Composition for improving NAD < + > level and application of composition in improvement of skin photoaging

Abstract

The invention belongs to the field of medicines and health foods, and particularly relates to a composition for improving the NAD < + > level and application of the composition in relieving skin light injury and light aging. The composition for improving the NAD < + > level comprises wheat polar fat and an oriental cherry extract, and can also comprise ergothioneine. All the components in the composition are reasonably matched and have a synergistic effect, the NAD < + > content of skin tissues and cells can be effectively increased, inflammatory factor generation caused by ultraviolet rays is effectively reduced, enrichment of active oxygen and advanced glycosylation end products is reduced, and light damage and light aging of the skin caused by the ultraviolet rays or blue light are relieved. The compound has a very good application prospect in products for preventing, relieving and / or treating light damage or / or light aging.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine and health food, and particularly relates to a composition for improving NAD+ level and application thereof in improving skin photoaging. BACKGROUND

[0002] NAD (Nicotinamide Adenine Dinucleotide) is also called coenzyme I, which is formed by nicotinamide, adenine and two riboses connected by phosphodiester bonds. NAD exists widely in various tissues of the body and is the most important electron carrier in cells. NAD has two forms, one is oxidized form NAD+, and the other is reduced form NADH. In a series of biological processes in cells, the two can be converted to each other.

[0003] NAD is a vital coenzyme in cells, which plays an important biological function in energy metabolism, activation of Sirtuins, participation in DNA damage repair and the like in cells. The most classic function of NAD+ is to participate in energy metabolism as a coenzyme, which plays a role of hydrogen and electron carrier in the mitochondrial respiratory chain and energy (ATP) production. In the catabolism of sugars, fats and amino acids, NAD+ is reduced to NADH by obtaining hydrogen ions (H + ) and electrons (e - ); NADH carries these electrons to the electron transport chain in mitochondria, and a large amount of ATP (cell energy currency) is produced through the process of oxidative phosphorylation. Sirtuins are a class of NAD+-dependent deacetylases, which are called "longevity proteins", and the activity of Sirtuins completely depends on the presence of NAD+. NAD+ acts as a coenzyme of Sirtuins, and provides electrons in the deacetylation reaction. Sirtuins remove the acetyl group on the substrate protein by consuming NAD+, so as to regulate the activity, stability and subcellular localization of the substrate protein. Participation in DNA damage repair is also an important biological function of NAD+. When DNA is damaged, a class of enzymes called PARPs (poly ADP ribose polymerase) are activated, which decompose NAD+ and add ADP-ribose chains (called PARylation) on the damaged DNA to recruit other repair proteins to complete DNA repair.

[0004] NAD+ levels in the body are regulated by multiple factors, with aging being the most significant factor contributing to the decline in NAD+ levels. This is primarily due to reduced NAD+ synthesis and increased consumption. NAD+ synthesis mainly occurs through two pathways: de novo synthesis and salvage synthesis. With age, the activity of key enzymes in the de novo synthesis pathway may decrease, leading to reduced NAD+ synthesis efficiency. Simultaneously, age-related factors such as chronic inflammation and accumulated DNA damage continuously activate NAD+-consuming enzymes, resulting in significant NAD+ depletion. Since DNA damage repair is one of the main pathways of NAD+ consumption, various external factors that easily cause DNA damage are also important factors leading to decreased NAD+ levels. For example, when ultraviolet radiation causes DNA damage to the skin (such as the formation of pyrimidine dimers), cells activate a DNA repair enzyme called PARP (poly-ADP-ribose polymerase) to repair this damage. PARP requires a large amount of NAD+ as a substrate to perform its repair function. A significant decrease in NAD+ levels can have a series of negative effects on skin cells, including impaired cellular energy metabolism, stunted cell proliferation and dysfunction, and accelerated photoaging of the skin. Studies have shown that NAD+ supplementation has a significant protective effect against UV-induced skin damage, including reducing oxidative stress, inflammation, DNA damage and apoptosis (Mengmeng Li et al., Int JPhysiol Pathophysiol Pharmacol, 2023; 15(2)41-49).

