A composition comprising docosahexaenoic acid, erucic acid and astaxanthin and uses thereof

Abstract

The present application relates to the field of food technology, and provide a kind of composition comprising docosahexaenoic acid, bird's nest acid and astaxanthin and its application, provide the composition including 1 bird's nest acid, 0.01~1 docosahexaenoic acid and 0.001~0.2 astaxanthin, the composition is regulated by the regulation of relevant genes, realizes the regulation of sleep.

Description

Technical Field

[0001] This invention relates to the field of food technology, and in particular to a composition comprising docosahexaenoic acid, sialic acid and astaxanthin and its application. Background Technology

[0002] We spend about one-third of our lives sleeping, and sleep quality is closely related to physical and mental health. In the fast-paced modern urban life, chronic stress, anxiety, depression, malnutrition, and other internal and external factors severely impact sleep quality. Sleep disorders can lead to various health problems, including obesity, diabetes, and hypertension. It has been reported that one of the main contributions of sialic acid to human health and nutrition is its ability to improve brain development and cognition in mammals. It is an important component of neurotrophic factors such as gangliosides and can improve brain function by modifying nerve adhesion cells. Therefore, based on the aforementioned problems, this invention provides a research scheme for improving sleep problems.

[0003] Current market products containing active ingredients have significant limitations: Firstly, their efficacy is limited; for example, astaxanthin preparations only target skin anti-oxidation, DHA products focus on cognitive enhancement, and sleep products rely on risky ingredients like cannabidiol (CBD) or traditional Chinese medicine with low absorption rates. Secondly, their mechanisms of action are unclear: existing products often rely on physiological indicators (such as SOD activity and cell viability) without validating pathways at the gene expression level, resulting in efficacy claims lacking molecular biological basis. More importantly, there is currently no synergistic solution that simultaneously addresses skin repair and sleep regulation. Summary of the Invention

[0004] This invention provides a composition containing docosahexaenoic acid, sialic acid, and astaxanthin, and its application, to address the shortcomings of existing oral beauty products, such as single efficacy and compliance risks associated with sleep-improving ingredients. This ternary compound system demonstrates at the molecular and biochemical levels that it can achieve effects such as sleep regulation and improved sleep quality. Furthermore, this composition also has synergistic potential in promoting collagen production, thus breaking through the barrier of single efficacy.

[0005] In a first aspect, the present invention provides a composition comprising, by weight parts: 1 part sialic acid, 0.01 to 1 part docosahexaenoic acid and 0.001 to 0.2 parts astaxanthin.

[0006] Sialic acid (SA) is the core active ingredient in bird's nest. Studies have shown that SA can regulate neurotransmitter balance, particularly promoting the synthesis and function of serotonin (5-HT). Serotonin is a precursor to melatonin, a key hormone regulating circadian rhythms and inducing sleep. Based on the synergistic effects of these three factors, this application hypothesizes that SA may indirectly inhibit the activity of the hypothalamic arousal center and reduce the expression of FOS in related neurons by enhancing serotonergic signaling, thereby promoting sleep.

[0007] Astaxanthin is a potent fat-soluble antioxidant that can effectively scavenge free radicals, and in this application, it was found that it can indirectly affect sleep.

[0008] DHA is the most abundant omega-3 long-chain polyunsaturated fatty acid in the brain and retina, and an important component of neuronal cell membrane phospholipids. Sufficient DHA levels help maintain cell membrane fluidity and signal transduction function. Based on the synergistic effects of these three factors, this application hypothesizes that DHA may indirectly affect the activity of sleep-related neural circuits by optimizing the membrane receptor environment and regulating neurotransmitter release, thereby influencing the FOS expression of wakefulness-promoting neurons.

[0009] The 0.01 to 1 part docosahexaenoic acid mentioned in this invention can be any value or a range of values ​​from 0.01 parts, 0.05 parts, 0.1 parts, 0.5 parts, 0.6 parts, 0.7 parts, 0.8 parts, 0.9 parts, and 1.0 parts.

[0010] The 0.001 to 0.2 parts of astaxanthin mentioned in this invention can be any value or a range of values ​​from 0.001 parts, 0.005 parts, 0.007 parts, 0.10 parts, 0.11 parts, 0.12 parts, 0.13 parts, 0.14 parts, 0.15 parts, 0.16 parts, 0.17 parts, 0.18 parts, 0.19 parts, and 0.20 parts.

[0011] Experimental verification shows that, under the dosage described above in this invention, a better anti-glycation effect can be obtained. Preferably, the mass ratio of sialic acid, docosahexaenoic acid and astaxanthin is 1:(0.05~1):(0.001~0.2).

[0012] The composition provided by the present invention comprises 1 part sialic acid, 0.05 to 0.5 parts docosahexaenoic acid and 0.007 to 0.2 parts astaxanthin.

[0013] The composition provided by the present invention specifically comprises: 1 part sialic acid, 0.05 parts docosahexaenoic acid and 0.007 parts astaxanthin.

[0014] Secondly, the present invention also provides the use of the composition in the preparation of fos gene repression products.

