Application of humic acid selenium in preparation of sunscreen and sunscreen

Abstract

This invention relates to the field of nanomedicine formulation technology, specifically disclosing the application of selenium humate in the preparation of sunscreens and the sunscreens themselves. The selenium humate is prepared by reacting sodium humate with sodium selenite. The selenium humate provided by this invention exhibits broad-spectrum absorption characteristics against UVA / UVB, significantly improving the sun protection factor of products. It effectively scavenge free radicals, inhibits the increase in reactive oxygen species levels in cells caused by ultraviolet radiation and the overexpression of inflammatory factors IL-6 and IL-8, significantly alleviates symptoms of ultraviolet damage to the skin, and promotes collagen synthesis in the dermis, possessing excellent integrated protection and repair functions.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine formulation technology, specifically to the application of selenium humate in the preparation of sunscreens and the sunscreens themselves. Background Technology

[0002] The skin is a vital barrier protecting the body from external damage and plays a crucial role in maintaining normal physiological functions. However, ultraviolet (UV) radiation can alter the structure and function of skin cells, causing oxidative stress and leading to the inactivation of various active substances. This impairs skin metabolism, resulting in signs of aging such as pigmentation and wrinkles. With rising global temperatures, the incidence of various skin diseases caused by UV radiation is increasing annually. Furthermore, 30% of newly diagnosed malignant tumors worldwide are skin tumors. Therefore, developing products that protect against UV damage is a pressing issue that needs to be addressed.

[0003] Currently, the most commonly used commercial sunscreens include physical sunscreens and chemical sunscreens. Chemical sunscreens absorb ultraviolet rays and convert them into heat energy, which may cause skin irritation or allergies, leading to inflammatory reactions, and are especially unsuitable for sensitive skin. Summary of the Invention

[0004] This invention provides the application of selenium humate in the preparation of sunscreens and the resulting sunscreen. The selenium humate provided by this invention exhibits broad-spectrum absorption characteristics against UVA / UVB, significantly improving the product's sun protection factor. It effectively scavenge free radicals, inhibits the increase in cellular reactive oxygen species levels and the overexpression of inflammatory factors IL-6 and IL-8 caused by ultraviolet radiation, significantly alleviates symptoms of ultraviolet damage to the skin, and promotes collagen synthesis in the dermis, possessing excellent integrated protection and repair functions.

[0005] This invention provides the application of selenium humate in the preparation of sunscreen agents, wherein the selenium humate is prepared by the following steps: Sodium humate and sodium selenite were added to a dilute nitric acid solution and reacted at 70℃~90℃ for 6 h~10 h. The pH of the reaction solution was adjusted to 6~8, and the supernatant was collected by centrifugation, dialyzed, and freeze-dried to obtain the selenium humate.

[0006] The selenium humate provided by this invention has broad-spectrum absorption characteristics for UVA / UVB, which can significantly improve the sun protection factor of the product, effectively scavenge free radicals, inhibit the increase of reactive oxygen species in cells caused by ultraviolet rays and the overexpression of inflammatory factors IL-6 and IL-8, significantly reduce the symptoms of ultraviolet damage to the skin, and promote the synthesis of collagen in the dermis, thus possessing excellent integrated protection and repair functions.

[0007] Furthermore, the mass ratio of sodium humate to sodium selenite is 0.5~2.5:0.5~2.5.

[0008] Furthermore, the molecular weight permeable by the dialysis is 3300 Da to 3700 Da; the freeze-drying conditions are: freeze-drying at -58℃ to -62℃ for 46 h to 50 h.

[0009] The present invention also provides a selenium humate sunscreen, wherein the selenium humate sunscreen comprises: selenium humate, an oil phase, an aqueous phase, and a preservative; the mass ratio of the oil phase, the aqueous phase, the preservative, and the selenium humate is 14~19:40~46:0.05~0.15:0.05~0.15; The oil phase comprises stearic acid, white petrolatum, glyceryl monostearate, and liquid paraffin; the aqueous phase comprises Tween-80, glycerol, and distilled water; and the preservative is methylparaben.

[0010] Furthermore, the mass ratio of stearic acid, white petrolatum, glyceryl monostearate, and liquid paraffin in the oil phase is 4~5:4~5:1~2:5~7; and the mass ratio of Tween-80, glycerol, and distilled water in the aqueous phase is 0.5~1.5:1.5~2.5:38~42.

[0011] Furthermore, the selenium humate sunscreen is used to promote the synthesis of collagen in the skin after UV damage.

[0012] Furthermore, the selenium humate sunscreen is used to scavenge hydroxyl free radicals or inhibit the increase in intracellular reactive oxygen species levels caused by ultraviolet radiation.