[0005] The cumulative damage to the skin caused by long-term ultraviolet (UV) exposure is mainly due to the overexpression of inflammatory factors, the accumulation of reactive oxygen species and advanced glycation end products (AGEs), which in turn lead to collagen degradation, premature skin aging, and a series of problems such as wrinkles, age spots, sagging, and changes in skin texture. UV radiation has become a major cause of skin damage and aging. By regulating the NAD+ content in skin cells and tissues, it is possible to effectively reduce oxidative stress, inflammation, DNA damage, and apoptosis, and inhibit photodamage and photoaging of the skin. Summary of the Invention

[0006] Based on this, the present invention provides a composition for increasing NAD+ levels. This composition is scientifically formulated, and multi-indicator, multi-level experimental studies have demonstrated the rational combination and synergistic effect of its various components to increase the NAD+ content of skin tissue and cells, effectively reducing UV-induced oxidative stress and inflammation, and alleviating photodamage and photoaging of the skin. Specifically, the technical solution adopted by the present invention is as follows:

[0007] A first aspect of the present invention provides a composition comprising, by weight, 1 part of wheat polar lipids and 1-10 parts of cherry blossom extract.

[0008] In some specific embodiments, the composition comprises, by weight, the following components: 1 part wheat polar lipids and 4 parts cherry blossom extract.

[0009] In some specific embodiments, the composition comprises, by weight, the following components: 1 part wheat polar lipids and 1 part cherry blossom extract.

[0010] In some specific embodiments, the composition comprises, by weight, the following components: 1 part wheat polar lipids and 10 parts cherry blossom extract.

[0011] In some specific embodiments, the composition further includes ergothionein and / or disodium pyrroloquinoline quinone (PQQ).

[0012] In some specific embodiments, the composition comprises, by weight, the following components: 1 part of wheat polar lipids, 1-10 parts of cherry blossom extract, and 1-20 parts of ergothioneine.

[0013] In some specific embodiments, the composition comprises, by weight, the following components: 1 part of wheat polar lipids, 4 parts of cherry blossom extract, and 1 part of ergothioneine.

[0014] In some specific embodiments, the composition comprises, by weight, the following components: 1 part of wheat polar lipids, 4 parts of cherry blossom extract, and 20 parts of ergothioneine.

[0015] In some specific embodiments, the composition comprises, by weight, the following components: 1 part wheat polar lipids, 4 parts cherry blossom extract, and 6 parts ergothioneine.

[0016] In some specific embodiments, the composition may further include chitosan oligosaccharide, yeast extract, γ-aminobutyric acid and / or collagen peptides.

[0017] A second aspect of the invention provides the use of the composition of the first aspect in the preparation of products that increase the body's NAD+ levels.

[0018] In some specific embodiments, increasing the body's NAD+ level refers to increasing the NAD+ content in human skin cells or tissues.

[0019] The third aspect of the invention provides the use of the composition of the first aspect in the preparation of products for preventing, alleviating and / or treating photodamage or / or photoaging.

[0020] In some specific implementations, the photodamage refers to skin redness, burning, pain, and / or peeling caused by ultraviolet and / or blue light.

[0021] In some specific implementations, photoaging refers to skin collagen degradation, wrinkle formation, decreased skin elasticity, pigmentation, and / or decreased skin radiance caused by ultraviolet and / or blue light.

[0022] In some specific implementations, the prevention, mitigation, and / or treatment of photodamage refers to reducing the expression of inflammatory factors, accumulation of reactive oxygen species, and / or accumulation of advanced glycation end products in skin tissues or cells caused by ultraviolet and / or blue light.

[0023] In some specific implementations, the prevention, mitigation, and / or treatment of photodamage refers to reducing skin redness, burning, pain, and / or peeling caused by ultraviolet and / or blue light.

[0024] In some specific implementations, the prevention, mitigation, and / or treatment of photoaging refers to reducing skin collagen degradation, wrinkle formation, decreased skin elasticity, pigmentation, and / or decreased skin radiance caused by ultraviolet and / or blue light.

[0025] A fourth aspect of the present invention provides a food product comprising the composition of the first aspect described above and food-acceptable excipients.

