Sea cucumber oil and preparation method and application thereof

Abstract

The application discloses sea cucumber oil and a preparation method and application thereof. The sea cucumber oil is prepared by using sea cucumber intestines as raw materials, and by using 95% ethanol and petroleum ether to soak and extract the sea cucumber oil, and then by using vacuum distillation. By constructing an acute skin ultraviolet radiation damage mouse model, the application verifies for the first time that the sea cucumber oil can effectively relieve the skin redness and keratin layer increase phenomenon caused by ultraviolet radiation, inhibit the ROS rise, reduce the degree of lipid peroxidation of the organism, improve the loss of collagen in the body, regulate the expression of inflammatory factors in the body, up-regulate the expression of the skin Nrf2 gene and the downstream antioxidant gene NQO1, reduce the expression of the negative feedback factor KEAP1, and play an antioxidant role. The sea cucumber oil has high safety, comprehensively and effectively utilizes the waste resources generated in the sea cucumber processing process, and has a good prospect in the cosmetics and medicines for repairing the acute skin light damage after ultraviolet radiation.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and specifically relates to a sea cucumber oil, its preparation method, and its application. Background Technology

[0002] Ultraviolet (UV) radiation refers to the light in sunlight with wavelengths ranging from 10 to 400 nm. Prolonged exposure to excessive UV radiation can cause erythema and wrinkles on the skin, promote photodamage and immune system imbalances, and increase the risk of skin cell damage and skin cancer. Sea cucumbers are rich in nutrients and have various pharmacological effects. Active substances in sea cucumbers, such as polypeptides, polysaccharides, and saponins, have been proven to have multiple effects, including lowering blood sugar and lipids, anti-inflammation, anti-oxidation, and anti-tumor activity. Sulfated fucoidan extracted from sea cucumbers can treat thromboembolic diseases, and fucoidan and fucoidan have fewer side effects, reducing the risk of complications such as bleeding and thrombocytopenia. Sea cucumber triterpenoids are potential drug prototypes for developing novel drugs with anti-tumor activity. Sea cucumber cerebrosides have the potential to protect the intestines from inflammation and tumors. Various types of unsaturated fatty acids (such as EPA and DHA) contained in sea cucumbers have good health benefits; for example, EPA can prevent hyperlipidemia and reduce the occurrence of thrombosis and atherosclerosis; DHA promotes brain development and prevents cancer and osteoporosis.

[0003] High UV exposure can cause DNA damage, including indirect oxidative DNA damage. Pharmacologically active compounds produced by sea cucumbers may become potential drug prototypes. They could be used in the pharmaceutical industry as therapeutic agents for photodamage and photoaging, or in the cosmetics industry as antioxidants to protect against UV damage. Although my country is rich in resources, research on photochemical protectants started relatively late and has mostly focused on plant-based photochemical protectants, with limited research on photodamage repair in marine organisms. In recent years, with the deepening of marine biological research and the improvement of research techniques, researchers have continuously discovered compounds from marine organisms that can repair UV damage. While sea cucumbers have been reported to have effects such as lowering blood lipids, blood sugar, and anti-aging, there are no reports of topical or oral application of sea cucumber oil for anti-photodamage or anti-photocarcinogenic effects. Sea cucumber oil is rich in anti-inflammatory and antioxidant unsaturated fatty acids such as EPA, DHA, and AA, suggesting that sea cucumber oil may have a positive effect on preventing skin photodamage and photocarcinogenicity, providing a basis for drug development and cosmetic applications. Summary of the Invention

[0004] The purpose of this invention is to provide a sea cucumber oil, its preparation method, and its application. The sea cucumber oil has good safety and a significant effect on repairing skin damage caused by ultraviolet radiation.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] This invention provides a method for preparing sea cucumber oil, which includes the following steps:

[0007] (1) Clean the sea cucumber intestines to remove impurities and set aside;

[0008] (2) Freeze-dry the cleaned sea cucumber intestines and then pulverize them;

[0009] (3) After soaking the crushed sea cucumber intestines, the solid and liquid are separated to obtain the liquid;

[0010] (4) Extract the liquid from step (3) with petroleum ether, add anhydrous sodium sulfate to the collected upper layer solution, filter, and then rotary evaporate the organic phase obtained by extraction to obtain sea cucumber oil.