[0015] Furthermore, this application can be used to: promote sleep; c-Fos belongs to the immediate early gene (IEG) family, a nuclear protein encoded by an early response gene. It is enriched and expressed in neurons, participating in signal transduction and regulation of important brain functions. When neurons are stimulated or activated, the c-Fos gene is rapidly induced to express, thus serving as a marker of neuronal activation. By detecting c-Fos expression levels, we can understand the activation state of neurons, and further study neuronal excitability, synaptic transmission, and the occurrence and development of neuropsychiatric diseases. Based on these characteristics, various studies have successfully linked many specific behaviors to specific brain regions, making c-Fos a popular marker of neural activity.

[0016] Studies have shown that the immediate early-onset gene c-Fos is strongly induced by spontaneous and forced wakefulness in many brain regions. In regions involved in the regulation of wakefulness, c-Fos gene expression increases significantly after drug stimulation or sleep deprivation, particularly in the medial preoptic area, where c-fos expression is causally related to sleep regulation (Chiara Cirelli, Maria Pompeiano et al. 1995, M Pompeiano, C Cirelli et al. 1995, Guohong Cai, Yifan Lu et al. 2022). These findings indicate that c-Fos gene expression is closely related to wakefulness and sleep states (Leonard M. Eisenman 2002), especially given the consistent finding in experiments across several species that c-Fos gene expression is very low in most brain regions during sleep (Chiara Cirelli 2000).

[0017] Therefore, the c-fos gene is suitable as a marker indicator for the calming and sleep-promoting effects of the composition of the present invention. By measuring the expression level of the c-fos gene, the effects of different substances and compositions on sleep can be obtained.

[0018] Thirdly, the present invention also provides the use of the composition in the preparation of sleep-promoting products.

[0019] The present invention also provides a food composition for anti-glycation, comprising the composition described above.

[0020] According to the food composition provided by the present invention, the composition accounts for more than 10% by mass in the food composition.

[0021] According to the food composition provided by the present invention, the docosahexaenoic acid is added in the form of DHA algal oil; preferably, the content of docosahexaenoic acid in the DHA algal oil is greater than or equal to 20%.

[0022] And / or, the astaxanthin is added in the form of algal oil; preferably, the algal oil is Haematococcus pluvialis, and the astaxanthin content in the Haematococcus pluvialis oil is greater than or equal to 5%.

[0023] The DHA algal oil described in this invention contains docosahexaenoic acid (DHA) of 20% or more, for example, any value or a range of values ​​from 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, and 60%.

[0024] The astaxanthin content in the Haematococcus pluvialis oil described in this invention is greater than or equal to 5%, for example, it can be any value or a numerical range composed of 5%, 10%, 15%, 20%, 25%, and 30%.

[0025] Experiments have shown that adding the anti-glycation active ingredient of the present invention in the form of the above-mentioned raw materials does not weaken the interaction between the components in the specific ratio of the present invention, and the above-mentioned raw materials can greatly reduce the cost of the food composition of the present invention and the stability of product quality.

[0026] The food composition provided by the present invention further includes: one or more of plant extracts, antioxidants, and emulsifiers; Preferably, the plant extract is selected from one or more of the following: long-stalked almond oil, tomato seed oil, maple seed oil, safflower seed oil, Dunaliella salina and its extract, inulin, grape seed extract, spearmint extract, licorice extract, ginger extract, Rhodiola rosea extract, and rosemary extract. Preferably, the antioxidant is selected from one or more of D-isoascorbic acid and its sodium salt, ascorbic acid, sodium ascorbate, calcium ascorbate, phospholipids, sodium lactate, tea polyphenols, and vitamin E; Preferably, the emulsifier is selected from one or more combinations of glycerides, sorbitol esters, xylitol esters, sucrose esters and propylene glycol esters.

[0027] In one specific embodiment, the plant extract consists of tomato seed oil and grape seed extract, more preferably, it consists of tomato seed oil and grape seed extract in a mass ratio of 62:5.

[0028] In one specific implementation, the antioxidant is ascorbic acid.

[0029] In one specific implementation, the emulsifier is a mono- and diglyceride fatty acid ester.

[0030] The addition of the aforementioned plant extracts, food additives, and other substances is primarily used to adjust the taste, consumer appeal, and stability of the food composition of this invention. Experimental verification has shown that these substances do not adversely affect the synergistic effect between the components in the specific proportions described above.

[0031] The food composition provided by the present invention comprises, by weight parts: 25-220 parts DHA algal oil, 120-130 parts sialic acid, 7-130 parts Haematococcus pluvialis oil, 0-100 parts plant extract, 0-10 parts antioxidant, and 0-5 parts emulsifier. The 25-220 parts of DHA algal oil mentioned in this invention can be any value or a range of values ​​from 25 parts, 50 parts, 100 parts, 180 parts, 185 parts, 190 parts, 195 parts, 200 parts, 205 parts, 210 parts, 215 parts, and 220 parts.

[0032] The 120-130 parts of sialic acid mentioned in this invention can be any value or a range of values ​​among 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, and 130 parts.

[0033] The 7-130 parts of Haematococcus pluvialis oil mentioned in this invention can be any value or a range of values ​​from 7, 10, 20, 50, 80, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, and 130 parts.

