medium containing 10% DMSO) and zero adjustment well (PBS). The sample concentration setting is shown in Table 1, and 3 replicate wells are set for each concentration. The specific operation steps are as follows:

[0178] (1) Inoculation: Digest the cells in the logarithmic growth phase and inoculate them into a 96-well plate. Place the plate in a 37° C., 5% CO 2 incubator and incubate for 18-24 h.

[0179] (2) Solution preparation: Prepare the test substances at different concentrations according to the concentration settings in Table 4.

[0180] Table 4. Sample cytotoxic concentration settings

[0181]

[0182] (3) Sampling: After the cells have grown for 18-24 hours, discard the supernatant, add culture medium containing different concentrations of the test substance, and place the culture plate in a 37°C, 5% CO2 incubator for 18-24 hours.

[0183] (4) Detection: After culturing the cells for 18-24 hours, discard the supernatant, add the prepared and filtered MTT (0.5 mg / mL), mix gently, and incubate at 37°C in the dark for 4 hours. After the incubation, discard the supernatant, add 150 μL of DMSO to each well, shake for 20 minutes, and read the OD570 nm value using a microplate reader.

[0184] (5) Relative cell viability calculation formula:

[0185] Relative cell viability = (OD of test sample well - OD of zero-adjusted well) / (OD of negative control well - OD of zero-adjusted well) × 100%.

[0186] 2.2.2 Detection of ROS expression in HFF-1 cells irradiated by UVA

[0187] (1) Inoculation: The cells were inoculated into 6-well plates and incubated in a 37°C, 5% CO2 incubator for 18-24 h.

[0188] (2) Solution preparation: Prepare the test substance and positive control according to Table 5.

[0189] (3) Sample administration: According to the experimental grouping and concentration settings in Table 5, after the cells were plated and grown in the 6-well plate for 18-24 hours, the samples were grouped and administered, with 3 replicate wells in each group. In Group 1, the blank control group and the negative control group were both added with cell culture medium containing 0.1% DMSO, and the positive control group was added with cell culture medium containing 0.001% vitamin E; in Group 2, the blank control and the negative control group were both added with cell culture medium, the positive control group was added with cell culture medium containing 0.01% vitamin C, and the sample group was added with cell culture medium containing samples of corresponding concentrations, and culture was continued in a 37°C, 5% CO2 incubator for 18-24 hours.

[0190] (4) Irradiation: After 18-24 h of culture, remove the culture medium, wash twice with 1 mL of pre-warmed PBS, remove the washing solution, add 1 mL of PBS, and set the group to be irradiated at 30 J / cm 2After reaching the irradiation dose, remove the PBS in the well, wash once with 1 mL of pre-warmed PBS, and add DMEM medium to continue culturing for 2 hours.

[0191] (5) ROS expression detection: After loading the cells with the fluorescent probe DCFH-DA, the cells were collected and tested by flow cytometry to read the fluorescence intensity value MFI.

[0192] (6) Data processing: All data obtained in the experiment were processed and plotted using Excel software. SPSS17.0 was used for statistical analysis, and one-way analysis of variance (ANOVA) was used for inter-group comparison. When P < 0.05, the difference was considered significant.

[0193] Table 5. Experimental groups and concentration settings

[0194]

[0195] 2.2.3 Evaluation of ROS expression detection results based on UVA irradiation in HFF-1 cells

[0196] The ROS expression in the negative control group increased significantly compared with the blank control group, while the ROS expression in the positive control group decreased significantly compared with the negative control group, indicating that the test system was established. The ROS expression in the sample group decreased significantly compared with the negative control group, indicating that the sample has an antioxidant effect.

[0197] 2.3 Experimental Results

[0198] The results are shown in Table 6 and Fig. 9 shown.

[0199] Table 6. ROS expression detection results

[0200]

[0201]

[0202] Among them, ▲▲ indicates that the difference is extremely significant compared with the SC (UVA-, 0.1% DMSO) group, P < 0.01;

[0203] △△ indicates that the difference is extremely significant compared with the BC (UVA-) group, P < 0.01;

[0204] ## indicates that the difference was extremely significant compared with the SC (UVA+, 0.1% DMSO) group, P < 0.01;

[0205] *Indicates significant difference compared with the NC (UVA+) group, P < 0.05;

[0206] **Indicates that the difference is extremely significant compared with the NC (UVA+) group, P<0.01.

