A solution rich in sargassum nanovesicles, and a preparation method and application thereof

Abstract

The application belongs to the technical field of cell biology, and particularly relates to a solution rich in Sargassum fusiforme nano-vesicles and a preparation method and application thereof. The solution rich in Sargassum fusiforme nano-vesicles is prepared by a differential centrifugation combined with hollow fiber membrane concentration, and the obtained solution rich in Sargassum fusiforme nano-vesicles can play an anti-inflammatory role, inhibit high-fat diet induced fatty liver and osteoporosis, and can also be used for preparing a mask with soothing, moisturizing and anti-wrinkle effects.

Description

Technical Field

[0001] This invention belongs to the field of cell biology technology, specifically relating to a solution rich in Sargassum fusiforme nanovesicles, its preparation method, and its application. Background Technology

[0002] Currently, differential centrifugation or a combination of differential centrifugation and ultracentrifugation are commonly used to prepare Sargassum fusiforme nanovesicles, which exhibit anti-inflammatory and osteoporosis-inhibiting effects. However, there is no method for large-scale preparation of solutions rich in Sargassum fusiforme nanovesicles using differential centrifugation combined with hollow fiber membrane concentration. Summary of the Invention

[0003] The purpose of this invention is to provide a solution rich in Sargassum fusiforme nanovesicles, its preparation method, and its applications. A solution rich in nanovesicles can be extracted on a large scale from Sargassum fusiforme using a differential centrifugation method combined with hollow fiber membrane concentration. The extracted solution can exert anti-inflammatory effects, inhibit fatty liver and osteoporosis induced by a high-fat diet, and can also be used to prepare face masks with soothing, moisturizing, and anti-wrinkle effects. Currently, nanovesicles can only be extracted from Sargassum fusiforme in small quantities, and there is a significant lack of methods for large-scale preparation of solutions rich in Sargassum fusiforme nanovesicles. The method provided in this invention fills this gap, and the extraction steps are simple, facilitating large-scale application.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

[0005] One of the technical solutions of this invention is to provide a method for preparing a solution rich in Sargassum fusiforme nanovesicles, comprising the following steps:

[0006] First, a Sargassum supernatant solution is prepared by crushing, grinding, and differential centrifugation; then, the Sargassum supernatant solution is concentrated using a hollow fiber membrane to obtain a solution rich in Sargassum nanovesicles.

[0007] Preferably, the centrifugal force of the differential centrifugation is 1000g, 3000g and 10000g respectively.

[0008] Preferably, the hollow fiber membrane has a pore size of 200 nm.

[0009] The second technical solution of the present invention provides a solution rich in Sargassum nanovesicles prepared according to the above-mentioned method for preparing a solution rich in Sargassum nanovesicles.

[0010] The third technical solution of the present invention provides an application of the above-mentioned solution rich in Sargassum fusiforme nanovesicles in the preparation of anti-inflammatory drugs.

[0011] The fourth technical solution of the present invention provides the application of the above-mentioned solution rich in Sargassum fusiforme nanovesicles in the preparation of drugs for treating or preventing fatty liver.

[0012] Fifth technical solution of the present invention: to provide the application of the above-mentioned solution rich in Sargassum fusiforme nanovesicles in the preparation of drugs for treating or preventing osteoporosis.

[0013] The sixth technical solution of the present invention provides an application of the above-mentioned solution rich in Sargassum fusiforme nanovesicles in the preparation of a face mask.

[0014] The facial mask prepared using the concentrated solution of Sargassum fusiforme nanovesicles of this invention has soothing, moisturizing and anti-wrinkle effects.

[0015] The beneficial technical effects of the present invention are as follows:

[0016] This invention prepares a solution rich in Sargassum fusiforme nanovesicles by differential centrifugation combined with hollow fiber membrane concentration. The obtained solution rich in Sargassum fusiforme nanovesicles can exert anti-inflammatory effects, inhibit fatty liver and osteoporosis induced by high-fat diet, and can also be used to prepare face masks with soothing, moisturizing and anti-wrinkle effects. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments 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.