[0013] Furthermore, the selenium humate sunscreen is used to inhibit the expression of inflammatory factors IL-6 and / or IL-8 induced by ultraviolet radiation.

[0014] Furthermore, the selenium humate sunscreen agent is prepared by the following steps: The oil phase components are mixed and heated to 70℃~90℃ and maintained for 10 min~20 min to allow them to fully melt; the aqueous phase components are mixed and heated to 70℃~90℃ to dissolve. Under stirring conditions, the oil phase at the same temperature is slowly added to the aqueous phase for emulsification; Add preservatives and selenium humate to the emulsified system and stir to mix thoroughly. Stop stirring when the temperature drops to 35℃~45℃ to obtain the selenium humate sunscreen.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: The selenium humate provided by this invention has strong absorption properties in the UVA and UVB bands, providing the skin with a basic physical / chemical ultraviolet filtering barrier. It can effectively quench harmful hydroxyl radicals (OH) generated after ultraviolet radiation to the skin, reduce oxidative stress, and has glutathione peroxidase (GPx) mimicry activity. It can catalyze endogenous antioxidants (such as glutathione) to remove peroxides on the skin surface, thereby enhancing the skin's own defense and repair capabilities from a biochemical level.

[0016] The selenium humate provided by this invention possesses good safety and formulation feasibility. Cytotoxicity tests show that selenium humate has no significant toxicity to skin-associated cells (L929, HaCaT) and exhibits good biocompatibility, providing a basis for its safety for topical use. The sunscreen agent is prepared using common oil-in-water or water-in-oil cream formulation techniques, with clearly defined process steps (as in Example 1). The excipients used (stearic acid, petrolatum, Tween-80, etc.) are all conventional pharmaceutical or cosmetic bases, facilitating industrial production and quality control.

[0017] The selenium humate sunscreen provided by this invention exhibits excellent biological protection and repair potential in skin models. In HaCaT (human epidermal keratinocyte) experiments, the active ingredient selenium humate significantly reduced UVB-induced intracellular reactive oxygen species (ROS) levels and effectively inhibited the overexpression of UVB-induced pro-inflammatory factors IL-6 and IL-8, demonstrating anti-inflammatory effects and alleviating photodamage. In mouse surface UV damage experiments, topical application of the selenium humate sunscreen significantly improved macroscopic symptoms. Visual scores showed that, compared to the control group exposed to UV light only, mice using the sunscreen exhibited significantly reduced erythema, edema, desquamation, and crusting, promoting skin repair in the animal model. Histological analysis (Masson staining and semi-quantitative collagen analysis) confirmed that mice using the sunscreen had significantly higher collagen content and denser collagen arrangement in their skin. This indicates that the sunscreen not only protects against damage but also promotes structural repair and remodeling of the skin after UV damage, possessing an integrated "protection-repair" function. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1The figures show the spectral and energy dispersive spectral analysis results of HA-Se. In the figure, a is the UV absorption spectrum of HA-Se; b is the FTIR spectrum of HA and HA-Se; c is the full-range XPS scan of HA-Se; d is the C 1s high-resolution XPS spectrum of HA-Se; e is the O 1s high-resolution XPS spectrum of HA-Se; and f is the Se 3d high-resolution XPS spectrum of HA-Se.

[0020] Figure 2 The figures show the SEM images of HA-Se and the results of selenium content determination. In the figure, a is the SEM image of HA; b is the SEM image of HA-Se; and c is the standard curve of selenium content.

[0021] Figure 3 The results are from the free radical scavenging activity assay.

[0022] Figure 4 The figures show cell survival after treatment with different concentrations of HA-Se. In the figure, a represents the survival of L929 cells; b represents the survival of HaCat cells.

[0023] Figure 5 The effects of HA-Se on the fluorescence intensity, ROS level, IL-6 concentration, and IL-8 concentration of HaCaT cells are shown in the figure. In the figure, a represents the fluorescence intensity of HaCaT cells after 24 h of culture following HA-Se treatment and UVB irradiation; b represents the ROS level in HaCaT cells after HA-Se treatment and UVB irradiation; c represents the IL-6 concentration in the HaCaT cell supernatant; and d represents the IL-8 concentration in the HaCaT cell supernatant.

[0024] Figure 6 The SPF values ​​for each group of sunscreen agents are shown.

[0025] Figure 7 The skin condition of mice in each group on the 14th day after surgery.

[0026] Figure 8 Visual scores were given for the skin appearance of mice in each group on postoperative day 14.

[0027] Figure 9 HE staining results of tissues from mice in each group on day 14 post-surgery.