[0011] Furthermore, in step (2), the freezing temperature is -50°C, the vacuum degree is 1Pa, and the final freeze-drying temperature of the sample is 25°C; and the sea cucumber intestines are pulverized to 80 mesh after freeze-drying.

[0012] Furthermore, in step (3), the crushed sea cucumber intestines are extracted using 95% ethanol; the ratio of the crushed sea cucumber intestines to 95% ethanol is 1:8~15, the extraction temperature is 25-30℃, and the extraction time is 16~20h.

[0013] Furthermore, in step (4), petroleum ether is used for extraction, wherein petroleum ether is added in a volume ratio of petroleum ether to ethanol of 1:1.

[0014] The present invention also provides sea cucumber oil prepared by the above preparation method, wherein the sea cucumber oil contains not less than 30% monounsaturated fatty acids and not less than 10% polyunsaturated fatty acids.

[0015] Furthermore, the sea cucumber oil is an orange-yellow liquid oil with stable properties, a fishy smell, an acid value of 27.8 mg / g, and a peroxide value of 0.67 nmol / kg.

[0016] The present invention also provides the application of the sea cucumber oil in the preparation of pharmaceuticals or cosmetics for repairing ultraviolet radiation damage.

[0017] Furthermore, when the sea cucumber oil is used to prepare internal medicines, the effective dose for repairing ultraviolet radiation damage is 2g / kg-8g / kg.

[0018] Furthermore, when the sea cucumber oil is used to prepare topical medicines or cosmetics, the effective amount added to repair ultraviolet radiation damage is 0.5%-2% of the volume of the topical medicine or cosmetic.

[0019] Furthermore, the topical medication includes sea cucumber oil cream; the specific preparation steps of the sea cucumber oil cream are as follows: a stearic acid, glyceryl monostearate, ethylparaben, and liquid paraffin are mixed to prepare an oil phase; a Tween 80, glycerin, and water are mixed to prepare an aqueous phase; the oil phase and the aqueous phase are heated to 80°C, sea cucumber oil is added to the oil phase, and the oil phase is slowly added to the aqueous phase while stirring. When emulsification reaches 50°C, stirring is stopped to obtain the sea cucumber oil cream.

[0020] Furthermore, the sea cucumber oil can alleviate skin erythema, wrinkles, and epidermal hyperplasia caused by ultraviolet radiation, reduce the production of infiltrating inflammatory cells, tighten intercellular spaces to relieve tissue edema, and repair disordered collagen fibers; it can inhibit ultraviolet-induced ROS generation, increase the activity of catalase and glutathione catalase, reduce the degree of lipid peroxidation in the body, and improve the level of oxidative stress in the body; it can significantly increase the content of hydroxyproline in the skin, inhibit ultraviolet-induced degradation of skin collagen, and improve skin photoaging caused by ultraviolet radiation.

[0021] Furthermore, the sea cucumber oil can regulate the expression of factors TNF-α, IL-1β, IL-6 and COX-2, thereby enhancing its immunomodulatory effect.

[0022] Furthermore, the sea cucumber oil can upregulate the mRNA expression levels of NRF2 and related antioxidant genes NQO1 and HO-1, reduce the expression of the negative feedback factor KEAP1, and exert antioxidant function.

[0023] Compared with existing technologies, the beneficial effects and advantages of the present invention are as follows:

[0024] This invention extracts sea cucumber oil using sea cucumber intestines as raw material, ensuring high safety and effectively utilizing waste resources generated during sea cucumber processing. The extracted sea cucumber oil contains 33.6% monounsaturated fatty acids and 10.8% polyunsaturated fatty acids, with an acid value of 27.8 mg / g and a peroxide value of 0.67 nmol / kg. This invention, through the construction of an acute ultraviolet radiation-induced skin injury mouse model, verifies for the first time that sea cucumber oil can effectively alleviate skin redness and swelling and increased stratum corneum caused by ultraviolet radiation, inhibit ROS increases, reduce lipid peroxidation, improve collagen loss, regulate the expression of inflammatory factors, upregulate the expression of the skin Nrf2 gene and its downstream antioxidant gene NQO1, and reduce the expression of the negative feedback factor KEAP1, thus exerting an antioxidant effect. It shows promising potential in cosmetics and pharmaceuticals for repairing acute skin photodamage after ultraviolet radiation. Attached Figure Description

[0025] Figure 1 The image shows the back of a mouse exposed to ultraviolet radiation; (A) skin of a mouse in the gavage group, and (B) skin of a mouse in the topical application group.