[0018] Figure 1 TEM image (A), particle size analysis results (B), potential analysis results (C), and effect on the relative mRNA expression levels of HFMCs prepared for Example 1 (D) of inflammatory factors IL-6, IL-1β, and TNF-α.

[0019] Figure 2 The effect of different treatment groups on the relative mRNA expression levels of inflammatory factors IL-6 (A), IL-1β (B), and TNF-α (C) in RAW264.7 cells in Experiment Example 3.

[0020] Figure 3 The ALT and AST enzyme activities in the serum of mice in each group of Experiment 4 are shown in Figure 4 (A), and the pathological results of H&E staining of the liver of mice in each group are shown in Figure 4 (B).

[0021] Figure 4The images shown are representative Micro-CT images of the distal femur of mice in each group of Experiment 5 (A), and the bone mineral density, bone volume fraction, trabecular thickness, number of trabeculae, and trabecular separation of each group of mice (B).

[0022] Figure 5 The results of H&E staining of the small intestine (A) and immunohistochemical staining of CD68 in the small intestine of mice in Experiment Example 6 (B) are shown.

[0023] Figure 6 The front (A) and back (B) of the face mask prepared for Experimental Example 7. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0025] It should be noted that any aspects not described in detail in this invention are conventional practices in the field and are not the focus of this invention.

[0026] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] Unless otherwise specified, room temperature in this invention refers to a temperature of 20±10℃.

[0030] Example 1

[0031] Preparation of solutions rich in Sargassum nanovesicles:

[0032] (1) Take the frozen seaweed out of the -30℃ freezer, wash it, thaw it, and accurately weigh an appropriate amount of seaweed using an electronic balance.

[0033] (2) Add PBS buffer that has been pre-cooled at 4 ℃ to Sargassum fusiforme. The ratio of Sargassum fusiforme mass to PBS buffer volume is 1 g: 5 mL. Put it into a blender and grind it. Grind it at the maximum speed for 5 times, each time for 1 min. Place it on ice for 5 min after each grinding to prevent overheating from damaging the components. Finally, pour the grinding liquid into several 50 mL centrifuge tubes.

[0034] (3) Centrifuge at 1000 g for 10 min and discard the precipitate;

[0035] (4) Transfer the supernatant obtained in step (3) to a 50 mL centrifuge tube, continue to centrifuge at 3000 g for 20 min, and discard the precipitate;

[0036] (5) Transfer the supernatant obtained in step (4) to a 50 mL centrifuge tube and continue to centrifuge at 10000 g for 40 min, then discard the precipitate;

[0037] (6) The supernatant obtained in step (5) was filtered through a hollow fiber membrane with a pore size of 200 nm to obtain a solution rich in Sargassum nanovesicles, which was named hollow fiber concentrate (HFMC).

[0038] Experimental Example 1

[0039] The HFMC prepared in Example 1 was characterized as follows:

[0040] To verify whether the Sargassum fusiforme solution obtained through hollow fiber concentration contained nanovesicles, the sample was negatively stained with phosphotungstic acid and observed under an electron microscope. The resulting TEM image (scale bar 200 nm) is shown below. Figure 1 In Figure A, the nanoparticles exhibit a saucer-like vesicle structure, indicating that hollow fiber concentration can yield a solution rich in Sargassum nanovesicles.

[0041] Take 100 μL of HFMC, dilute it 5000 times, and determine its particle size using NTA. The results are shown in the figure. Figure 1 In section B, the average particle size of the HFMC is 170.3 nm.

[0042] 10 μL of the purified HFMC was diluted with 9.99 mL of PBS, and its charge was detected using a Zeta potentiometer. The results are shown in [Figure number missing]. Figure 1 In the middle C, the charge of HFMC is shown to be -39.10 mV.

[0043] Experimental Example 2

[0044] The effects of HFMC prepared in Example 1 on inflammatory factors in RAW264.7 cells were investigated.

[0045] (1) Grouping: NC (blank control group), LPS stimulation group, LPS stimulation + HFMC group. Cells were seeded according to the group, with 50,000 cells seeded in each well of a 24-well plate.