[0034] Preferably, the food composition comprises, by weight parts: 25 parts DHA algal oil, 125 parts sialic acid, 7.5 parts Haematococcus pluvialis oil, 67 parts plant extracts, 2.5 parts nutritional fortifiers, and 15.5 parts food additives.

[0035] Preferably, the food composition comprises, by weight parts: 200 parts DHA algal oil, 125 parts sialic acid, 120 parts Haematococcus pluvialis oil, 67 parts plant extracts, 2.5 parts nutritional fortifiers and 15.5 parts food additives.

[0036] In one specific embodiment, the plant extract may consist of 62 parts tomato seed oil and 5 parts grape seed extract.

[0037] Fourthly, the present invention also provides a capsule, the contents of which comprise the food composition described above.

[0038] The capsule wall material of this invention effectively protects the stability of the contents, preventing degradation, inactivation, or adverse reactions due to external factors such as light and high temperature. Furthermore, the wall material can be designed to have specific release properties to achieve better absorption, depending on product requirements. Optional wall materials include gelatin, sodium alginate, and other plant polysaccharides. Other excipients, such as chelating agents and binders, can also be added as needed.

[0039] According to the capsule provided by the present invention, the contents include 200 parts of DHA algal oil, 125 parts of sialic acid, 120 parts of Haematococcus pluvialis oil, 62 parts of tomato seed oil, 5 parts of grape seed extract, 2.5 parts of ascorbic acid and 15.5 parts of mono- and diglyceride fatty acids.

[0040] This invention provides a composition containing docosahexaenoic acid, sialic acid, and astaxanthin, and its application in regulating sleep. This composition regulates sleep and improves sleep quality through mechanisms such as downregulating fos gene expression and upregulating NPAS3 gene expression. Furthermore, it can also regulate collagen-promoting and repair-related genes COL24A1, FGF2, and FGF5 to achieve the dual functions of promoting collagen production and regulating sleep. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0042] In a specific embodiment of the present invention, a composition is first provided, comprising, by mass parts: 1 part sialic acid, greater than or equal to 0.01 parts and less than 0.5 parts docosahexaenoic acid and 0.001 to 0.2 parts astaxanthin.

[0043] In a specific embodiment of the present invention, a composition is also provided, comprising, by mass parts: 1 part sialic acid, greater than or equal to 0.05 parts and less than 0.5 parts docosahexaenoic acid and 0.007 to 0.2 parts astaxanthin.

[0044] In a specific embodiment of the present invention, a composition is also provided, comprising, by mass parts: 1 part sialic acid, 0.05 parts docosahexaenoic acid and 0.007~0.0075 parts astaxanthin; or comprising: 1 part sialic acid, 0.65 parts docosahexaenoic acid and 0.1~0.11 parts astaxanthin.

[0045] In a specific embodiment of the present invention, the use of the composition in the preparation of a product for regulating the expression of sleep-related genes is also provided, wherein the composition is the composition described above or a composition comprising the following components: The composition, by weight parts, includes: 1 part sialic acid, 0.5 to 1 part docosahexaenoic acid, and 0.001 to 2 parts astaxanthin; preferably, by weight parts, it includes: 1 part sialic acid, 0.5 to 1 part docosahexaenoic acid, and 0.01 to 0.3 parts astaxanthin. The adjustment includes one or more of the following combinations: (1) fos gene repression; (2) Upregulate the NPAS3 gene.

[0046] In a specific embodiment of the present invention, an application of the composition in the preparation of a sleep-promoting product is also provided, wherein the composition is the composition described above or a composition comprising the following components: The composition comprises, by weight parts: 1 part sialic acid, 0.5 to 1 part docosahexaenoic acid and 0.001 to 2 parts astaxanthin; preferably, by weight parts: 1 part sialic acid, 0.5 to 1 part docosahexaenoic acid and 0.01 to 0.3 parts astaxanthin.

[0047] In some specific embodiments, the application is used to promote sleep; the sleep promotion includes one or more of the following combinations: (1) Improve the sleep onset rate of subjects; (2) Shorten the sleep latency of the subjects; (3) Prolong the sleep duration of the subjects; (4) Reduce the number of times the subjects wake up at night; (5) Improve the continuity of deep sleep in subjects.

[0048] In a specific embodiment of the present invention, an application of the composition in the preparation of products that promote collagen production, resist skin aging, or promote skin repair is also provided, wherein the composition is the composition described above or a composition comprising the following ingredients: The composition comprises, by weight parts: 1 part sialic acid, 0.5 to 1 part docosahexaenoic acid and 0.001 to 2 parts astaxanthin; preferably, by weight parts: 1 part sialic acid, 0.5 to 1 part docosahexaenoic acid and 0.01 to 0.3 parts astaxanthin.

[0049] In a specific embodiment of the present invention, a food composition is also provided, comprising the composition described above or a composition comprising the following ingredients: The composition, by weight parts, includes: 1 part sialic acid, 0.5 to 1 part docosahexaenoic acid, and 0.001 to 2 parts astaxanthin; preferably, by weight parts, it includes: 1 part sialic acid, 0.5 to 1 part docosahexaenoic acid, and 0.01 to 0.3 parts astaxanthin. The composition constitutes more than 10% of the total mass of the food composition.