[0028] Figure 10 Masson staining results of tissues from mice in each group on day 14 post-surgery.

[0029] Figure 11 The collagen volume fraction of each group of mice on the 14th day after surgery. Detailed Implementation

[0030] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0031] Example 1: Application of selenium humate in the preparation of sunscreen agents.

[0032] 3 g of humic acid powder (purchased from Beijing Bowei Shennong Technology Co., Ltd.) was dissolved in 100 mL of water. The pH was adjusted to 8.0 with 1 mol / L NaOH. After centrifugation at 5000 r / min for 5 min, the supernatant was collected and freeze-dried at -60℃ for 48 h to obtain sodium humate (HA-Na). 0.5 g of sodium humate powder was added to 100 mL of 0.5% HNO3 and stirred for 30 min. Then, 0.5 g of Na2SeO3 was added, and the reaction was carried out at 80℃ for 8 h. After the reaction, the temperature was lowered to 25℃, and the pH was adjusted to 7.0 with 1 mol / L Na2CO3. After centrifugation at 3500 r / min for 5 min, the supernatant and precipitate were collected separately. The supernatant was dialyzed using a 3500 Da dialysis bag for 24 h to obtain dialysate one. The precipitate was dissolved in 40 mL of water, centrifuged at 3500 r / min for 5 min, and the supernatant was collected. This supernatant was then dialyzed using a 3500 Da dialysis bag for 24 h to obtain dialysate two. The dialysate 1 and dialysate 2 were combined and freeze-dried at -60℃ for 48 h to obtain selenium humate (HA-Se).

[0033] 4.5 g stearic acid, 4.5 g white petrolatum, 1.5 g glyceryl monostearate, and 6 g liquid paraffin were used as the oil phase; 1 g Tween-80, 2 g glycerol, and 40 g distilled water were used as the aqueous phase. The oil phase was heated in a beaker, and the aqueous phase was heated and dissolved in another beaker. The mixture was stirred during heating. Once the temperature reached 80°C, heating was continued for 15 minutes to ensure complete melting. The oil phase at the same temperature was slowly added to the aqueous phase, allowing it to emulsify for 8 minutes. Then, 0.1 g methylparaben was added as a preservative, followed by 0.1 g selenium humate, stirring continuously until fully mixed. Finally, the mixture was stirred and cooled at 25°C. Stirring was stopped when the temperature dropped to 40°C, and the mixture was bottled to obtain the selenium humate sunscreen (HA-Se sunscreen).

[0034] Example 2: Application of selenium humate in the preparation of sunscreen agents.

[0035] Take 1 g of sodium humate powder, add 200 mL of 0.5% HNO3, stir for 30 min, then add 1 g of Na2SeO3, and react at 80℃ for 8 h. After the reaction is complete, cool to 25℃, adjust the pH to 7.0 with 1 mol / L Na2CO3, then centrifuge at 3500 r / min for 5 min and collect the supernatant. Dialyze using a 3500 Da dialysis bag for 24 h. Dissolve the solid after centrifugation in 80 mL of water, centrifuge at 3500 r / min for 5 min, collect the supernatant and dialyze, then freeze-dry at -60℃ for 48 h to obtain HA-Se.

[0036] The preparation process of selenium humate sunscreen is the same as in Example 1.

[0037] Example 3: Application of selenium humate in the preparation of sunscreen agents.

[0038] Take 2.5 g of sodium humate powder, add 500 mL of 0.5% HNO3, stir for 30 min, then add 2.5 g of Na2SeO3, and react at 80℃ for 8 h. After the reaction is complete, cool to 25℃, adjust the pH to 7.0 with 1 mol / L Na2CO3, then centrifuge at 3500 r / min for 5 min and collect the supernatant. Dialyze using a 3500 Da dialysis bag for 24 h. Dissolve the solid after centrifugation in 200 mL of water, centrifuge at 3500 r / min for 5 min and collect the supernatant for dialysis. Freeze-dry at -60℃ for 48 h to obtain HA-Se.

[0039] The preparation process of selenium humate sunscreen is the same as in Example 1.

[0040] Example 4: Application of selenium humate in the preparation of sunscreen agents.

[0041] Take 0.5 g of low molecular weight humic acid powder (purchased from Shanghai Maclean Biochemical Technology Co., Ltd.), add 100 mL of 0.5% HNO3, stir for 30 min, then add 0.5 g of Na2SeO3, and react at 80℃ for 8 h. After the reaction is complete, cool to 25℃, adjust the pH to 7.0 with 1 mol / L Na2CO3, then centrifuge at 3500 r / min for 5 min and collect the supernatant. Dialyze using a 3500 Da dialysis bag for 24 h. Dissolve the solid after centrifugation in 40 mL of water, centrifuge at 3500 r / min for 5 min and collect the supernatant for dialysis, then freeze-dry at -60℃ for 48 h to obtain HA-Se.