[0046] (2) After 6 hours, the cells adhered to the wall. A solution rich in Sargassum nanovesicles was added to the LPS-stimulated + HFMC pores, with a particle count of 1.5 × 10⁻⁶. 10 particles / mL. After co-incubating with cells for 24 h, LPS (1 μg / mL) was added to the wells of the LPS-stimulated group and the LPS-stimulated + HFMC group.

[0047] (3) After stimulation for 6 h, observe that no cell death has occurred, collect the samples and extract RNA. Aspirate the cell culture medium, wash twice with PBS, and add 300 μL of TRlzol reagent to each well. Transfer the cells to the corresponding EP tubes.

[0048] (4) Allow the mixture to settle on ice for 5 min to fully decompose, then add 1 / 5 volume of chloroform and immediately vortex for 15 s to mix thoroughly.

[0049] (5) Settle on ice for 5 min, set at 4 ℃, 14000 rpm, and centrifuge for 12 min.

[0050] (6) Pre-cool isopropanol and prepare the corresponding number of 1.5 mL enzyme-removing EP tubes, and label them.

[0051] (7) Use a pipette to transfer an appropriate volume of supernatant (do not aspirate the white precipitate on the centrifuge tube wall or the TRlzol reagent at the bottom of the tube) into the above-labeled enzyme-free EP tube.

[0052] (8) Add an equal volume of isopropanol to precipitate the RNA. After mixing by inverting the container, precipitate on ice for 10 min.

[0053] (9) Centrifuge at 14000 rpm at 4℃ for 30 min, and carefully discard the supernatant.

[0054] (10) Add 80% ethanol (prepared with DEPC water) to wash the precipitate, centrifuge at 14000 rpm at 4℃ for 15 min, carefully discard the supernatant, and dry the precipitate at 42℃ for about 3 minutes.

[0055] (11) Add 10 μL of DEPC water to dissolve the RNA.

[0056] (12) The reverse transcription and qPCR steps are as follows, referring to the Nanjing Novizan kit (NO: R223-01): I. Reverse transcription

[0057] ① Genomic DNA was removed. The reagents used are shown in Table 1.

[0058] Table 1. Reagents for removing genomic DNA

[0059]

[0060] After gently mixing with a pipette, place the EP tube into the PCR instrument and set the program to run at 42 °C for 2 min.

[0061] ② Prepare the reverse transcription reaction system, the composition of which is shown in Table 2.

[0062] Table 2 Reverse transcription reaction system

[0063]

[0064] Gently pipette the mixture to mix it thoroughly, then briefly centrifuge the EP tube at 2000 rpm for 10 seconds before placing it in the PCR instrument for the next step.

[0065] ③ Reverse transcription reaction, conditions are shown in Table 3.

[0066] Table 3 Reverse transcription reaction conditions

[0067]

[0068] After the PCR procedure is completed, the cDNA samples are stored in a -20°C freezer.

[0069] II. Real-time quantitative PCR (qRT-PCR)

[0070] The steps are as follows, referring to the Nanjing Novizan reagent kit (NO: Q311-02):

[0071] Primer sequences are shown in Table 4.

[0072] Table 4 Primer Sequences

[0073]

[0074] ① Prepare the reaction system, the composition of which is shown in Table 5.

[0075] Table 5 Reaction System

[0076]

[0077] Label the 96-well PCR plate and add 20 μL of the above mixture to each well in sequence. Balance the PCR plate in a centrifuge and collect the reaction solution after a short centrifugation.

[0078] ②PCR reaction (operated on a CFX96 Real-Time PCR System instrument), the reaction procedure is shown in Table 6.

[0079] Table 6 PCR reaction procedure

[0080]

[0081] The relative expression levels of genes after the reaction were calculated according to 2. -△△Ct ( △ Ct value = Ct 目的 -Ct 内参 , △△ Ct value = △ Ct 实验组 - △ Ct 对照组 The relative expression levels of the genes were calculated, with the GADPH gene used as a control gene, to detect the mRNA expression levels of IL-6, IL-1β, and TNF-α in cells (results are shown in [link to results]). Figure 1 (D, ** indicates P<0.01, *** indicates P<0.001) RT-qPCR results showed that the relative mRNA expression levels of IL-6, IL-1β and TNF-α in RAW cells of the LPS-stimulated + HFMC group were significantly lower than those of the LPS-stimulated group alone, indicating that HFMC has an inhibitory effect on inflammation.