[0050] In some specific embodiments, the food composition further includes one or more of the following: plant extracts, antioxidants, and emulsifiers; Preferably, the plant extract is selected from one or more of the following: long-stalked almond oil, tomato seed oil, maple seed oil, safflower seed oil, Dunaliella salina and its extract, inulin, grape seed extract, spearmint extract, licorice extract, ginger extract, Rhodiola rosea extract, and rosemary extract. Preferably, the antioxidant is selected from one or more of D-isoascorbic acid and its sodium salt, ascorbic acid, sodium ascorbate, calcium ascorbate, phospholipids, sodium lactate, tea polyphenols, and vitamin E; Preferably, the emulsifier is selected from one or more combinations of glycerides, sorbitol esters, xylitol esters, sucrose esters and propylene glycol esters.

[0051] In a specific embodiment of the present invention, a capsule is also provided, the contents of which include the food composition described above.

[0052] The technical solution of the present invention will be verified in detail below with specific implementation examples.

[0053] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0054] The DHA algal oil in the following embodiments of the present invention is a commercially available product, wherein the content of docosahexaenoic acid is 40%.

[0055] The Haematococcus pluvialis oil in the following embodiments of the present invention is a commercially available product, wherein the astaxanthin content is 10%.

[0056] The sialic acid in the following embodiments of the present invention is a commercially available product, wherein the purity of the sialic acid is 98%.

[0057] Example 1 Composition This embodiment provides a composition comprising, by weight, 1 part sialic acid, 0.05 parts docosahexaenoic acid and 0.007 parts astaxanthin.

[0058] Example 2 Composition This embodiment provides a composition comprising, by weight, 1 part sialic acid, 0.65 parts docosahexaenoic acid and 0.11 parts astaxanthin.

[0059] Example 3 Composition This embodiment provides a composition comprising, by mass parts, 1 part sialic acid, 1 part docosahexaenoic acid and 0.3 parts astaxanthin.

[0060] Example 4 Food Composition This embodiment provides a food composition for anti-glycation, which, by weight, consists of 25 parts DHA algal oil, 125 parts sialic acid, 7 parts Haematococcus pluvialis oil, 62 parts tomato seed oil, 5 parts grape seed extract, 2.5 parts vitamin C, and 15.5 parts mono- and diglyceride fatty acid esters.

[0061] Example 5 Food Composition This embodiment provides a food composition for anti-glycation, which, by weight, consists of 200 parts DHA algal oil, 125 parts sialic acid, 120 parts Haematococcus pluvialis oil, 62 parts tomato seed oil, 5 parts grape seed extract, 2.5 parts vitamin C, and 15.5 parts mono- and diglyceride fatty acid esters.

[0062] Example 5 Capsules This embodiment provides a capsule comprising a wall material and contents; the contents are the food composition of Example 3.

[0063] Test Example 1: RT-qPCR Experiment 1. SH-SY5Y neural cell culture (1) Remove or discard the original culture medium in the culture bottle.

[0064] (2) Wash the cells twice with PBS.

[0065] (3) Add 1 mL of 0.25% EDTA-containing trypsin solution to the bottle.

[0066] (4) Place the culture flask in a 37°C incubator or at room temperature (25°C) for digestion. After 1-2 minutes, place the culture flask under a microscope for observation. When the cytoplasm shrinks and the intercellular spaces increase, add 2 mL of complete culture medium to stop the digestion.

[0067] (5) Use a pipette to gently blow the cells off the bottle wall repeatedly to form a cell suspension. Centrifuge at 1000 rpm for 5 min. After centrifugation, remove the supernatant and resuspend in 1 mL of complete culture medium.

[0068] (6) After counting with a counting plate, the inoculate into new culture bottles and place them in an incubator with 5% CO2 and a temperature of 37°C for incubation.

[0069] 2. Incubation of SH-SY5Y cell raw materials (1) SH-SY5Y cells in logarithmic growth phase were digested using standard methods, and after counting, the cell density was adjusted to 5 × 10⁶ cells using complete culture medium (DMEM + 15% FBS + 1% P / S). 5 cells / mL.

[0070] (2) Add 2 mL of cell suspension (approximately 1 × 10⁻⁶) to each well of a 6-well plate. 6 (cells / well), cultured overnight at 37°C with 5% CO2 to allow cells to adhere and grow to 70-80% confluence.

[0071] (3) Discard the old culture medium and wash each well once with 1 mL PBS.

[0072] (4) Group-based drug administration: Negative control group: 2 mL of complete DMEM medium; Sialic acid (SA) group (with added SA): SA was added to the culture medium to a final concentration of 0.5%; DHA group (with added DHA): DHA was added to the culture medium to a final concentration of 0.025%; Astaxanthin group (with added astaxanthin): Astaxanthin was added to the culture medium to a final concentration of 0.0036%; Example 1 group (with the composition of Example 1 added): The culture medium was supplemented with a final concentration of 0.16% SA, 0.0083% DHA and 0.0012% astaxanthin; Designed based on preliminary experimental results.

[0073] (5) Incubate at 37℃ and 5% CO2 for 48 hours.