[0042] The preparation process of selenium humate sunscreen is the same as in Example 1.

[0043] Example 5: Application of a selenium humate sunscreen.

[0044] I. Experimental Methods Taking Example 1 as an example, the performance of the HA-Se and selenium humate sunscreen prepared in this invention was investigated, and the following experimental scheme was designed: 1. Microscopic morphological analysis of HA-Se The morphology of humic acid raw powder and HA-Se was characterized using a ZEISS GeminiSEM 300 scanning electron microscope. Before testing, the samples were attached to the sample stage with conductive adhesive and sputtered with gold for 60 seconds. The magnification ranged from 0.5 kX to 50 kX.

[0045] 2. Spectroscopic and energy-dispersive X-ray spectroscopy analysis The ultraviolet absorption spectra of humic acid and HA-Se were obtained using an ultraviolet spectrophotometer. Before the test, the samples were prepared as 1 mg / mL aqueous solutions and water was used as a control for the determination.

[0046] Fourier transform infrared spectroscopy was used to characterize the infrared spectra of humic acid and HA-Se. The infrared spectra of humic acid and HA-Se were determined using the potassium bromide (KBr) pellet method with potassium bromide as background. Humic acid and HA-Se were ground with KBr powder at a mass ratio of 1:50 in an agate mortar, placed into a pelleting mold, and pressed into pellets at 12 MPa for 5 min using a hydraulic press. The pressed sample pellets were then removed and inserted into the infrared sample optical path for spectral scanning.

[0047] The chemical elemental composition of HA-Se was investigated using X-ray photoelectron spectroscopy (XPS, Thermo Scientific K-Alpha, USA). 1 mg of HA-Se and 2 mg of high-purity graphite powder were placed in a mold and compressed into a pellet at 8 MPa for 1 min. After compression, the pellet was attached to a sample tray, and the sample was placed in the sample chamber of the XPS instrument at a pressure less than 2.0 × 10⁻⁻¹. 7 At mbar, the sample is sent into the analysis chamber with a spot size of 400 μm, an operating voltage of 12 kV, and a filament current of 6 mA. The full spectrum scan pass energy is 150 eV with a step size of 1 eV, and the narrow spectrum scan pass energy is 50 eV with a step size of 0.1 eV.

[0048] 3. Selenium content determination The selenium content of HA-Se was determined using the o-phenylenediamine method. 20 mg of HA-Se was first digested with 2 mL of concentrated nitric acid for 30 min, followed by digestion at 100 °C for 30 min. Then, 5 mL of concentrated hydrochloric acid was added, and digestion continued until the total solution volume was 1 mL. After digestion, the solution was transferred to a volumetric flask, and 2 mL of 5% EDTA-2Na and 2 mL of 2% o-phenylenediamine were added. The pH was adjusted to 2 with 0.1 mol / L HCl, and the reaction was carried out in the dark for 1 h. Then, 5 mL of toluene was added, and the mixture was extracted using a separatory funnel. The UV spectrum of the extracted toluene was measured. The selenium content was then calculated by substituting the results into the selenium content standard curve.

[0049] 4. Free radical scavenging activity assay Take 2 mL of HA-Se sample solutions (prepared with pure water) at concentrations of 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, and 2.0 mg / mL, respectively, into five colorimetric tubes. First, add 2 mL of 6 mmol / L FeSO4 solution, followed by 2 mL of 6 mmol / L H2O2 solution. Mix well and let stand for 10 min. Then, add 2 mL of freshly prepared 6 mmol / L salicylic acid ethanol solution. React at 37℃ for 30 min, and measure the absorbance at 510 nm (zeroing with pure water). Ascorbic acid and humic acid were used as positive controls, and the reaction was repeated in triplicate. Calculate the scavenging rate of OH free radicals using the following formula:

[0050] Clearance rate (%) = (A 空白 -A 样品 +A 对照 ) / A 空白 ×100%; In the formula: A 空白 Use 2.0 mL of pure water instead of the sample solution for absorbance; A 样品 A represents the absorbance of the sample group and the positive control group; 对照 The absorbance of 2.0 mL of 95% ethanol solution instead of 6 mmol / L salicylic acid ethanol solution.