[0026] Figure 2 HE staining pathological morphology of mouse dorsal skin; (A) HE staining pathological morphology of the gavage group, (B) HE staining pathological morphology of the smear group.

[0027] Figure 3 The effect of sea cucumber oil on the ROS content in the skin of UV-induced mice was investigated. Among them, (A) the effect of oral administration of sea cucumber oil on the ROS content in the skin of UV-induced mice, and (B) the effect of topical application of sea cucumber oil on the ROS content in the skin of UV-induced mice.

[0028] Figure 4 The effect of sea cucumber oil on UV-induced oxidative stress levels in mouse skin was investigated. Specifically, (A) the effect of oral administration of sea cucumber oil on UV-induced oxidative stress levels in mouse skin, and (B) the effect of topical application of sea cucumber oil on UV-induced oxidative stress levels in mouse skin.

[0029] Figure 5 The effect of sea cucumber oil on HYP content in the skin of UV-induced mice was investigated. Among them: (A) the effect of oral administration of sea cucumber oil on HYP content in the skin of UV-induced mice; (B) the effect of topical application of sea cucumber oil on HYP content in the skin of UV-induced mice.

[0030] Figure 6 The study investigated the effects of sea cucumber oil on the mRNA expression levels of UV-induced skin inflammatory factors in mice. Specifically, (A) the effect of oral administration of sea cucumber oil on the mRNA expression levels of UV-induced skin inflammatory factors in mice, and (B) the effect of topical application of sea cucumber oil on the mRNA expression levels of UV-induced skin inflammatory factors in mice.

[0031] Figure 7 The study investigated the effects of sea cucumber oil on the mRNA expression levels of NRF2, NQO1, and KEAP1 genes in the skin of UV-induced mice. Specifically, (A) the effect of oral administration of sea cucumber oil on the mRNA expression levels of NRF2, NQO1, and KEAP1 genes in the skin of UV-induced mice, and (B) the effect of topical application of sea cucumber oil on the mRNA expression levels of NRF2, NQO1, and KEAP1 genes in the skin of UV-induced mice. Detailed Implementation

[0032] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited to the scope of the examples described.

[0033] Example 1: Preparation of sea cucumber oil

[0034] 1. The method for preparing sea cucumber oil in this invention includes the following steps:

[0035] (1) Clean the sea cucumber intestines to remove impurities and set aside;

[0036] (2) Freeze-dry the sea cucumber intestines after cleaning in step (1) at a freezing temperature of -50℃ and a vacuum of 1Pa. The final freeze-drying temperature of the sample is 25℃.

[0037] (3) Grind the freeze-dried sea cucumber intestines from step (2) into 80 mesh;

[0038] (4) Add 95% ethanol to the crushed sea cucumber intestines in step (3) at a material-to-liquid ratio of 1:10 (W / V), extract at 25°C, and separate the solid and liquid after 16 hours of extraction.

[0039] (5) Extract the liquid obtained in step (4) with petroleum ether. Add petroleum ether at a ratio of 1:1 (petroleum ether: ethanol), shake, and extract three times.

[0040] (6) Collect the upper layer solution, add anhydrous sodium sulfate, filter, extract the organic phase, and then evaporate it at 30°C to obtain sea cucumber oil.

[0041] 2. Composition determination of sea cucumber oil

[0042] Dissolve the sample in 0.5 mL of chromatographically pure hexane, then add 2 mL of methylation reagent (i.e., chromatographically pure methanol containing 1% sulfuric acid, freshly prepared). Shake to mix thoroughly, then purge with nitrogen and seal. Incubate in a 70°C water bath for 1 hour, gently shaking every 15 minutes. Remove and rapidly cool to room temperature. Add 1 mL of water, allow to stand, and collect the supernatant. Wash with water until the pH is neutral (tested with pH paper), then dry with anhydrous sodium sulfate.