[0074] 3. RNA extraction Total RNA extraction from cells and tissues (Trizol method) Total RNA extraction from cells and tissues was performed using the Trizol lysis method. EP tubes and pipette tips were RNase-free, and the process was conducted in a fume hood. The specific steps are as follows: (1) Sample preparation: After incubation, the SH-SY5Y cell samples were washed three times with PBS, and 600 μL of Trizol was added to each well to lyse the cells. The cells were collected by pipetting and adhering.

[0075] (2) After the tissue / cells are fully lysed, transfer them into a 1.5 mL RNase-free EP tube, add 200 µL of chloroform at a ratio of 200 µL chloroform / 600 µL Trizol, mix by inverting for 15 s, let stand at room temperature for 5 min, and centrifuge at 12000 rpm at low temperature (4 °C) for 15 min.

[0076] (3) After centrifugation, the sample will show three layers: the bottom layer is the organic phase, the middle layer is the protein phase, and the top layer is the colorless aqueous phase (mainly containing RNA). Carefully aspirate the top aqueous phase into a new RNase-free EP tube (do not aspirate the middle white layer).

[0077] (4) Add an equal volume of isopropanol, gently shake up and down to mix 6-8 times, place on ice for 10 min, and centrifuge at 12000 rpm and low temperature (4℃) for 15 min.

[0078] (5) Discard the supernatant; the white, transparent precipitate is RNA. Add 1 mL of pre-cooled 75% ethanol washing buffer (prepared with DEPC water) to each tube, gently invert to wash the precipitate thoroughly, and centrifuge at 12,000 rpm at low temperature (4°C) for 5 min. Repeat twice.

[0079] (6) After discarding the supernatant, place it in a fume hood to dry for about 30 minutes.

[0080] (7) Add an appropriate amount of DEPC water according to the amount of RNA precipitation, and incubate it in a 55°C water bath for 7 minutes until it is fully dissolved in DEPC water.

[0081] (8) Determine the RNA concentration and purity, requiring the OD260 / OD280 ratio to be between 1.8 and 2.0. Take about 500 ng of RNA for reverse transcription experiments, and store the remaining RNA at -80℃ for subsequent experiments.

[0082] 4. Reverse transcription RNA reverse transcription was performed according to the instructions of the Vazyme kit, as detailed in Table 1 below.

[0083] Table 1

[0084] To achieve a final RNA concentration of 1000 μg / mL, determine the amount of RNA sample and ddH2O to be added.

[0085] Gently pipette to mix, then incubate at 50°C for 15 min and 85°C for 5 s using a PCR instrument.

[0086] The reaction product can be used immediately for qPCR, or stored at -20°C and used within six months; for long-term storage, it is recommended to aliquot and store at -80°C. Repeated freeze-thaw cycles should be avoided with cDNA.

[0087] Total RNA was isolated from transfected cells using Trizol reagent (Invitrogen) according to the manufacturer's instructions, as described above. Reverse transcription polymerase chain reaction (RT-PCR) was performed using a cDNA reverse transcription kit (HiScript II Q RT SuperMix for qPCR (+gDNA wiper)). Primers for detecting spliced ​​and unspliced ​​mRNA were designed based on previous studies and are listed in the table below. The levels of spliced ​​and unspliced ​​mRNA were detected using the SYBR Green Master Mix Kit (Vazyme, China), and the fold change was calculated using the 2-ΔΔCt method. GAPDH (human) was used as an endogenous control. The reaction system and conditions involved are shown in Tables 2-3 below. The test results are shown in Table 4.

[0088] Table 2 Reaction System

[0089] Table 3 Reaction conditions

[0090] Table 4

[0091] in, This indicates a significant difference, with a statistical significance level of p < 0.05. This indicates a highly significant difference, with a significance level of p < 0.001.

[0092] The data above show that when astaxanthin is combined with DHA and SA, it can significantly reduce the content of the fos gene (p < 0.001), indicating that the combination of astaxanthin with DHA and SA forms a new mechanism of action and produces a synergistic effect.

[0093] Test Example 2 Transcriptome Experiment The transcriptome experiment involved three sets of experiments: the first was to investigate the effect of the composition on genes related to sleep, using SH-SY5Y neural cell culture; the second was to investigate the effect of the composition on genes promoting collagen production, using HSF cell culture; and the third was to investigate the effect of the composition on genes related to repair efficacy, using Hcat cell culture.

[0094] 1. Incubation of SH-SY5Y cell raw materials was performed in the same manner as in test case 1, step 2. 2. The incubation steps (1) to (3) of HSF cell raw materials are the same as step 2 of test case 1, except for the drug addition step: Negative control group: 2 mL of complete DMEM medium; Sialic acid (SA) group (with added SA): SA was added to the culture medium to a final concentration of 0.1%; DHA group (with added DHA): DHA was added to the culture medium to a final concentration of 0.065%; Astaxanthin group (with added astaxanthin): Astaxanthin was added to the culture medium at a final concentration of 0.011%; Example 2 group (with the composition of Example 2 added): The culture medium was supplemented with a final concentration of 0.03% SA, 0.022% DHA and 0.004% astaxanthin; The incubation steps are as follows: incubate at 37℃ and 5% CO2 for 24 hours.

[0095] 3. The Hacat cell raw material incubation steps (1) to (2) are the same as step 2 of test example 1. The difference is that after the cells are cultured, 0.1% hydrogen peroxide is added to create a model for 2 hours. Then the old culture medium is discarded, and each well is gently washed with 1 mL PBS before the drug is added.