[0207] From Table 6 and Fig. 9 As shown, the ROS scavenging effect of the cerium oxide microspheres in Example 1 is comparable to that of the VE control and VC control, indicating that the cerium oxide microspheres have excellent antioxidant effects. Compared with the UVA non-irradiated (UVA-) group, the UVA irradiated (UVA+) group caused a significant increase in ROS expression; and compared with the SC (UVA+, 0.1% DMSO) group, the PC (VE) group can significantly reduce ROS expression, indicating that this test system is established. According to the results of the cytotoxicity test and cell morphology, the concentrations of cerium oxide microsphere dispersions on HFF-1 cells were finally selected as 0.0313%, 0.0625%, 0.1%, and 0.125%. The test results show that compared with the NC (UVA+) group, the cerium oxide microsphere dispersion can significantly reduce the expression of ROS at a concentration of 0.0313%, 0.0625%, 0.1%, and 0.125%. Based on the UVA irradiation human skin fibroblast damage model, the cerium oxide microsphere dispersion at concentrations of 0.0313%, 0.0625%, 0.1%, and 0.125% can all exert an antioxidant effect by reducing ROS expression.

[0208] 3. Inhibition of matrix metalloproteinase-1 activity (MMP-1)

[0209] Skin aging is divided into endogenous aging and exogenous aging. The former is also called natural aging, which is a natural programmed aging process. The latter is skin aging caused by external environmental factors such as ultraviolet rays, smoking, wind, and exposure to chemicals. Human skin fibroblasts are the main cells in the dermis of the skin, rich in collagen, and their unique biological characteristics 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 dermal cells. Dermal fibroblasts synthesize type I procollagen intracellularly and secrete it to the extracellular space. Under the action of terminal procollagen peptidase, the terminal peptides are separated and polymerized to form collagen fibers. Once collagen is lacking in the skin, the collagen fibers will cross-link and solidify, causing skin aging. ECM is an intricate network composed of macromolecules. The main components include collagen, elastin, non-collagen (matrix) glycoproteins and proteoglycans. Matrix metalloproteinase-1 (MMP-1) can degrade ECM, causing the loss of skin collagen, so the overexpression of MMP-1 is one of the important causes of skin aging.

[0210] This test uses human skin fibroblasts (HFF-1) as the research object. After adding the product, the product is tested to see how it affects the MMP-1 content in the cell supernatant, thereby evaluating the firming and anti-wrinkle effects of the product. MMPs degrade collagen and elastin in the dermis, and are the main enzymes that cause aging symptoms such as wrinkles and fine lines on the skin. Collagen and elastin have always been regarded as an important indicator for evaluating aging, and MMP-1 is the main enzyme that degrades type I and type III collagen in the dermis. When MMP-1 is overexpressed, it specifically degrades extracellular matrix components, destroys the normal structure of collagen and elastin, and thus causes skin aging. By testing the ability of the test substance to inhibit the activity of matrix metalloproteinases, the anti-aging effect of the test substance can be evaluated.

[0211] 3.1 Test materials and equipment

[0212] 3.1.1 Test materials

[0213] Human skin fibroblasts (HFF-1) were purchased from the Stem Cell Bank of the Chinese Academy of Sciences. Fetal bovine serum (FBS, BI), DMEM high glucose medium (DMEM, purchased from Gibco), dimethyl sulfoxide (DMSO, purchased from Sigma), phosphate buffered saline (PBS, purchased from Gibco), TGFβ1 (PEPROTECH), thiazolyl blue (MTT, Lab), matrix metalloproteinase-1 (total) ELISA kit (MMP-1, purchased from Boster).

[0214] 3.1.2 Test equipment

[0215] Clean bench (purchased from Yatai Cologne), inverted microscope (purchased from Leica), carbon dioxide incubator (purchased from Thermo), electric suction assist device (purchased from Eppendorf), and microplate reader (purchased from Tecan).

[0216] 3.2 Test methods

[0217] 3.2.1 Toxicity assay based on HFF-1 cells

[0218] This experiment uses the MTT method to detect cell activity and screen the maximum safe concentration of cell samples. The experiment sets up negative control (culture medium), positive control (culture medium containing 10% DMSO) and zero adjustment well (PBS). The sample concentration setting is shown in Table 1, and 3 replicate wells are set for each concentration. The specific operation steps are as follows:

[0219] (1) Inoculation: Digest the cells in the logarithmic growth phase and inoculate them into a 96-well plate. Place the plate in a 37° C., 5% CO 2 incubator and incubate for 18-24 h.