[0043] The samples prepared by the above method were placed in dedicated gas chromatography vials, and the fatty acid composition of sea cucumber intestines was analyzed using an Agilent 7890 / 5975-GC / MS. An HP-5-MS capillary column (30m × 0.25mm × 0.25um) was used as the carrier gas, with ammonia. Fatty acid methyl ester analysis conditions: initial temperature 50℃, held for 1 min; rapidly increased to 170℃ at a rate of 50℃ / min, then to 300℃ at a rate of 4℃ / min, and finally to 320℃ at a rate of 40℃ / min, held for 3.6 min. Mass spectrometry was performed using an EI source (70eV), Scan mode, with a scan range of 50 to 550 m / z and a solvent delay of 4 min. In the fatty acid analysis, based on the retention times and mass spectra of each component in the GC-MS, identification was performed using fatty acid standards and the NISTO8 library. The composition and relative content of each fatty acid in the sample were calculated using the peak area normalization method.

[0044] The results are shown in Table 1. The fatty acids contained 33.6% monounsaturated fatty acids and 10.8% polyunsaturated fatty acids, including 2.91% cis-5,8,11,14-eicosatetraenoic acid (EPA), 1.61% arachidonic acid (AA), and 0.924% cis-4,7,10,13,16,19-docosahexaenoic acid (DHA).

[0045] Table 1: Composition of Sea Cucumber Oil

[0046] ;

[0047] .

[0048] Example 2: Cream Preparation

[0049] (1) Preparation of sea cucumber oil base cream

[0050] Add 5g stearic acid, 5g glyceryl monostearate, and 0.1g ethylparaben to 10g liquid paraffin to obtain the oil phase of the cream; add 1g Tween 80 and 10g glycerin to a beaker, and add water to 80g to obtain the aqueous phase of the cream; heat the oil and aqueous phases to 80℃, and slowly add the oil phase to the aqueous phase while stirring. When emulsification reaches 50℃, stop stirring and cool to room temperature to obtain the sea cucumber oil-based cream.

[0051] (2) Preparation of 0.5% sea cucumber oil cream

[0052] Add 5g stearic acid, 5g glyceryl monostearate, and 0.1g ethylparaben to 10g liquid paraffin to obtain the oil phase of the cream. Add 1g Tween 80 and 10g glycerin to a beaker, and add water to 79.5g to obtain the aqueous phase of the cream. Heat the oil and aqueous phases to 80℃, and add 0.5g sea cucumber oil to the oil phase while stirring. After mixing evenly, slowly add the oil phase to the aqueous phase while stirring. When emulsification reaches 50℃, stop stirring and cool to room temperature to obtain a 0.5% sea cucumber oil cream.

[0053] (3) Preparation of 1% sea cucumber oil cream

[0054] Add 5g stearic acid, 5g glyceryl monostearate, and 0.1g ethylparaben to 10g liquid paraffin to obtain the oil phase of the cream. Add 1g Tween 80 and 10g glycerin to a beaker, and add water to 79g to obtain the aqueous phase of the cream. Heat the oil and aqueous phases to 80℃, and add 1g sea cucumber oil to the oil phase while stirring. After mixing evenly, slowly add the oil phase to the aqueous phase while stirring. When emulsification reaches 50℃, stop stirring and cool to room temperature to obtain a 1% sea cucumber oil cream.

[0055] (4) Preparation of 2% sea cucumber oil cream

[0056] Add 5 g of stearic acid, 5 g of glycerol monostearate, and 0.1 g of ethylparaben to 10 g of liquid paraffin to obtain the oil phase of the cream; add 1 g of Tween 80 and 10 g of glycerol to a beaker, and add water to make up to 78 g to obtain the water phase of the cream; heat the oil phase and the water phase to 80 °C, add 2 g of sea cucumber oil to the oil phase while stirring, and after mixing evenly, slowly add the oil phase to the water phase while stirring. When emulsifying to 50 °C, stop stirring and cool to room temperature to obtain 2% sea cucumber oil cream.

[0057] (5)Preparation of 2% Vitamin E Cream

[0058] Add 5 g of stearic acid, 5 g of glycerol monostearate, and 0.1 g of ethylparaben to 10 g of liquid paraffin to obtain the oil phase of the cream; add 1 g of Tween 80 and 10 g of glycerol to a beaker, and add water to make up to 78 g to obtain the water phase of the cream; heat the oil phase and the water phase to 80 °C, add 2 g of vitamin E to the oil phase while stirring, and after mixing evenly, slowly add the oil phase to the water phase while stirring. When emulsifying to 50 °C, stop stirring and cool to room temperature to obtain 2% vitamin E cream.