[0082] Experimental Example 3

[0083] The active components of the HFMC prepared in Example 1 were examined:

[0084] 1. To evaluate the active components of HFMC, a targeted metabolomics analysis of HFMC was first performed. The experimental methods are as follows:

[0085] Sample processing:

[0086] (1) Take 1 mL of SF-NVs sample for lyophilization, place it in an EP tube, and add 500 μL of 80 vol% methanol aqueous solution;

[0087] (2) Vortex oscillation, let stand in ice bath for 5 min, centrifuge at 15000 g and 4 ℃ for 15 min;

[0088] (3) Take a certain amount of supernatant and dilute it with mass spectrometry grade water until the methanol content is 53% vol%.

[0089] (4) Centrifuge at 15000 g and 4 ℃ for 20 min, collect the supernatant, and analyze it by LC-MS.

[0090] Chromatographic conditions are shown in Table 7:

[0091] Table 7 Chromatographic conditions

[0092]

[0093] The mass spectrometry conditions are as follows:

[0094] 1) Positive ion mode

[0095] Mass spectrometry conditions: Curtain Gas: 35 psi; Collision Gas: Medium; Ion Spray Voltage: 5500 V; Temperature: 550 °C; Ion Source Gas 1: 60; Ion Source Gas 2: 60.

[0096] 2) Negative ion mode

[0097] Mass spectrometry conditions: Curtain Gas: 35psi; Collision Gas: Medium; IonSpray Voltage: -4500V; Temperature: 550℃; Ion Source Gas 1:60; Ion Source Gas 2:60.

[0098] Metabolite identification methods:

[0099] Multiple reaction monitoring (MRM) was employed. First, compounds were qualitatively identified based on parameters such as Q1 (mother ion), Q3 (daughter ion), retention time, declustering voltage, and collision energy. Then, quantification was performed based on the Q3 (daughter ion) signal. Raw mass spectrometry data were processed using SCIEX OS V1.4 software, with peak selection based on a minimum peak height of 500 nm, a signal-to-noise ratio of 5, and a smoothing point count of 1. Integration and correction were then performed. The peak area of ​​the daughter ion at a specific retention time for each compound represented its relative content. Finally, all peak area data were exported to obtain the qualitative and quantitative results of the metabolites. The results are shown in Table 8. The major metabolites with relatively high content included menotriose, eugenol 3-O-galactoside group, diosgenin, elemol, and artemisinic acid.

[0100] Table 8. Major metabolites with high content

[0101]

[0102] 2. Investigate whether the main small molecule compounds have anti-inflammatory effects:

[0103] (1) Grouping and plating: The experiment was set up with the following groups: blank control group (NC), LPS stimulation group, LPS stimulation + mesotriose group, LPS stimulation + eugenol 3-O-galactoside group, LPS stimulation + diosgenin group, LPS stimulation + elemol group, and LPS stimulation + artemisinic acid group. Cells were plated at 5 × 10⁶ cells per well. 4 The cells were inoculated at a density of 100 cells per well in a 24-well plate.

[0104] (2) Compound pretreatment and LPS stimulation: After cell culture for 6 h and cell adhesion, pine glycoside (final concentration 100 μg / mL), eugenol-3-O-galactoside (100 μg / mL), diosgenin (final concentration 100 μg / mL), elemol (final concentration 25 μg / mL), or artemisinic acid (final concentration 100 μg / mL) were added to the corresponding treatment wells and co-incubated with the cells for 24 h. Subsequently, except for the blank control group, all other groups were stimulated with LPS (final concentration 1 μg / mL). Subsequent experimental procedures were the same as in Experiment 2. The effects of different treatment groups on the relative mRNA expression levels of inflammatory factors IL-6 (A), IL-1β (B), and TNF-α (C) in RAW264.7 cells are as follows: Figure 2 As shown in the figure. RT-qPCR results showed that in macrophages, only elemol treatment could significantly inhibit LPS-induced expression of IL-6, IL-1β and TNF-α mRNA; while mesotriose, syringin-3-O-galactoside, diosgenin and artemisinic acid had no significant effect on the expression of these cytokines.