[0026] In some specific embodiments, the acceptable excipients in the food include any one or more combinations of fillers, lubricants, disintegrants, thickeners, acidulants, sweeteners, and preservatives.

[0027] In some specific implementation schemes, the food is a health food, a regular food, or a dietary supplement.

[0028] A fifth aspect of the present invention provides a cosmetic product comprising the composition of the first aspect described above and cosmetically acceptable additives.

[0029] In some specific embodiments, the cosmetic is an essence water, essence lotion, sleeping mask, body lotion, facial cleanser, pressed powder, massage cream, loose powder, sunscreen, or hand cream.

[0030] In some specific embodiments, the additive includes any one or more combinations of surfactants, thickeners, conditioning agents, humectants, preservatives, chelating agents, fragrances, and water.

[0031] Advantages of the invention

[0032] Compared with the prior art, the composition of this application has at least the following beneficial effects: The present invention provides a composition for increasing the NAD+ level of the body. The components in this composition are rationally combined and work synergistically to effectively increase the NAD+ content of skin tissue and cells, effectively reduce the generation of inflammatory factors caused by ultraviolet radiation, reduce the accumulation of reactive oxygen species and advanced glycation end products, and alleviate photodamage and photoaging of the skin caused by ultraviolet radiation or blue light. Attached Figure Description

[0033] Figure 1 The images show the imaging results of HaCaT-mCherry-FiNad cell lines after 30 min of different sample treatments.

[0034] Figure 2 The images show the imaging results of HaCaT-mCherry-cpYFP cell lines after 30 min of different sample treatments.

[0035] Figure 3 The image shows a typical diagram of the caudal fin area of ​​zebrafish without sample treatment. Note: The area within the yellow dashed box is the analysis area. Specific implementation methods

[0036] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0037] Furthermore, unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Those skilled in the art will understand that the examples are described by way of illustration and are not intended to limit the scope of protection claimed by the invention. All disclosures and other references mentioned herein are incorporated herein by reference in their entirety.

[0038] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, to better understand this invention, definitions and explanations of relevant terms are provided below.

[0039] As used herein, the alternative range of the terms “and / or” and “and / or” includes any one of two or more of the related listed items, as well as any and all combinations of the related listed items, including any two related listed items, any more related listed items, or a combination of all the related listed items.

[0040] As used in this article, “one or more” means any one, two or more of the listed items.

[0041] As used in this article, terms such as "first aspect" and "second aspect" are for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, terms such as "first" and "second" serve only as a non-exhaustive enumeration and should be understood as not constituting a closed limitation on quantity.

[0042] In this application, numerical ranges are referred to as continuous unless otherwise specified, and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when multiple ranges are provided to describe a feature or characteristic, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0043] As used in this article, the term "nicotinamide adenine dinucleotide (NAD)," also known as coenzyme I, is composed of nicotinamide, adenine, and two ribose sugars linked by phosphodiester bonds. NAD is widely distributed in various tissues and cells of the body and is the most important electron carrier in cells. NAD exists in two forms: the oxidized form NAD+ and the reduced form NADH.

[0044] As used in this article, the term "wheat polar lipids" refers to natural lipid substances extracted from wheat flour. Their main components include phytoceramides (primarily ceramide-1, ceramide-2, ceramide-3, and ceramide-4) and DGDG (digalactosylglycerol), with phytoceramide content exceeding 3% and DGDG content exceeding 40%. They have significant effects in maintaining the skin barrier, moisturizing, and anti-aging.

[0045] As used in this article, the term "cherry blossom extract" refers to an extract made from the flowers of Japanese late-blooming cherry blossoms or Kanzan cherry blossoms. Its main components are plant polyphenols such as gallic acid, caffeoyl glucose, and quercetin glucoside. Gallic acid accounts for more than 5% by mass, caffeoyl glucose for more than 2% by mass, and quercetin glucoside for less than 0.2% by mass. Experiments have demonstrated its significant efficacy in improving glycosylation reactions and reducing AGEs content.