[0051] 5. GPx activity assay Mix 50 µL of 2 mg / mL HA-Se sample solution and 450 µL of GSH solution (1 mM, dissolved in carbonate buffer, pH=8.0) in the dark for 60 min. A negative control group (500 µL GSH solution) and a positive control group (450 µL GSH solution and 50 µL 0.1 M H2O2) were established. After incubation at 37°C for 10 min, 450 µL of TRIS-HCl buffer (50 mM, pH=8) and 100 µL of 5,5′-dithiobisnitrobenzene acid (DTNB, 10 mM) were added to the mixture. After reacting for 30 min, the absorbance of the supernatant was measured at 410 nm. The enzyme activity was calculated using the following formula:

[0052] Enzyme activity (U / mg) = (C 阳 -C 样 V / mt; In the formula: C 阳 The remaining GSH concentration and C in the positive control group 样 V represents the remaining GSH concentration in the HA-Se sample group, V represents the enzymatic reaction volume, t represents the enzymatic incubation time, and m represents the mass of HA-Se in the reaction system.

[0053] 6. Biocompatibility of HA-Se with HaCaT cells In vitro cell biocompatibility was assessed using the CCK-8 colorimetric assay for L929 and HaCaT cells. Cells were seeded in 96-well cell culture plates at a density of 5 × 10⁶ cells per well. 3 Cells were cultured at 37°C and 5% CO2. After co-incubating the cells with HA-Se at concentrations of 0 μg / mL, 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL for 24 h, 10 μL of CCK-8 solution (2 mg / mL) was added to each well. After reacting for 1 h, the OD value was measured at 450 nm using a microplate reader.

[0054] 7. ROS Measurement HaCaT cells were cultured in DMEM medium and incubated at 37°C with 5% CO2 until the cell density reached 90%. HaCaT cells were then seeded into 6-well plates at a density of 5 × 10⁶ cells per well. 4 Cells were incubated in DMEM medium for 24 h, then the medium was discarded. HA-Se at a final concentration of 100 μg / mL was dispersed in DMEM medium and added to 6-well plates containing HaCaT cells. After incubation for 24 h, the cells were irradiated with UVB at a total dose of 50 mJ / cm². 2Irradiation was stopped, cells were collected, and 2 mL of 5 μmol / L DCFH-DA (serum-free dilution) was added. The cells were incubated in a 37°C cell culture incubator for 20 min in the dark. Cells were then washed three times with Procyon serum-free medium to thoroughly remove any untreated DCFH-DA. Cells were then resuspended in PBS to measure fluorescence intensity. Cells not treated with UVB were used as a blank control group, and the experiment was repeated three times. ROS levels were calculated using the following formula:

[0055] ROS level = FI 样 / FI 空 ; Where: FI 样 The fluorescence intensity measured for the HA-Se group, FI 空 The fluorescence intensity was measured for the blank control group.

[0056] 8. Enzyme-linked immunosorbent assay (ELISA) for identifying inflammatory factors To assess cytokine production, HaCaT cells were seeded in 6-well plates at 5 × 10⁶ cells per well. 4 Cells were collected and processed according to the ROS assay protocol. After 24 h of UVB irradiation, the supernatant was collected by centrifugation at 1000 g for 5 min, and the concentrations of IL-6 and IL-8 were measured according to the Solarbio IL-6 (SEKH-0013) and IL-8 (SEKH-0016) ELISA kit instructions.

[0057] 9. UV protection performance testing 3M pore-filled adhesive tape was affixed to the light-transmitting side of a clean quartz cuvette. Using a cuvette with the 3M pore-filled tape but no sunscreen applied as a control, the sample was zeroed. 8 mg of HA-Se sunscreen was accurately weighed and applied to the surface of the cuvette with the tape using a glass rod. The application was then spread evenly using a medical latex glove. The transmittance (T) of UVB per 1 nm in the wavelength range of 290 nm to 320 nm was measured using a Shimadzu UV-2600 microscope. Sunscreens containing humic acid (prepared using humic acid powder instead of HA-Se, with the remaining preparation process the same as in Example 1) and sunscreens without HA-Se (only lacking HA-Se, with the remaining preparation process the same as in Example 1) were used as controls. The sun protection factor (SPF) of the HA-Se sunscreen was calculated using the following formula:

[0058] ; Where Eλ is the solar spectral irradiance obtained with reference to QX / T 368-2016, Sλ is the CIE erythema spectral effectiveness obtained with reference to GB / T 21005-2007, and Tλ is the transmittance of HA-Se sunscreen.