[0105] Test Example 4

[0106] The effect of HFMC prepared in Example 1 on alleviating liver injury induced by a high-fat diet was investigated.

[0107] Eight-week-old SPF-grade C57 / B6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. After a one-week acclimatization period in the animal facility, the mice were randomly divided into four groups (NCD+PBS group, NCD+HFMC group, HFD+PBS group, and HFD+HFMC group), with eight mice in each group. The NCD group was fed a 10kcal% fat maintenance diet (control group), while the HFD group was fed a 60kcal% fat high-fat diet (high-fat diet group). This feeding regimen continued for 16 weeks. Starting in week 7, oral gavage was initiated (administering 2.55 × 10¹¹ particles of HFMC or an equivalent amount of PBS), once every other day for nine consecutive weeks. Throughout the experiment, the mice's normal activity and water intake were not restricted, and the temperature was maintained at a constant 24°C with a 12-hour diurnal cycle.

[0108] All mice were fasted for 16 hours prior to dissection. Blood, liver, and femur samples were collected from the mice for further research.

[0109] 1. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzyme activities detection:

[0110] The reagent kit was purchased from Nanjing Jiancheng Biotechnology Institute, and the assay was performed according to the kit instructions. Serum samples can be tested directly.

[0111] (1) Add 5 μL of pre-warmed matrix solution at 37°C to the test wells and control wells of the 96-well plate. Add 5 μL of the sample to be tested to the test well. Note that after each sample is aspirated from the test well, insert the pipette tip into the matrix solution at the bottom of the plate and repeatedly aspirate and mix. However, be careful not to aspirate air bubbles. React at 37°C for 30 min.

[0112] (2) Add 20 μL of 2,4-dinitrophenylhydrazine solution to each well, and then add 5 μL of the test sample to the control well. React at 37℃ for 20 min.

[0113] (3) Then add 200 μL of 0.4 mol / L sodium hydroxide solution to each well, gently shake the 96-well plate horizontally to mix, let it stand at room temperature for 15 minutes, and measure the OD value of each well with a microplate reader at a wavelength of 510 nm. Substitute the absolute OD value (OD value of the measured well minus the OD value of the control well) into the standard curve to calculate the corresponding AST / ALT activity units.

[0114] The results showed that the HFD diet significantly increased serum AST and ALT levels in mice, while oral administration of HFMC significantly alleviated the increase in AST and ALT levels. Figure 3 In the diagram, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0115] 2. Liver H&E staining

[0116] Liver tissues were collected and fixed overnight in 4% formaldehyde solution, then dehydrated with 70% ethanol, cleared with xylene, and embedded in paraffin. All liver tissues were cut into 5 μm thick sections, dewaxed with xylene, and stained with hematoxylin and eosin (HE). After drying, the sections were observed and photographed under an optical microscope (Nikon).

[0117] The results of liver H&E staining in each group of mice (scale bar 50 µm) are shown below. Figure 3 The results showed that a high-fat diet led to lipid vacuolation and inflammatory accumulation in the liver, while HFMC treatment significantly alleviated lipid vacuolation and inflammation caused by a high-fat diet.

[0118] Experimental Example 5

[0119] The effect of HFMC prepared in Example 1 on alleviating high-fat diet-induced bone loss was investigated.

[0120] After completing Experiment 3, the femur of the mouse was collected, fixed in 4% formaldehyde solution overnight, and then scanned with Micro-CT.