[0046] As used in this article, the term "ergothioneine (EGT)" is a natural sulfur-containing amino acid derivative with the molecular formula C9H. 15N3O2S, with a molecular weight of 229.3, contains a 2-mercaptoimidazole ring linked to a trimethylated α-amino acid in its molecular structure. Ergothionein exists in two isomers, the thiol form and the thione form, which can interconvert under physiological conditions. Ergothionein was first isolated from the ergot fungus *Clavicepspurpure* in 1909 and is now mainly extracted from fungi or grains, or prepared through microbial fermentation (such as with or without genetically modified *E. coli*, *Trichoderma reesei*, etc.).

[0047] Unless otherwise specified, the experiments and methods described in the embodiments are performed in accordance with conventional methods well known in the art and described in various references.

[0048] Furthermore, unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Those skilled in the art will understand that the examples are described by way of illustration and are not intended to limit the scope of protection claimed by the invention. All disclosures and other references mentioned herein are incorporated herein by reference in their entirety.

[0049] The wheat polar lipids used in these embodiments were purchased from Seppic Co., Ltd., France; the cherry blossom extract used in these embodiments was purchased from Guangdong Qingyunshan Pharmaceutical Co., Ltd. It should be understood that the plant source and extraction process affect the composition of plant extracts, and thus their bioactivity. Plant extracts obtained using the same or similar plant sources and extraction processes will have the same or comparable composition and bioactivity. Therefore, the wheat polar lipids and cherry blossom extracts of this invention are not limited to the aforementioned manufacturers. They can be sourced from other manufacturers, but wheat polar lipids or cherry blossom extracts with the same or similar plant sources and extraction processes as the aforementioned manufacturers, and with the same or similar composition and bioactivity, can be used.

[0050] The ergothioneine used in this embodiment of the invention was purchased from Shanghai Ergothioneine Biotechnology Group Co., Ltd. It should be understood that ergothioneine is a chemically defined component, and the ergothioneine used in this invention is not limited to the aforementioned manufacturer and can also be obtained from other manufacturers.

[0051] Example 1

[0052] A composition comprising, by weight, 1 part wheat polar lipids and 4 parts cherry blossom extract.

[0053] Example 2

[0054] A composition comprising, by weight, 1 part wheat polar lipids and 1 part cherry blossom extract.

[0055] Example 3

[0056] A composition comprising, by weight, 1 part wheat polar lipids and 10 parts cherry blossom extract.

[0057] Example 4

[0058] A composition comprising, by weight, 1 part wheat polar lipids, 4 parts cherry blossom extract, and 1 part ergothioneine.

[0059] Example 5

[0060] A composition comprising, by weight, 1 part wheat polar lipids, 4 parts cherry blossom extract, and 6 parts ergothioneine.

[0061] Example 6

[0062] A composition comprising, by weight, 1 part wheat polar lipids, 4 parts cherry blossom extract, and 20 parts ergothioneine.

[0063] Example 7

[0064] A composition comprising, by weight, 1 part wheat polar lipids, 4 parts cherry blossom extract, and 4 parts PQQ.

[0065] Comparative Example 1

[0066] A composition comprising: 1 wheat polar lipid.

[0067] Comparative Example 2

[0068] A composition comprising: 1 part cherry blossom extract.

[0069] Comparative Example 3

[0070] A composition comprising 1 part ergothioneine.

[0071] Experimental Example 1: Cell Experiment

[0072] This embodiment uses a fluorescent protein probe genetically encoded by mCherry-FiNad (disclosed in the paper "Illuminating NAD+ Metabolism in Live Cells and In Vivo Using a Genetically Encoded Fluorescent Sensor" published by Yejun Zou et al. in *DEVELOPMETAL CELL*) to specifically detect the real-time dynamic changes of NAD+ in living human keratinocytes. The structure of this fluorescent probe is: mcherry-T-Rex(1-77)-T-Rex(78-189)-cpYFp-T-Rex(190-211), which contains two fluorescent proteins, mcherry and cpYFp, as well as the T-Rex protein (a protein derived from bacteria that can specifically bind to NAD (including its oxidized form NAD+ and reduced form NADH)). When NAD+ levels in cells increase, the fluorescence of channel 488 of the mCherry-FiNad probe intensifies, while the fluorescence of channel 561 remains unchanged. Therefore, the ratio of the fluorescence value of channel 488 (F488) to the fluorescence value of channel 561 (F561), R488 / 561 (F488 / F561), can be used to detect changes in NAD+ levels in cells or living organisms. mCerry-cpYFP, ​​as a control probe for mCerry-FiNad, does not respond to NAD+ and can be used to calibrate non-specific changes caused by pH disturbances.