[0220] (2) Solution preparation: Prepare test substances of different concentrations according to the concentration settings in Table 7.

[0221] Table 7. Sample cytotoxic concentration settings

[0222]

[0223] (3) Sampling: After the cells have grown for 18-24 hours, discard the supernatant, add culture medium containing different concentrations of the test substance, and place the culture plate in a 37°C, 5% CO2 incubator for 18-24 hours.

[0224] (4) Detection: After culturing the cells for 18-24 hours, discard the supernatant, add the prepared and filtered MTT (0.5 mg / mL), mix gently, and incubate at 37°C in the dark for 4 hours. After the incubation, discard the supernatant, add 150 μL of DMSO to each well, shake for 20 minutes, and read the OD570 nm value using a microplate reader.

[0225] (5) Relative cell viability calculation formula:

[0226] Relative cell viability = (OD of test sample well - OD of zero-adjusted well) / (OD of negative control well - OD of zero-adjusted well) × 100%.

[0227] 3.2.2 HaCaT cell-based scratch test

[0228] (1) Inoculation: First, draw two vertical lines on the back of a 6-well plate with a marker pen and a ruler. Inoculate the cells into the 6-well plate and incubate in a 37°C, 5% CO2 incubator overnight.

[0229] (2) Solution preparation: Prepare the test substance and positive control according to Table 8.

[0230] (3) Scratching: After the cells have been plated and grown for 18-24 h in the 6-well plate, use a 1 mL pipette tip with a ruler to scratch each well perpendicular to the horizontal line on the back. Make two scratches in each well, discard the supernatant, and wash three times with PBS to remove the scratched cells.

[0231] Table 8. Experimental groups and concentration settings

[0232]

[0233] (4) Drug administration and photography: Add the prepared samples to the corresponding positions of the 6-well plate for drug administration, take photos under a 5× microscope at 0 h (immediately), and continue to culture the culture plate at 37°C and 5% CO2 for 18-24 h.

[0234] (5) Photography and image analysis: After 24 hours of cell culture, take another photo under a 5× microscope. The photo position should be the same as at 0 hours, and then perform migration area analysis.

[0235] (6) Calculation of cell migration rate: Calculated according to the cell migration rate formula, the calculation formula is as follows: Cell migration rate (%) = (A0-A 24 ) / A0×100

[0236] A0 refers to the blank area when taking the photo at 0h, A 24 Refers to the blank area of ​​the photo taken 24 hours a day.

[0237] (7) Data processing: All data obtained in the experiment were processed and plotted using Excel software. SPSS17.0 was used for statistical analysis, and one-way analysis of variance (ANOVA) was used for inter-group comparison. The difference was considered significant when P < 0.05.

[0238] 3.2.3 Evaluation of scratch test results based on HaCaT cells

[0239] Compared with the negative control group, the cell migration rate of the positive control group increased significantly, indicating that the experimental system was established. Compared with the negative control group, the cell migration rate of the sample group increased significantly, indicating that the sample can promote cell migration and achieve the effect of barrier damage repair.

[0240] 3.3 Experimental Results

[0241] The results of MMP-1 expression detection based on natural HFF-1 cells are shown in Table 9. Fig.10 As shown, compared with the NC group, the expression of MMP-1 content in the PC (TGFβ1) group was significantly reduced, indicating that this experimental system is established.

[0242] Table 9. MMP-1 expression detection results

[0243]

[0244]

[0245] From Table 9 and Fig.10 The results show that, compared with the NC group, the cerium oxide microsphere dispersion in Example 1 can significantly reduce the expression of MMP-1 when the sample concentrations are 0.0313%, 0.0625%, 0.1%, and 0.125% on HFF-1 cells. Based on the results of the natural HFF-1 cell MMP-1 expression test, it can be seen that the cerium oxide microsphere dispersion has certain firming and anti-wrinkle effects. And when the cerium oxide microsphere dispersion is sampled on HFF-1 cells at concentrations of 0.0625% and 0.1%, the inhibitory effect on MMP is close to the PC (TGF-β1) reference level.