[0121] Representative Micro-CT images of the distal femur of mice in each group are shown below. Figure 4In the study, the results showed that mice treated with high-fat diets (HFMC) exhibited significant improvements in bone microstructure compared to the untreated high-fat diet group. Bone mineral density, bone volume fraction, trabecular thickness, trabecular number, and trabecular separation were observed in each group. Figure 4 In the B-cell assay, the results showed that HFMC treatment significantly restored the decrease in bone mineral density and bone volume fraction caused by high-fat diet and effectively increased trabecular bone thickness. Furthermore, the number of trabecular bones showed an increasing trend, while trabecular separation decreased, although these changes did not reach statistical significance (* indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001).

[0122] Experimental Example 6

[0123] The study investigated the alleviating effect of HFMC prepared in Example 1 on intestinal microenvironment inflammation and homeostasis imbalance induced by a high-fat diet.

[0124] 1. Small intestine H&E staining

[0125] The experimental procedure is the same as in Example 4, and the experimental results are shown below. Figure 5 In Figure A (scale bar 50 µm), the experimental results showed that, compared with the HFD group, the HFMC group had restored mucosal structure, increased number of intact crypts, and significantly increased number of goblet cells producing mucus.

[0126] 2. Small intestinal CD68 immunohistochemistry

[0127] After collecting samples from mice in Experiment 4, small intestinal tissue was fixed overnight in 4 vol% formaldehyde solution, followed by dehydration in 70 vol% ethanol and embedding in paraffin. The paraffin-embedded small intestinal tissue was dewaxed, hydrated, and then subjected to antigen retrieval and non-specific site blocking. Sections were incubated overnight at 4 °C with anti-CD68 primary antibody. After washing, HRP-labeled secondary antibody was added for incubation. Antigen staining was performed using 3,3′-diaminobenzidine (DAB) substrate; the reaction was terminated immediately with tap water when a pale yellow color was observed. Finally, sections were counterstained with hematoxylin, dehydrated with graded ethanol, cleared with xylene, and mounted with neutral resin for microscopic observation and evaluation. The results are shown below. Figure 5 In section B (scale bar 50 µm), the experimental results showed that a large number of CD68 molecules were observed in the lamina propria and submucosa of the small intestine in the high-fat diet group. + Macrophage infiltration, with positive signals appearing as densely distributed, dark brown granules with enlarged cell bodies, indicating a significant intestinal inflammatory response; while after HFMC intervention, CD68... + The number of cells was significantly reduced, the positive staining became lighter, and the distribution became sparser, confirming that HFMC can improve chronic intestinal inflammation.

[0128] Experimental Example 7

[0129] The soothing, moisturizing, and anti-wrinkle effects of the face mask prepared using the HFMC obtained in Example 1 were investigated.

[0130] Mask preparation:

[0131] (1) Accurately weigh polyacrylic acid, aluminum hydroxyl, EDTA (ethylenediaminetetraacetic acid), CMC (carboxymethyl cellulose), and masking agent according to the formula (Table 7);

[0132] (2) Weigh out the glycerin according to the formula and place it in the mixing pot;

[0133] (3) Add (1) to (2) and stir for 8 minutes until uniform to obtain the oil phase raw material for later use;

[0134] (4) Weigh the purified water, tartaric acid, and preservative according to the formula and stir well;

[0135] (5) The Sargassum nanovesicle solution is weighed according to the formula and added to (4) and stirred evenly to obtain the aqueous raw material for later use;

[0136] (6) Add the aqueous phase (5) to the oil phase (3) and mix under vacuum;

[0137] (7) Vacuum stirring;

[0138] (8) Coating and film covering, air drying, cutting, boxing, labeling, and packing.

[0139] The front (A) and back (B) of the prepared face mask are shown below. Figure 6 .

[0140] Table 9. Facial Mask Formula (mass fraction)

[0141]

[0142] The soothing effects of the prepared face mask were tested:

[0143] (1) Dilute the extract of the mask to be tested to a 20 vol.% solution (sample group), and the positive control is dipotassium glycyrrhizate solution;

[0144] (2) Add reagents to the pretreated sample solution according to the steps to ensure complete reaction;

[0145] (3) Measure the absorbance of the reagent after the reaction is complete;

[0146] (4) Record the data and calculate the hyaluronidase inhibition rate and statistical differences.