[0059] Example 3

[0060] Select 30 male SPF-grade KM mice (purchased from Jinan Pengyue Laboratory Animal Breeding Co., Ltd.), license number: SCXK (Lu) 20190003. Randomly divide 42 male KM mice into 6 groups, with 7 mice in each group. Shave the hair on the back of the mice locally in an area of 2 cm × 2 cm, and wash it with 8% sodium sulfide. Fix two series-connected 8-watt UVB lamps 20 cm away from the back of the mice, with an ultraviolet radiation intensity of 0.53 mW / cm², once a day, for 10 min each time. Administer the drug 30 min before each irradiation, and perform intragastric administration to the mice at 0.1 ml / 10 g, as shown in Table 2. The experimental mice are raised in the Animal Center of Haoyuan, Qingdao University, with a light cycle of (12L:12 D), 23 - 24 °C, and a relative humidity of 60 - 65%. During the whole experimental process, the diet and drinking water of each group of mice are not restricted. At the same time, all mice are fasted for 12 h before sampling without water restriction.

[0061] Select male SPF-grade KM mice (purchased from Jinan Pengyue Biotechnology Co., Ltd.), and randomly divide 42 male KM mice (28 - 30 g) into 6 groups, with 7 mice in each group. Shave the hair on the back of the mice locally in an area of 2 cm × 2 cm, and wash it with 8% sodium sulfide. Fix two series-connected 8-watt UVB lamps 20 cm away from the back of the mice, with an ultraviolet radiation intensity of 0.53 mW / cm², once a day, for 10 min each time. Administer the drug 30 min before each irradiation, and apply it at 0.1 g / 10 g cream, as shown in Table 3. As Figure 1As shown, after two weeks of ultraviolet irradiation, the mice's skin showed obvious redness and swelling, as well as slight wrinkles, thus establishing an acute ultraviolet-induced skin injury model. Throughout the experiment, the mice in each group were not restricted in their food or water intake. However, all mice were fasted for 12 hours prior to sample collection, although water was permitted.

[0062] Table 2: Drug administration regimens for mice in the gavage group

[0063]

[0064] Table 3: Dosing regimen for mice in the smear group

[0065]

[0066] After anesthetizing mice, blood was collected from the fundus. The blood was allowed to stand at room temperature for 30 minutes, then centrifuged at 7500 rpm / min for 30 minutes at 4°C. The supernatant was collected and stored at -20°C for subsequent parameter measurements. After anesthetizing mice, a 2 cm × 2 cm piece of skin from the back was quickly taken and fixed in tissue fixative or rapidly frozen in liquid nitrogen.

[0067] Example 4: HE staining of mouse dorsal skin

[0068] Skin tissue (3–5 μm thick) was excised using a cryostat and stained with an HE kit according to the instructions. In short, the samples were fixed in 10% (v / v) formalin fixative, thoroughly rinsed with phosphate-buffered saline (PBS), and stained with HE. Finally, the sections were scanned using a laser scanning confocal microscope.

[0069] Figure 2 The results showed that, compared with the normal group, the model group had significantly increased epidermal thickness, thicker stratum corneum, and an increased number of infiltrating inflammatory cells. Increased intercellular spaces indicated tissue edema. There was an increase in amorphous substances and disordered collagen fibers at the junction of the epidermis and dermis. After gavage and topical application of sea cucumber oil, the stratum corneum was significantly thinned, and the number of infiltrating inflammatory cells decreased. The junction of the epidermis and dermis became clearer, indicating that sea cucumber oil has a repairing effect on the skin. Therefore, sea cucumber oil can alleviate skin erythema, wrinkles, and epidermal hyperplasia caused by ultraviolet radiation, reduce the production of infiltrating inflammatory cells, tighten intercellular spaces to relieve tissue edema, and repair disordered collagen fibers.