[0246] 4. Inhibitory effect of IL-1α inflammatory factor

[0247] There are three common types of skin inflammation: skin irritation, allergic reaction and sensitive reaction. Among them, skin irritation refers to the process in which the skin is exposed to external irritants, activating the inflammatory response mediated by the innate immune response, and ultimately leading to skin damage. Macrophages are an important line of defense in the immune system of living organisms. They can cause innate and acquired immune responses. When stimulated by external stimuli, macrophages will express cellular inflammatory factors, and excessive amounts will cause immune disorders in the body. Lipopolysaccharide (LPS) is one of the components of the cell wall of Gram-negative bacteria. When LPS acts on macrophages, it induces an inflammatory response in macrophages, resulting in the production of a large number of cellular inflammatory factors, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6).

[0248] Skin inflammation is also one of the causes of skin aging. Oxidative stress caused by excessive reactive oxygen can trigger inflammation. The occurrence of inflammation is often accompanied by oxidative stress, which can maintain the continuous occurrence of inflammation, forming a vicious cycle of oxidative stress-inflammation-oxidative stress-inflammation. ROS in inflammation activates the NF-κB signaling pathway, thereby promoting the expression of inflammatory factors (TNF-α, IL-6, IL-8, IL-1α), exacerbating the body's inflammatory response, and the presence of cerium oxide can reduce the production of this proinflammatory cytokine and play an anti-inflammatory role. In addition, cerium oxide can activate the nitric oxide synthase (iNOS) pathway by inhibiting c-Jun N-terminal kinase (JNKs), thereby preventing the formation of gaseous inflammatory mediator NO, and can also achieve resistance to inflammatory responses. This experiment is based on the LPS-induced mouse macrophage (RAW264.7) inflammation model to evaluate the product's ability to reduce the expression and secretion of cellular inflammatory factors, and then predict its ability to inhibit further inflammatory responses. The indicators tested in this experiment are introduced as follows:

[0249] Interleukin-6 (IL-6) is identified as a B cell growth factor. It can be induced by TNF-α and IL-1β. Its biological effects are similar to IL-1β. It can stimulate the production of other cytokines, induce specific and nonspecific immune responses in the body, and cause fever. It plays an important role in immune regulation and inflammatory response. It is considered to be one of the main endogenous mediators that cause fever during inflammatory response. Once the content of inflammatory factors in skin cells increases and the skin temperature is higher than normal, the skin inflammatory response will be aggravated.

[0250] Interleukin-1α (IL-1α), mainly produced by monocytes and macrophages, plays a key role in inflammation and immune response. Interleukin-1α and interleukin-1β bind to the same cell surface receptor and induce the same biological response.

[0251] 4.1 Test materials and equipment

[0252] 4.1.1 Test materials

[0253] Mouse macrophages (RAW264.7, purchased from the Cell Resource Center, Institute of Basic Medicine, Chinese Academy of Medical Sciences), fetal bovine serum (FBS BI), DMEM high glucose medium (DMEM, purchased from Gibco), dimethyl sulfoxide (DMSO, purchased from Sigma), phosphate buffered saline (PBS, purchased from Gibco), thiazolyl blue (MTT, Lab), interleukin 6 enzyme-linked immunosorbent assay kit (MouseIL 6ELISA kit Boster), mouse interleukin-1α enzyme-linked immunosorbent assay kit (MouseIL 1αELISAkit Boster).

[0254] 4.1.2 Test equipment

[0255] Ultraclean bench (purchased from Yatai Cologne), inverted microscope (purchased from Leica), carbon dioxide incubator (purchased from Thermo), electric suction assist device (purchased from Eppendorf), cell waste liquid extraction pump (purchased from Qilin Bell), and microplate reader (purchased from Tecan).

[0256] 4.2 Test methods

[0257] 4.2.1 Toxicity assay based on RAW264.7 cells

[0258] This experiment uses the MTT method to detect cell activity and screen the maximum safe concentration of cell samples. The experiment sets up negative control (culture medium), positive control (culture medium containing 5% DMSO) and zero adjustment well (PBS). The sample concentration setting is shown in Table 1, and 3 replicate wells are set for each concentration. The specific operation steps are as follows:

[0259] (1) Inoculation: Digest the cells in the logarithmic growth phase and inoculate them into a 96-well plate. Place the plate in a 37.5% CO2 incubator and incubate for 18-24 hours.

[0260] (2) Solution preparation: Prepare test substances of different concentrations according to the concentration settings in Table 10.

[0261] (3) Sampling: After the cells have grown for 18-24 hours, discard the supernatant, add culture medium containing different concentrations of the test substance, and place the culture plate in a 37°C, 5% CO2 incubator for 18-24 hours.