[0059] 10. Determination of the ability of HA-Se sunscreen to resist ultraviolet damage on the surface of mice. To evaluate the ability of HA-Se sunscreen to protect against UV damage on the skin of mice, Kunming mice (purchased from Henan Skebers Biotechnology Co., Ltd.) were divided into four groups (n=6 per group): blank control group (NC), UV control group (UV), humic acid group (HA, sunscreen containing humic acid), HA-Se sunscreen group (HA-Se sunscreen), and avobenzone positive control group (PC). Fifteen minutes before daily UVB irradiation, the test substance was evenly applied to the bare skin (approximately 4 cm × 2 cm) on the back of the neck of the mice using a cotton swab. The blank control group and the UV control group were treated with sunscreen without HA-Se, at a concentration of 2 mg / cm². One day before irradiation, all hair on the back of the neck was shaved with a razor, followed by thorough removal of vellus hair with depilatory cream, ensuring a bare area of ​​4 cm × 2 cm. Mice were then placed in a UVB lamp box (two 40 W UVB 313 nm lamps) at a distance of 20 cm, with an irradiation intensity of 0.7 mW / cm², for 30 minutes daily for a total of 14 days, resulting in a total irradiation dose of 17.64 J / cm². Water and food were suspended during irradiation and resumed 0.5 h after irradiation. Skin erythema, edema, and desquamation were visually assessed during the experiment. After the experiment, a pea-sized piece of skin from the irradiated area on the back was harvested, subcutaneous fat and connective tissue were removed, and the skin was fixed in 4% paraformaldehyde.

[0060] After rinsing the fixed tissue twice with distilled water for 5 minutes each time, it was sequentially dehydrated by soaking in 75% ethanol, 85% ethanol, 95% ethanol I, 95% ethanol II, anhydrous ethanol I, and anhydrous ethanol II at 25°C for 2 hours each. Then, it was soaked in xylene I and xylene II at 25°C for 1 hour each until the tissue was translucent. Subsequently, it was placed in molten paraffin at 60°C and soaked in paraffin I and paraffin II in a 60°C oven for 2 hours each. The paraffin-soaked tissue blocks were placed in an embedding frame, fresh molten paraffin was added, and the observation side was adjusted downwards. The blocks were placed at 25°C for 30 minutes to allow the paraffin to solidify into a wax block. The wax block was fixed on a microtome and the thickness was adjusted to 4 μm for continuous sectioning. The cut wax strips were placed in a 45°C water bath to flatten naturally. The flattened wax sections were picked up with a glass slide and finally baked in a 60°C oven for 2 hours. After cooling to 25°C, the paraffin sections were obtained and could be used for subsequent staining.

[0061] HE staining: Paraffin sections were baked in a 60℃ oven for 2 h, then dewaxed by immersion in xylene I and xylene II for 10 min each, followed by gradient hydration by immersion in anhydrous ethanol I, anhydrous ethanol II, 95% ethanol, 85% ethanol, and 75% ethanol for 5 min each, and rinsed twice with distilled water for 1 min each time; immersed in hematoxylin stain at room temperature for 8 min, and rinsed lightly with distilled water for 1 min; immersed in 1% hydrochloric acid-alcohol for 3 s for differentiation, and rinsed twice with distilled water for 1 min each time; immersed in 0.5% ammonia for 1 min for blue reversion, and rinsed with distilled water for 2 min; immersed in 0.5% water-soluble eosin stain at 25℃ for 4 min, and rinsed lightly with distilled water for 20 s; then dehydrated by gradient hydration by immersion in 75% ethanol for 10 s, 85% ethanol for 30 s, 95% ethanol I and 95% ethanol II for 1 min each, and anhydrous ethanol I and anhydrous ethanol II for 2 min each; and finally immersed in xylene I and xylene II for 2 min each. Minify the transparent material, add a drop of neutral resin to cover the coverslip and seal it. After drying at 25°C, observe it under a microscope.

[0062] Masson staining: Paraffin sections were baked in a 60℃ oven for 2 h. Following the same steps as HE staining, dewaxing, gradient hydration, and rinsing with distilled water were completed. Hematoxylin staining solution was soaked at 25℃ for 4 min, followed by a gentle rinse with distilled water for 1 min. Differentiation was achieved by soaking in 1% hydrochloric acid-alcohol solution for 2 s, followed by rinsing twice with distilled water for 1 min each time. 0.5% ammonia solution was soaked for 30 s to achieve blue reversion, followed by rinsing with distilled water for 2 min and draining. Ponceau S-Acid Fuchsin staining solution was soaked at 25℃ for 8 min, followed by a quick gentle rinse with distilled water for 10 s. Differentiation was achieved by soaking in 1% phosphomolybdic acid solution for 4 min. After discarding the solution, the sections were immediately soaked in 1% aniline blue staining solution at 25℃ for 6 min, followed by rinsing twice with 0.5% glacial acetic acid solution for 30 s each time. A quick gentle rinse with distilled water for 10 s was then performed. The same gradient dehydration and xylene clearing steps as HE staining were then followed. Neutral resin was added to cover the slide and the slide was sealed. The slide was then air-dried at 25℃ and observed under a microscope.