[0070] Example 5: Determination of ROS content in mouse skin

[0071] Sterilize the tweezers and scissors needed for the experiment in an autoclave beforehand. Place a small amount of liquid nitrogen in an incubator. After removing the skin sample from the freezer, quickly place it in the incubator. Use tweezers to remove the sample from the liquid nitrogen and quickly place it into a sterile centrifuge tube pre-cooled with liquid nitrogen. Cut the sample into small pieces with scissors, add physiological saline according to the ratio, add 4 grinding beads to each EP tube, and grind into a homogenate using a grinder. Centrifuge the homogenate at 10000g / min for 15 minutes, collect the supernatant, and transfer it to a new EP tube for the next step of the experiment. Store the remaining sample frozen at -20℃. Thaw at room temperature, ensuring the sample is thawed evenly and thoroughly, avoiding repeated freeze-thaw cycles.

[0072] The kit is an ELISA kit (double-antibody one-step sandwich enzyme-linked immunosorbent assay). The microwells are pre-coated with reactive oxygen species (ROS) antibodies. Samples, standards, and HRP-labeled detection antibodies are added sequentially to the microwells, which are then incubated at 37°C. The samples are thoroughly washed after the assay. The assay uses TMB for color development; TMB turns blue under the catalysis of peroxidase, and yellow in an acidic environment. The intensity of the color reflects the amount of reactive oxygen species (ROS) in the sample. The absorbance (OD value) of the tissue sample is measured at 450 nm using a microplate reader to calculate the sample concentration.

[0073] The results are as follows Figure 3 As shown, the ROS content in the back skin of the model group significantly increased after UV irradiation, indicating that UV radiation penetrated the skin surface and induced excessive ROS production. In the gavage group, ROS content decreased after treatment with vitamin E and low- and medium-dose sea cucumber oil, but the difference was not statistically significant. P >0.05), the ROS content in the skin decreased significantly after high-dose gavage treatment ( P <0.05). The results of the topical application group and the gavage group were consistent. After treatment with vitamin E and low and medium doses of sea cucumber oil, the ROS content decreased, but the difference was not statistically significant. P >0.05), the ROS content in the skin decreased significantly after high-dose gavage treatment ( P <0.05).

[0074] Example 6: Measurement of Oxidative Stress Levels

[0075] Sterilize the tweezers and scissors needed for the experiment in an autoclave beforehand. Place a small amount of liquid nitrogen in an incubator. After removing the skin sample from the freezer, quickly place it in the incubator. Use tweezers to remove the sample from the liquid nitrogen and quickly place it into a sterile centrifuge tube pre-cooled with liquid nitrogen. Cut the sample into small pieces with scissors, add physiological saline according to the ratio, add 4 grinding beads to each EP tube, and grind into a homogenate using a grinder. Centrifuge the homogenate at 10000g / min for 15 minutes, collect the supernatant, and transfer it to a new EP tube for the next step of the experiment. Store the remaining sample frozen at -20℃. Thaw at room temperature, ensuring the sample is thawed evenly and thoroughly, avoiding repeated freeze-thaw cycles.

[0076] According to the instructions of the Beijing Solarbio Catalase (CAT) Activity Assay Kit, the absorbance at 240 nm of the reaction solution was measured as a function of time, and the CAT activity was calculated based on the change in absorbance.

[0077] According to the instructions of the Beijing Solarbio Glutathione peroxidase (GSH-Px / GPX) activity assay kit, the absorbance value at 412 nm was measured, and the GPX activity was calculated.

[0078] According to the instructions of the Beijing Solarbio malondialdehyde (MDA) content detection kit, the absorbance values ​​at 532nm and 600nm were measured to calculate the MDA content and detect the lipid oxidation level.

[0079] The results are as follows Figure 4 As shown, after ultraviolet radiation treatment, the activities of CAT and GPX in the model mice in the gavage group and the smear group were significantly reduced. P <0.01), after treatment with sea cucumber oil, the CAT activity in both the gavage group and the topical application group increased significantly ( P <0.01), GPX activity was significantly increased in the high-dose gavage group ( P <0.01), the GPX activity in the sea cucumber oil treatment group increased significantly after treatment. P <0.01). Compared with the normal group, the MDA content in both the gavage group and the smear group of mice was significantly increased ( P <0.01), the MDA content in both the gavage group and the topical application group treated with sea cucumber oil decreased significantly. P The value was <0.01), and it decreased in a dose-dependent manner, indicating that sea cucumber oil can improve the level of oxidative stress induced by ultraviolet radiation and repair acute photodamage to mouse skin.