[0262] (4) Detection: After 18-24 hours of cell culture, discard the supernatant, add the prepared and filtered MTT (0.5 mg / mL), mix gently, and incubate at 37°C in the dark for 4 hours. After the incubation, discard the supernatant, add 150 μL of DMSO to each well, shake for 20 minutes, and read the OD 570nm absorbance value using a microplate reader.

[0263] (5) Relative cell viability calculation formula:

[0264] Relative cell viability = (OD of test sample well - OD of zero-adjusted well) / (OD of negative control well - OD of zero-adjusted well) × 100%.

[0265] Table 10. Sample cytotoxic concentration settings

[0266]

[0267] 4.2.2 Detection of inflammatory factors in RAW264.7 cells induced by LPS

[0268] (1) Inoculation: The cells were inoculated into a 24-well plate and incubated in a 37°C, 5% CO2 incubator for 18-24 h.

[0269] (2) Solution preparation: Prepare the test substance and positive control according to Table 11.

[0270] (3) Sampling: According to the experimental grouping and concentration setting in Table 11, after the cells were plated and grown in the 24-well plate for 18-24 hours, the samples were grouped and sampled, and 3 replicate wells were set for each treatment group. In Group 1, the blank control and negative control groups were added with cell culture medium containing 0.1% DMSO, and the positive control group was added with cell culture medium containing 0.001% dexamethasone; in Group 2, the blank control and negative control groups were added with cell culture medium, and the sample group was added with cell culture medium containing samples of corresponding concentrations, and cultured in a 37°C, 5% CO2 incubator for 18-24 hours.

[0271] Table 11. Experimental groups and concentration settings

[0272]

[0273] It should be noted that in Table 11, BC represents blank control; NC represents negative control; and SC represents negative control and blank control corresponding to positive substances.

[0274] (4) LPS induction: After 18-24 h of culture, the culture medium in the plate was aspirated and the cells were washed once with PBS. Cell culture medium was added to the blank control group, and cell culture medium containing LPS was added to the negative control group, sample group, and positive control group. The cells were cultured in a 37°C, 5% CO2 incubator for another 18-24 h, and the supernatant was collected.

[0275] (5) Detection of inflammatory factor content: Take the cell supernatant from each well and detect the cell inflammatory factor content according to the operating instructions of the ELISA kit.

[0276] (6) Data processing: All data obtained in the experiment were processed and plotted using Excel software. SPSS17.0 was used for statistical analysis, and one-way analysis of variance (ANOVA) was used for inter-group comparison. When P < 0.05, the difference was considered significant.

[0277] 4.2.3 Evaluation of the results of LPS-induced inflammatory factors detection in RAW264.7 cells

[0278] The expression of inflammatory factors in the negative control group was significantly higher than that in the blank control group, and the expression of inflammatory factors in the positive control group was significantly lower than that in the negative control group, indicating that the experimental system was established. The expression of inflammatory factors in the sample group was significantly lower than that in the negative control group, indicating that the sample has the ability to inhibit further inflammatory reactions.

[0279] 4.3 Experimental Results

[0280] The results of the LPS-induced RAW264.7 cell model test are as follows:

[0281] The results of the inflammatory factor IL-6 expression test are shown in Table 12 and Fig.11 As shown, compared with the LPS-uninduced (LPS-) group, the LPS-induced (LPS+) group caused a very significant increase in the expression of IL-6 (P<0.01), indicating that the LPS-induced model was successful. Compared with SC (0.1% DMSO, LPS+), the PC (dexamethasone) group dexamethasone at a sample concentration of 0.001% can significantly downregulate the expression of IL-6 (P<0.01), indicating that this experimental system is established. Compared with the NC (LPS+) group, the dispersion of cerium oxide microspheres in Example 1 had a significant inhibitory effect on the expression of IL-6 at a concentration of 0.0078%.

[0282] Table 12. Detection results of inflammatory factor IL-6 expression

[0283]

[0284] It should be noted that the method quantification limit (lowest concentration of the standard curve) is 15.6 pg / mL and the detection limit is 1 pg / mL.

[0285] The results of the inflammatory factor IL-1α expression test are shown in Table 13 and Fig.12 As shown in the figure, compared with the LPS-uninduced group, the LPS-induced (LPS+) group caused a very significant increase in the expression of IL-1α (P<0.01), indicating that the LPS-induced model was successful.

[0286] Table 13. Detection results of inflammatory factor IL-1α expression

[0287]

[0288] Among them, the method quantification limit (lowest concentration of the standard curve) was 4.69pg / mL, and the detection limit was 1pg / mL.