[0096] The steps for administering the medication are as follows: Negative control group: 2 mL of complete DMEM medium; Sialic acid (SA) group (with added SA): SA was added to the culture medium to a final concentration of 0.01%; DHA group (with added DHA): DHA was added to the culture medium to a final concentration of 0.01%; Astaxanthin group (with added astaxanthin): Astaxanthin was added to the culture medium at a final concentration of 0.003%; Example 3 group (with the composition of Example 3 added): The culture medium was added with a final concentration of 0.003% SA, 0.003% DHA and 0.001% astaxanthin; The incubation steps are as follows: incubate at 37℃ and 5% CO2 for 24 hours.

[0097] 3. Experimental Procedure The experimental workflow for transcriptome sequencing includes several steps: RNA extraction, RNA detection, library construction, and sequencing.

[0098] 3.1 RNA Sample Detection High-quality RNA is fundamental to the success of the entire project. To ensure RNA quality, samples are tested using the following methods, and library construction can only proceed after the RNA has passed the tests.

[0099] (1) Qubit 4.0 fluorometer / MD microplate reader: high-precision measurement of RNA concentration; (2) Qsep400 Bioanalyzer: Accurately detects RNA integrity.

[0100] 3.2 Document Library Construction Eukaryotic transcriptome mRNA is obtained by utilizing the structural feature of polyA tails found in most eukaryotic mRNAs. Oligo (dT) magnetic beads are used to enrich mRNAs with polyA tails. Subsequently, fragmentation buffer is used to break the RNA into short fragments. Using these short fragments as templates, first-stranded cDNA is synthesized using six-base random hexamers. Then, buffer, dNTPs (dTTP, dATP, dGTP, and dCTP), and DNA polymerase are added to synthesize second-stranded cDNA. The double-stranded cDNA is then purified using DNA purification beads. The purified double-stranded cDNA undergoes end repair, A-tailing, and ligation with sequencing adapters. Fragment size selection is then performed using DNA purification beads, and finally, PCR enrichment is performed to obtain the final cDNA library.

[0101] 3.3 Document Quality Inspection After the library is constructed, the library quality is tested. Sequencing can only be performed after the test results meet the requirements. The test methods are as follows: (1) Use the Qubit dye method for preliminary quantification; (2) Use a fragment analyzer to test the insert size of the library. The next step of the experiment can only be performed after the insert size meets the expectations.

[0102] 3.4 Sequencing After the libraries pass the library inspection, different libraries are pooled according to the target amount of data to be sequenced, then loaded onto the sequencing chip, and sequenced using a high-throughput sequencer.

[0103] 3.5 Bioinformatics Analysis Workflow The transcriptome data was filtered to obtain Clean Data, which was then aligned with a specified reference genome to obtain Mapped Data. Alternative splicing analysis was performed on the resulting Mapped Data. Differential expression analysis, functional annotation of differentially expressed genes, and functional enrichment were conducted based on gene expression levels in different samples or sample groups. Data analysis revealed that the NPAS3 gene, which regulates sleep, showed significant differences in expression across groups. Additionally, the COL24A1, FGF2, and FGF5 genes, which regulate collagen production, also showed significant differences in expression across groups. Except for the negative control group, the p-values ​​for the relative content data in all experimental groups were <0.05. These p-values ​​only indicate the reliability of the transcriptome experimental results.

[0104] The relative results of the NPAS3 gene, which plays a regulatory role in sleep, in SH-SY5Y cells are shown in the table below.

[0105] Table 5

[0106] The association between the NPAS3 gene and sleep is primarily manifested through its structural homology and functional link with the core clock gene NPAS2. NPAS2 is a core circadian rhythm transcription factor in the brain (especially in the prefrontal cortex) that functions parallel to the CLOCK protein, directly regulating gene expression related to the sleep-wake cycle. NPAS3 and NPAS2 belong to the same bHLH-PAS protein family and are highly similar in protein structure. Therefore, NPAS3 is thought to indirectly participate in the regulation of downstream genes of the circadian rhythm through similar molecular mechanisms or by forming heterodimers with NPAS2. This relationship allows NPAS3 to act as a co-regulator, influencing the circadian rhythm stability of neural circuits (especially the cortical-hippocampal circuit involved in cognition and emotion), thereby exerting an indirect but important regulatory effect on sleep quality and structure.

[0107] The data above show that astaxanthin, DHA, and SA have a slight promoting effect on the expression of the NPAS3 gene; however, the combined effect of the three substances significantly increases the expression of the NPAS3 gene compared to the simple superposition or individual effects of each substance. This indicates that the composition improves sleep structure and quality by targeting and significantly upregulating the expression of sleep rhythm-related genes, addressing the root cause of regulating the brain's biological clock and circadian rhythm.

[0108] The relative results of the COL24A1 gene, which plays a regulatory role in promoting collagen production in HSF cells, are shown in the table below.

[0109] Table 6

[0110] COL24A1 encodes the α1 chain of type XXIV collagen, belonging to the non-fibrillary collagen (FACIT family). This protein is mainly distributed in bone, tendons, articular cartilage, and some nerve tissues, participating in the formation of the supramolecular network of the extracellular matrix. Its structure contains typical collagen domains (Gly-XY repeats) and non-collagenous insertion regions, playing a role in maintaining the spatial order of connective tissue and regulating the interaction between cells and the matrix.