[0080] Example 7: Determination of Skin Aging Level in Mice

[0081] Sterilize the tweezers and scissors needed for the experiment in an autoclave beforehand. Place a small amount of liquid nitrogen in an incubator. After removing the skin sample from the freezer, quickly place it in the incubator. Use tweezers to remove the sample from the liquid nitrogen and quickly place it into a sterile centrifuge tube pre-cooled with liquid nitrogen. Cut the sample into small pieces with scissors, add physiological saline according to the ratio, add 4 grinding beads to each EP tube, and grind into a homogenate using a grinder. Centrifuge the homogenate at 10000g / min for 15 minutes, collect the supernatant, and transfer it to a new EP tube for the next step of the experiment. Store the remaining sample frozen at -20℃. Thaw at room temperature, ensuring the sample is thawed evenly and thoroughly, avoiding repeated freeze-thaw cycles.

[0082] Hydroxyproline is a component of collagen in the body. Measuring hydroxyproline content determines tissue collagen content and skin aging levels. Weigh approximately 0.2g of sample into a glass tube, and cut the tissue into small pieces for digestion. Loosen the cap slightly. Add 2mL of extraction buffer, boil or bake at 110℃ for 2 to 6 hours until no large visible clumps remain. After cooling, adjust the pH to 6-8 with approximately 1mL of 10mol / L NaOH (avoiding excessive acidity or alkalinity), then bring the volume to 4mL with distilled water. Finally, centrifuge at 16000rpm, 25℃ for 20min (if impurities remain after centrifugation, remove them by filtration). Collect the supernatant for analysis. Measure the absorbance at 560nm according to the instructions of the Beijing Solarbio Hydroxyproline (HYP) Content Detection Kit to calculate the hydroxyproline content.

[0083] The results are as follows Figure 5 As shown, compared with the normal group, the hydroxyproline content in the gavage group and the smear group was significantly decreased. P <0.01), after treatment with sea cucumber oil, the HYP content in the high-dose gavage group increased significantly ( P <0.05), while there were no significant changes in the low- and medium-dose gavage groups; the HYP content increased significantly in the medium- and high-dose topical application groups ( P <0.01), with no significant change at low doses. In conclusion, sea cucumber oil can inhibit UV-induced degradation of skin collagen and delay skin aging.

[0084] Example 8: Measurement of mRNA expression levels of inflammatory factors and the NRF2 pathway

[0085] 1. Extraction of total RNA from back skin tissue

[0086] Total RNA was extracted from mouse back skin tissue using a total RNA extraction kit, and the RNA purity, concentration, and integrity were determined.

[0087] 2. Reverse transcription of RNA

[0088] The RNA reverse transcription reaction system is shown in Table 4.

[0089] Table 4: PCR reaction system

[0090]

[0091] PCR reaction conditions: 42 ℃ for 30 min, followed by 85 ℃ for 5 s, and the resulting cDNA was stored at -20 ℃ for later use.

[0092] 3. Real-time quantitative PCR

[0093] Following the instructions for TransStart® Top Green qPCR SuperMix (+Dye II), add the reagents as shown in Table 5 to 100 μL eight-tube bundles, mix well, and centrifuge.

[0094] Table 5: qRT-PCR reaction system

[0095]

[0096] qPCR was performed using a Roche fluorescence spectrometer, with the following program settings:

[0097] ;

[0098] Using GADPH as an internal reference, Ct (2 -△△CT Calculate the expression level of the target gene.

[0099] Table 6: qRT-PCR primer sequences

[0100]

[0101] The results are as follows Figure 6 As shown, after ultraviolet radiation irradiation, the expression levels of TNF-α, IL-6, IL-1β, and COX-2 genes in the skin tissue of both the gavage group and the topical application group significantly increased. P <0.01) indicates that inflammation occurred in mice after exposure to ultraviolet radiation. The expression level significantly decreased after treatment with sea cucumber oil. P <0.01), and it is dose-dependent, with high doses showing the most significant downregulation of gene expression, suggesting the application of sea cucumber oil in the treatment of inflammation.