[0063] Semi-quantitative analysis of collagen: Semi-quantitative analysis of collagen in Masson-stained sections was performed. Using ImageJ software, five non-overlapping fields of view were randomly selected from three biological replicates and stained without compression under a uniform magnification (200×) using an optical microscope. After importing the images into the software, the corresponding fluorescence channels were separated, and only collagen fiber areas were highlighted. The total area of ​​collagen fibers and the total area of ​​the tissue were measured, and the proportion of collagen fiber area was calculated using the following formula:

[0064] Collagen fiber area ratio = (total collagen fiber area / total tissue area) × 100%.

[0065] II. Test Results 1. Microscopic morphological analysis of HA-Se Morphological observation of humic acid and HA-Se was performed using scanning electron microscopy, such as... Figure 2 As shown in a and b, the surface of humic acid is relatively flat and smooth with regular fracture surfaces, while the surface of HA-Se exhibits flower-like curls and the fracture surfaces are mostly irregular curls. This may be due to the selenization of humic acid, which alters its surface.

[0066] 2. Spectroscopic and energy-dispersive X-ray spectroscopy analysis The characterization results of the ultraviolet spectrophotometer are as follows: Figure 1 a. A full-spectrum scan was performed in the range of 200 nm to 800 nm. The results showed that HA-Se had large absorption values ​​in both the UVA (320 nm to 400 nm) and UVB (280 nm to 320 nm) regions, with the absorption peak located in the UVB region, indicating that HA-Se has strong ultraviolet absorption capabilities.

[0067] FTIR spectroscopy ( Figure 1 b) shows the characteristic functional group distribution on the HA-Se surface. HA-Se reacts with humic acid at 1133 cm⁻¹. -1 and 622 cm -1 Compared to humic acid, it exhibits more characteristic absorption peaks, with a peak value at 622 cm⁻¹. -1 The vibration is attributed to Se-OC, with a peak value at 1133 cm⁻¹. -1 The tensile vibrations at 743 cm⁻¹ belong to the O-Se-O group. Sodium selenite at 743 cm⁻¹... -1 The presence of a characteristic absorption peak at this location, while humic acid and HA-Se show no characteristic absorption peaks at this location, indicates that humic acid and sodium selenite underwent a chemical reaction rather than simple physical adsorption, further proving that humic acid was successfully selenized.

[0068] Elemental composition and chemical state of HA-Se were analyzed by XPS. The XPS peaks at 58.2, 284.8, and 531.3 were attributed to Se, C, and O, respectively. Figure 1 In the C 1s spectrum, the peaks at 284.8 eV, 286.7 eV, and 288.3 eV are attributed to -CC / C=C, -CO / CN / C-Se, and -C=O, respectively. Figure 1 In the O 1s spectrum, the peaks at 531.3 eV, 532.9 eV, and 535.8 eV are attributed to the Auger peaks of -C=O, -C-OH / OCO, and Na, respectively. Figure 1 (e); the chemical valence state of Se compounds is usually Se. 4+ and Se 6+ In the Se 3d spectrum, the peak position is at 59.0 eV ( Figure 1 f), indicating that Se is Se 4+Therefore, in HA-Se, Se is Se. 4+ It exists in the form of selenite groups.

[0069] 3. Selenium content and activity determination like Figure 2 As shown in c, the established standard curve for selenium content is y = 0.0739 + 0.02174x (where x is the selenium concentration and y is the measured OD value). The absorbance of HA-Se was measured to be 0.718. Substituting these values ​​into the formula, the selenium content was calculated to be 7.5 mg / g.

[0070] like Figure 3 As shown, HA, HA-Se, and Vc all exhibited a concentration-dependent scavenging trend. At high concentrations, HA-Se showed significantly higher free radical scavenging ability than HA, indicating that the introduction of Se has a more significant scavenging effect on OH free radicals and possesses the potential for further development into an antioxidant material. The GPx activity of HA-Se was determined using the DTNB method, and the calculated GPx enzyme activity of HA-Se was 78.79 U / mg, demonstrating a certain free radical scavenging ability.