[0111] The relative results of FGF2 and FGF5 genes, which play a regulatory role in promoting repair in Hacat cells, are shown in the table below.

[0112] Table 7

[0113] Table 8

[0114] Both FGF2 and FGF5 are members of the fibroblast growth factor family. FGF2 is a potent repair-promoting factor. It significantly stimulates fibroblast proliferation and angiogenesis, accelerates granulation tissue formation, and has a clear role in skin wound healing, tissue regeneration, and improving the skin barrier, making it a classic repair-related factor. FGF5 primarily regulates the hair follicle cycle. In terms of repair, inhibiting FGF5 expression can effectively prolong the hair follicle growth phase, thereby reducing abnormal hair loss and promoting hair growth, and is often used for repairing hair damage.

[0115] Transcriptomic results from fibroblasts and keratinocytes showed that astaxanthin, along with DHA and SA, had a slight promoting effect on the expression of COL24A1, FGF2, and FGF5 genes. The combination of these three substances significantly increased the expression of COL24A1, FGF2, and FGF5 genes compared to the individual effects of each substance. This indicates that the combination has strong potential in promoting collagen production and skin repair.

[0116] Test Example 3: Mouse Sleep Experiment Fifty healthy male ICR mice, 6 weeks old and weighing 20±2g, were acclimatized for one week under an environment of 24±2℃, 50%–60% relative humidity, 12 hours of alternating light and dark, and free access to water and food. They were then randomly divided into a control group (pure water) and four experimental groups: DHA group (0.9mg / day by gavage), SA group (0.9mg / day by gavage), astaxanthin group (0.9mg / day by gavage), and Example 2 group (0.9mg / day by gavage), with 10 mice in each group. The corresponding solutions were administered to the mice in each experimental group by gavage.

[0117] (1) Direct sleep test Each group was administered the drug once daily by gavage for 7 consecutive days. After the last administration, the mice's sleep status was observed to conduct a direct sleep test. The results showed that the number of mice falling asleep in each dose group and the blank group was 0, indicating that the samples had no direct hypnotic effect on mice at the doses set in the experiment.

[0118] (2) Sodium pentobarbital hypnosis test Each group was administered the drug once daily by gavage for 7 consecutive days. A subthreshold dose-induced hypnotic test using sodium pentobarbital was conducted 45–60 minutes after the last administration. Mice were intraperitoneally injected with sodium pentobarbital solution (36 mg / kg, injection volume 0.1 mL / 10 g), and the sleep rate of each group was recorded within 30 minutes, using the disappearance of the righting reflex as an indicator. The experimental results are shown in Table 7. Table 7 shows that the sleep rate in all extract groups was higher than that in the normal group, indicating that the extract can improve the sleep rate of mice. The combined extract group had the highest sleep rate, at 70%.

[0119] Table 9. Effects of the extract on the sleep rate of mice after subthreshold doses of sodium pentobarbital.

[0120] (3) Sodium pentobarbital sleep test Each group was administered the drug once daily by gavage for 21 consecutive days. A suprathreshold dose sleep test of sodium pentobarbital was conducted 45–60 minutes after the last administration. Mice were intraperitoneally injected with sodium pentobarbital solution (50 mg / kg, injection volume 0.1 mL / 10 g). The disappearance and recovery of the righting reflex were used as indicators, and the sleep latency and sleep duration were recorded. The experimental results are shown in Table 8. Table 8 shows that compared with the control group, the sleep latency of experimental group 2 and group SA was significantly shortened. Compared with the control group (55.83 ± 7.82) min, the sleep duration of all groups increased to varying degrees, but only group 2 showed significant differences in both sleep latency and sleep duration.

[0121] Table 10

[0122] Test Example 4: Pittsburgh Sleep Assessment The Pittsburgh Sleep Quality Index (PSQI) consists of 19 self-reported items and 5 peer-reported items. Only the self-reported items are scored. The 19 self-reported items are divided into 7 factors, each scored from 0 to 3, where "0" indicates no difficulty, "1" indicates mild difficulty, "2" indicates moderate difficulty, and "3" indicates severe difficulty. The sum of the scores for each factor component constitutes the total PSQI score, ranging from 0 to 21. A higher score indicates poorer sleep quality.

[0123] Ten participants with sleep quality problems were selected using the Pittsburgh Sleep Quality Index (PSE) (scores range from 15 to 21). Volunteers possessed 2-3 of the following characteristics: 1. Prolonged sleep latency: It takes more than 40 minutes to fall asleep; 2. Sleep maintenance disorder: waking up more than twice a night or waking up early in the morning; 3. Poor sleep quality: shallow sleep, frequent dreams; 4. Short total sleep time: usually less than 5 hours; 5. Daytime residual effects: feeling dizzy, lethargic, drowsy, and weak the next morning.