[0102] NRF2 pathway factor expression as follows Figure 7 As shown, compared with the normal group, the NRF2 expression in the gavage group and the smear group was significantly increased ( P <0.01 is because when cells are subjected to oxidative stress, NRF2 spontaneously responds to external signals by upregulating its own expression to enhance antioxidant capacity and resist damage to the body. After treatment with sea cucumber oil, NRF2 significantly increased compared to the model group ( P<0.01), indicating that sea cucumber oil can further activate NRF2, protecting cells from damage. Upon oxidative stress, the KEAP1-NRF2 binding is unstable, releasing NRF2 into the cell nucleus, where it binds to ARE and activates the transcription of the downstream antioxidant gene NQO1. Compared to the normal group, NQO1 expression was increased in both the gavage and topical application groups (model groups). P <0.01), after treatment with sea cucumber oil, the expression of NQO1 in the gavage group further increased in a dose-dependent manner ( P <0.01), the expression of mRNA increased in the low and medium dose groups, but the difference was not statistically significant. P >0.05), the high-dose topical group showed a significant increase ( P <0.01). A negative feedback mechanism exists between KEAP1 and NRF2; upregulation of NRF2 inhibits KEAP1 gene expression and promotes KEAP1 degradation. Therefore, KEAP1 expression was significantly reduced after sea cucumber oil treatment. P <0.01).

[0103] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A type of sea cucumber oil, characterized in that, The sea cucumber oil is an orange-yellow liquid oil with stable properties and a fishy smell. Its acid value is 27.8 mg / g, and its peroxide value is 0.67 nmol / kg. The sea cucumber oil contains 33.6% monounsaturated fatty acids and 10.8% polyunsaturated fatty acids, including 2.91% cis-5,8,11,14-eicosatetraenoic acid, 1.61% arachidonic acid, and 0.924% cis-4,7,10,13,16,19-docosahexaenoic acid. The method for preparing the sea cucumber oil includes the following steps: (1) Clean the sea cucumber intestines to remove impurities and set aside; (2) The cleaned sea cucumber intestines were freeze-dried and pulverized; the freezing temperature was -50℃, the vacuum degree was 1Pa, and the final freeze-drying temperature of the sample was 25℃; and the sea cucumber intestines were pulverized to 80 mesh after freeze-drying. (3) After soaking the crushed sea cucumber intestines, the solid and liquid are separated to obtain the liquid; the crushed sea cucumber intestines are soaked in 95% ethanol; the ratio of the crushed sea cucumber intestines to the liquid of 95% ethanol is 1:8~15, the soaking temperature is 25-30℃, and the soaking time is 16~20h. (4) Extract the liquid from step (3), add anhydrous sodium sulfate to the collected upper layer solution, filter, and evaporate the organic phase obtained by rotary evaporation to obtain sea cucumber oil. The extraction is carried out using petroleum ether, wherein petroleum ether is added in a volume ratio of petroleum ether to ethanol of 1:

1.

2. The application of the sea cucumber oil according to claim 1 in the preparation of pharmaceuticals or cosmetics for repairing ultraviolet radiation damage, characterized in that, When the sea cucumber oil is used to prepare internal medicines, the effective dosage for repairing UV radiation damage is 2g / kg-8g / kg; when the sea cucumber oil is used to prepare external medicines or cosmetics, the effective addition amount for repairing UV radiation damage is 0.5%-2% of the volume of the external medicine or cosmetic; the sea cucumber oil can alleviate skin erythema, wrinkles, and epidermal hyperplasia caused by UV radiation, reduce the production of infiltrating inflammatory cells, tighten intercellular spaces to relieve tissue edema, and repair disordered collagen fibers; it can inhibit UV-induced ROS generation, increase the activity of catalase and glutathione peroxidase, reduce the degree of lipid peroxidation in the body, and improve the level of oxidative stress in the body; it can significantly increase the content of hydroxyproline in the skin, inhibit UV-induced degradation of skin collagen, and improve skin photoaging caused by UV radiation.

3. The application according to claim 2, characterized in that, The topical medication includes sea cucumber oil cream; the specific preparation steps of the sea cucumber oil cream are as follows: a stearic acid, glyceryl monostearate, ethylparaben, and liquid paraffin are mixed to prepare an oil phase; a Tween 80, glycerin, and water are mixed to prepare an aqueous phase; the oil phase and the aqueous phase are heated to 80°C, sea cucumber oil is added to the oil phase, and the oil phase is slowly added to the aqueous phase while stirring. When the emulsification reaches 50°C, stirring is stopped to obtain the sea cucumber oil cream.

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