[0071] 4. Biocompatibility, ROS and inflammatory factor assays like Figure 4 As shown in a and b, the prepared HA-Se exhibits good biocompatibility with L929 cells and HaCaT cells. The levels of ROS and IL-6 and IL-8 inflammatory factors were measured using the kit. The results showed that the ROS levels and IL-6 and IL-8 inflammatory factor levels in the HA-Se group were significantly lower than those in the UVB group. Figure 5 (a~d). These results indicate that HA-Se has a certain protective effect against UVB-induced cell damage and can effectively inhibit UVB-induced overexpression of IL-6 and IL-8, thereby exerting a significant anti-inflammatory effect.

[0072] 5. Determination of UV protection performance and resistance to UV damage Figure 6 The results showed that there was no significant difference in SPF values ​​between sunscreens containing humic acid and those without HA-Se, while the SPF value of HA-Se sunscreens was significantly higher than the former two. This indicates that the addition of selenium can significantly improve the UV protection capability of the cream.

[0073] like Figure 7 As shown, normal mice have smooth skin without scabs; mice exposed to ultraviolet light exhibit thickened skin, redness, and scabs. Compared to the UV control group, mice in the HA-Se sunscreen group showed less scab formation, less redness, and relatively more rosy skin. Visual assessment results indicate that the HA-Se sunscreen group mice experienced less skin damage. Figure 8 ).like Figure 9 Analysis of H&E sections showed that the HA-Se sunscreen group had fewer inflammatory factors compared to the UV control group, indicating a relatively lower degree of skin damage and a certain degree of relief compared to the positive control group; Figure 10 Analysis of Masson staining results showed that the HA-Se sunscreen group contained more collagen, consistent with semi-quantitative collagen analysis. Figure 11 Collagen plays an important role in skin remodeling and recovery. The results showed that the mice in the HA-Se sunscreen group had better recovery after UV irradiation, indicating that the material has a certain ability to resist and repair UV damage. It also has certain advantages over the positive control group. Furthermore, the collagen in the HA-Se sunscreen group was denser, indicating better recovery and stronger resistance to UV damage.

[0074] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0075] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of selenium humate in the preparation of sunscreen agents, characterized in that, The selenium humate is prepared by the following steps: Sodium humate and sodium selenite were added to a dilute nitric acid solution and reacted at 70℃~90℃ for 6 h~10 h. The pH of the reaction solution was adjusted to 6~8, and the supernatant was collected by centrifugation, dialyzed, and freeze-dried to obtain the selenium humate.

2. The application according to claim 1, characterized in that, The mass ratio of sodium humate to sodium selenite is 0.5~2.5:0.5~2.

5.

3. The application according to claim 1, characterized in that, The molecular weight permeable by the dialysis is 3300 Da to 3700 Da; the freeze-drying conditions are: freeze-drying at -58℃ to -62℃ for 46 h to 50 h.

4. A selenium humate sunscreen, characterized in that, The selenium humate sunscreen is composed of: selenium humate as described in claim 1, an oil phase, an aqueous phase, and a preservative; the mass ratio of the oil phase, aqueous phase, preservative, and selenium humate is 14~19:40~46:0.05~0.15:0.05~0.

15. The oil phase comprises stearic acid, white petrolatum, glyceryl monostearate, and liquid paraffin; the aqueous phase comprises Tween-80, glycerol, and distilled water; and the preservative is methylparaben.

5. The selenium humate sunscreen agent according to claim 4, characterized in that, The mass ratio of stearic acid, white petrolatum, glyceryl monostearate, and liquid paraffin in the oil phase is 4~5:4~5:1~2:5~7; the mass ratio of Tween-80, glycerol, and distilled water in the aqueous phase is 0.5~1.5:1.5~2.5:38~42.

6. The selenium humate sunscreen agent according to claim 4, characterized in that, The selenium humate sunscreen is used to promote the synthesis of collagen in the skin after UV damage.

7. The selenium humate sunscreen agent according to claim 4, characterized in that, The selenium humate sunscreen is used to scavenge hydroxyl free radicals and / or inhibit the increase in intracellular reactive oxygen species levels caused by ultraviolet radiation.

8. The selenium humate sunscreen agent according to claim 4, characterized in that, The selenium humate sunscreen is used to inhibit the expression of inflammatory factors IL-6 and / or IL-8 induced by ultraviolet radiation.

9. The method for preparing the selenium humate sunscreen agent according to claim 4, characterized in that, Includes the following steps: The oil phase components are mixed and heated to 70℃~90℃ and maintained for 10 min~20 min; the aqueous phase components are mixed and heated to 70℃~90℃. Under stirring conditions, the oil phase at the same temperature is slowly added to the aqueous phase for emulsification; Add preservatives and selenium humate to the emulsified system and mix well; The humic acid selenium sunscreen is obtained by cooling the temperature to 35℃~45℃.

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