[0124] After taking the composition of Example 2 continuously for 30 days, sleep quality was recorded using a unified wristband. Data from day 0 and the last day were collected (if there were objective reasons (such as illness, work, etc.) that caused significant differences in sleep conditions compared to the usual day, data from adjacent days with sleep conditions consistent with the usual day were used for analysis, and so on). The analysis is as follows: (1) 80% of the subjects had an extended effective sleep time, and the average sleep time of all subjects increased from 5.3 hours to 6.11 hours; (2) 50% of the subjects had a shorter time to fall asleep, and the average time to fall asleep for all subjects decreased from 33.9 minutes to 28.7 minutes; (3) 60% of the subjects experienced a decrease in the average number of nighttime awakenings, and the average number of nighttime awakenings for all subjects decreased from 1.6 times to 0.6 times; (4) The percentage of REM sleep decreased in 70% of the subjects, and the percentage of REM sleep decreased from 32.1% to 28.2% for all subjects; (5) 50% of the subjects had improved deep sleep continuity scores, and the deep sleep continuity scores of all subjects increased from 64.5 to 70.1.

[0125] The results showed that the product prepared in Example 2 could increase the effective sleep time of the subjects, shorten the time it took for the subjects to fall asleep, reduce the average number of times the subjects woke up at night, reduce the proportion of REM sleep in the subjects, and improve the continuity of deep sleep in the subjects.

[0126] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composition, characterized in that, By weight, it includes: 1 part sialic acid, ≥0.01 parts and ≤0.5 parts docosahexaenoic acid, and 0.001~0.2 parts astaxanthin.

2. A composition, characterized in that, By weight, it includes: 1 part sialic acid, ≥0.05 parts and ≤0.5 parts docosahexaenoic acid, and 0.007~0.2 parts astaxanthin.

3. A composition, characterized in that, By weight, it includes: 1 part sialic acid, 0.05 parts docosahexaenoic acid and 0.007 to 0.0075 parts astaxanthin; or it includes: 1 part sialic acid, 0.65 parts docosahexaenoic acid and 0.1 to 0.11 parts astaxanthin.

4. The use of a composition in the preparation of a product for regulating the expression of sleep-related genes, characterized in that, The composition is the composition according to any one of claims 1 to 3 or a composition comprising the following components: The composition, by weight parts, includes: 1 part sialic acid, 0.5 to 1 part docosahexaenoic acid, and 0.001 to 2 parts astaxanthin; preferably, by weight parts, it includes: 1 part sialic acid, 0.5 to 1 part docosahexaenoic acid, and 0.01 to 0.3 parts astaxanthin. The adjustment includes one or more of the following combinations: (1) fos gene repression; (2) Upregulate the NPAS3 gene.

5. The use of a composition in the preparation of a sleep-promoting product, characterized in that, The composition is any one of claims 1 to 3 or a composition comprising the following components: The composition comprises, by weight parts: 1 part sialic acid, 0.5 to 1 part docosahexaenoic acid and 0.001 to 2 parts astaxanthin; preferably, by weight parts: 1 part sialic acid, 0.5 to 1 part docosahexaenoic acid and 0.01 to 0.3 parts astaxanthin.

6. The application according to claim 4 or 5, characterized in that, The application is used to promote sleep; the sleep promotion includes one or more of the following combinations: (1) Improve the sleep onset rate of subjects; (2) Shorten the sleep latency of the subjects; (3) Prolong the sleep duration of the subjects; (4) Reduce the number of times the subjects wake up at night; (5) Improve the continuity of deep sleep in subjects.

7. The use of a composition in the preparation of products that promote collagen production, combat skin aging, or promote skin repair, characterized in that, The composition is any one of claims 1 to 3 or a composition comprising the following components: The composition comprises, by weight parts: 1 part sialic acid, 0.5 to 1 part docosahexaenoic acid and 0.001 to 2 parts astaxanthin; preferably, by weight parts: 1 part sialic acid, 0.5 to 1 part docosahexaenoic acid and 0.01 to 0.3 parts astaxanthin.

8. A food composition, characterized in that, This includes the composition according to any one of claims 1 to 3, or a composition comprising the following components: The composition, by weight parts, includes: 1 part sialic acid, 0.5 to 1 part docosahexaenoic acid, and 0.001 to 2 parts astaxanthin; preferably, by weight parts, it includes: 1 part sialic acid, 0.5 to 1 part docosahexaenoic acid, and 0.01 to 0.3 parts astaxanthin. The composition constitutes more than 10% of the total mass of the food composition.

9. The food composition according to claim 8, characterized in that, Also includes: One or more of the following: plant extracts, antioxidants, and emulsifiers; Preferably, the plant extract is selected from one or more of the following: long-stalked almond oil, tomato seed oil, maple seed oil, safflower seed oil, Dunaliella salina and its extract, inulin, grape seed extract, spearmint extract, licorice extract, ginger extract, Rhodiola rosea extract, and rosemary extract. Preferably, the antioxidant is selected from one or more of D-isoascorbic acid and its sodium salt, ascorbic acid, sodium ascorbate, calcium ascorbate, phospholipids, sodium lactate, tea polyphenols, and vitamin E; Preferably, the emulsifier is selected from one or more combinations of glycerides, sorbitol esters, xylitol esters, sucrose esters and propylene glycol esters.

10. A capsule, characterized in that, The contents of the capsule include the food composition according to any one of claims 8 to 9.

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