[0147] The test results represent the inhibition rate of the sample against hyaluronidase. Compared with the control substance with soothing effects, if the inhibition rate of the positive control group is higher than that of the blank control group and the difference is statistically significant (P<0.05), the test results are valid. If the inhibition rate of the test sample is higher than that of the blank control group and the difference is statistically significant (P<0.05), it indicates that the test sample has soothing effects. The experiment was conducted in three parallel experiments, and the average value of the three parallel experiments was used as the test results.

[0148] Table 10 Hyaluronidase Inhibition Rate in Each Group (Evaluation of Soothing Efficacy)

[0149]

[0150] The moisturizing effect of the prepared face mask was tested:

[0151] (1) Use ultrapure water to prepare 10 vol.% glycerol (control group), and dilute the extract of the mask to be tested to 20 vol.% solution (sample group).

[0152] (2) Weigh the mass of each container as m. 空 ;

[0153] (3) Using 10 vol.% glycerol as the standard control, the sample added was 5 g, and the total mass after adding the reagent was weighed as the initial total sample mass m0;

[0154] (4) Weigh the total mass m of the test sample at 2h, 4h and 8h respectively. t ;

[0155] (5) Record the experimental data and calculate the moisturizing rate and relative moisturizing rate at each time point of action.

[0156] (6) Results analysis: Calculate the moisturizing rate and relative moisturizing rate of the test samples.

[0157] Moisture retention rate = (m t -m 空 ) / (m0-m 空 ) × 100%.

[0158] Relative moisturizing rate % = P (测试样品的保湿率) / P (标准对照的保湿率) ×100%.

[0159] Table 11 Moisturizing rate of each group (moisturizing efficacy assessment)

[0160]

[0161] The anti-wrinkle efficacy of the prepared face mask was tested:

[0162] (1) Dilute the extract of the mask to be tested into a 20 vol.% solution (sample group), and the positive control is epigallocatechin gallate (EGCG).

[0163] (2) Add reagents to the pretreated sample solution according to the steps to ensure complete reaction;

[0164] (3) Measure the absorbance of the reagent after the reaction is complete;

[0165] (4) Record the data and calculate the elastase inhibition rate and statistical differences.

[0166] The test results represent the inhibition rate of the sample against elastase. Compared with the control group with anti-wrinkle effect, if the inhibition rate of the positive control group is higher than that of the blank control group and the difference is statistically significant (P<0.05), the test results are valid. If the inhibition rate of the test sample is higher than that of the blank control group and the difference is statistically significant (P<0.05), it indicates that the test sample has anti-wrinkle effect. The experiment was conducted in three parallel experiments, and the average value of the three parallel experiments was used as the test result.

[0167] Table 12 Elastase inhibition rate of each group (anti-wrinkle efficacy assessment)

[0168]

[0169] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a solution rich in Sargassum fusiforme nanovesicles, characterized in that, Includes the following steps: First, the Sargassum fusiforme supernatant solution was prepared by crushing, grinding, and differential centrifugation. The Sargassum supernatant solution was then concentrated using a hollow fiber membrane to obtain a solution rich in Sargassum nanovesicles.

2. The method for preparing a solution rich in Sargassum nanovesicles according to claim 1, characterized in that, The centrifugal forces of the differential centrifuges are 1000g, 3000g and 10000g respectively.

3. The method for preparing a solution rich in Sargassum nanovesicles according to claim 1, characterized in that, The hollow fiber membrane has a pore size of 200 nm.

4. A solution rich in Sargassum nanovesicles prepared by the method of preparing a solution rich in Sargassum nanovesicles according to any one of claims 1 to 3.

5. The use of the solution rich in Sargassum nanovesicles as described in claim 4 in the preparation of anti-inflammatory drugs.

6. The use of the solution rich in Sargassum nanovesicles as described in claim 4 in the preparation of a medicament for treating or preventing fatty liver.

7. The use of the solution rich in Sargassum nanovesicles as described in claim 4 in the preparation of a medicament for treating or preventing osteoporosis.

8. The application of the solution rich in Sargassum nanovesicles as described in claim 4 in the preparation of a face mask.

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