[0289] Depend on Figure 11-12 As shown in the results of Tables 12-13, compared with SC (0.1% DMSO, LPS+), the PC (dexamethasone) group dexamethasone at a sample concentration of 0.001% can significantly downregulate the expression of IL-1α (P<0.01), indicating that this test system is established. Compared with the NC (LPS+) group, the dispersion of cerium oxide microspheres in Example 1 has a significant inhibitory effect on the expression of IL-1α at a concentration of 0.0078%.

[0290] Compared with the LPS-uninduced (LPS-) group, the LPS-induced (LPS+) group caused a significant increase in the expression of IL-6 and IL-1α; and compared with the SC (0.1% DMSO, LPS+) group, the PC (dexamethasone) group can significantly downregulate the expression of IL-6 and IL-1α, indicating that this test system is established. According to the results of the cytotoxicity test and cell morphology, the concentrations of cerium oxide microspheres added to the cells in Example 1 were selected as 0.0078%, 0.0156%, and 0.0313%. The test results show that compared with the NC (LPS+) group, the dispersion of cerium oxide microspheres in Example 1 had a significant inhibitory effect on the expression of IL-6 and IL-1α at a concentration of 0.0078%.

[0291] From the above experimental results, we can see that Ce 3+ When the mass proportion in cerium oxide microspheres is as high as 30%-38%, the catalytic performance and antioxidant performance of cerium oxide microspheres can be improved; when it is applied in the field of cosmetics, it has excellent antioxidant and anti-aging effects.

[0292] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0293] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a cosmetic ingredient having antioxidant and anti-aging effects, characterized in that: The cosmetic ingredient is cerium oxide microspheres, which contain Ce 3+ , the Ce 3+ The molar proportion of cerium oxide in the cerium oxide microspheres is 30%-38%, and the preparation method comprises the following steps: Mixing a cerium-containing precursor with water to prepare a precursor solution; the cerium-containing precursor includes one or more of cerium nitrate hexahydrate and cerium nitrate; the mass volume ratio of the cerium-containing precursor to the water is (20-120) g: (10-60) mL; Adding a pH adjuster to the precursor solution to prepare a first solution; the pH adjuster includes acetic acid; the mass volume ratio of the cerium-containing precursor to the pH adjuster is (20-120) g: (20-120) mL; Adding a dispersion medium to the first solution to prepare a second solution; the mass proportion of the cerium-containing precursor in the second solution is 3%-7%; the dispersion medium includes ethylene glycol, and the mass volume ratio of the cerium-containing precursor to the dispersion medium is (20-120) g: (600-700) mL; The second solution is subjected to a heating reaction; the heating reaction is carried out at a pressure of 0.5Mpa-4Mpa, a temperature of 160°C-170°C, and a time of 140min-180min; the heating reaction is carried out in a Hastelloy pressure-resistant reactor with a volume of 1L; The heated reaction product is filtered, and the filtered suspension is washed to neutrality and dried to prepare the cerium oxide microspheres.

2. The method for preparing a cosmetic ingredient having antioxidant and anti-aging effects according to claim 1, characterized in that: The mass proportion of the cerium-containing precursor in the second solution is 5%-7%.

3. The method for preparing a cosmetic ingredient having antioxidant and anti-aging effects according to claim 1, characterized in that: The filtration treatment is performed using a ceramic filter membrane.

4. The method for preparing a cosmetic ingredient having antioxidant and anti-aging effects according to claim 3, characterized in that: The average pore size of the ceramic filter membrane is 100nm-500nm.

5. The method for preparing a cosmetic ingredient having antioxidant and anti-aging effects according to claim 1, characterized in that: The Ce 3+ The molar proportion in the cerium oxide microspheres is 36%-38%.

6. The method for preparing a cosmetic ingredient having antioxidant and anti-aging effects according to any one of claims 1 to 4, characterized in that: The average particle size of the cerium oxide microspheres is 0.2 μm-3 μm.

7. The method for preparing a cosmetic ingredient having antioxidant and anti-aging effects according to claim 6, characterized in that: The average particle size of the cerium oxide microspheres is 0.4 μm-3 μm.

8. Use of cerium oxide microspheres prepared by the method for preparing a cosmetic ingredient with antioxidant and anti-aging effects as claimed in any one of claims 1 to 7 in preparing cosmetics with free radical scavenging effects.

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