[0073] 1. Group settings

[0074] The experiment included a blank control group, and experimental groups 1 (composition of Example 1), 2 (composition of Example 4), 3 (composition of Example 6), 4 (composition of Example 7), 5 (comparative example 1), 6 (comparative example 2), 7 (comparative example 3 low dose), and 8 (comparative example 3 high dose).

[0075] Samples were prepared using complete culture medium to achieve the following concentrations in each experimental group: cherry blossom extract 0.03906%, wheat polar lipids 0.00975%; ergothioneine 0.00975% in experimental groups 2 and 7; ergothioneine 0.195% in experimental groups 3 and 8; and PQQ 0.03906% in experimental group 4. All concentrations were based on the presence of the specified component in the experimental group.

[0076] 2. Experimental Methods

[0077] 2.1 Cell Construction

[0078] HaCaT cells were seeded in 6-well plates at a density of approximately 50% and a cell count of approximately 1-3 × 10⁵ cells, and cultured overnight. The culture medium was discarded, and 2 mL of fresh medium was added. mCherry-FiNad and mCherry-cpYFP lentiviruses were added dropwise at MOIs of 100:1, 30:1, 10:1, 5:1, and 1:1, respectively. Polybrene was then added to a final concentration of 8 μg / mL. Cells were incubated for 6 hours, after which the medium was changed. Cells infected with appropriate MOIs were selected for subsequent experiments. Selection criteria included strong fluorescence, high positivity rate, and no obvious cell damage or death.

[0079] After the selected cells have grown to confluence in the plate, they are passaged into 6cm dishes and selected with 0.5μg / mL puromycin for 1-2 weeks. Simultaneously, a dish of wild-type cells is prepared and treated with 0.5μg / mL puromycin until all wild-type cells die, at which point selection is stopped. The resulting cell lines are the stable HaCaT cell lines expressing the mCherry-Finad and mCherry-cpYFP probes. Four-well imaging-specific culture dishes are coated with 5μg / cm² fibronectin in an incubator for 2 hours, washed twice with PBS, and the stable cell lines are seeded into 30% of the samples in four-well imaging-specific glass dishes. After culturing for 12 hours, the medium is replaced with fresh medium and equilibrated for 2 hours.

[0080] Images were acquired using a rotating confocal microscope system with excitation and emission wavelengths of Ex488 / Em528 and Ex561 / Em645, respectively. The fluorescence images of the two channels were processed using ImageJ software to calculate the ratio, and the cells were then given pseudocolor after the ratio processing.

[0081] 2.3 Sample processing machine detection

[0082] Before adding the sample, the culture medium in the petri dish was replaced with fresh complete medium, and the cells were equilibrated in a cell incubator for 2 hours. After adding the sample and processing for 25 minutes, the sample was loaded onto the microscope. The fluorescence values ​​of the probe in channels 488 and 561 were recorded 30 minutes after sample addition using a rotating laser confocal microscope, and the fluorescence ratio of the two channels (R488 / 561) was calculated. The higher the R488 / 561 ratio, the higher the NAD+ content in the cells, and the stronger the sample's ability to increase NAD+.

[0083] 3. Experimental Results

[0084] Table 1. Statistical analysis results of probe imaging (standardized).

[0085]

[0086] Cell fluorescence imaging results as follows Figures 1-2As shown in Table 1, the statistical quantitative analysis of the cell imaging results indicates that, after 30 minutes of treatment, both low and high doses of ergothioneine significantly enhanced NAD+ in HaCaT cells. Cherry blossom polyphenols and wheat polar lipids also increased NAD+ levels in cells, but their effects were not as significant as those of ergothioneine. The combined use of cherry blossom polyphenols and wheat polar lipids exhibited a synergistic effect. The synergistic effect of ergothioneine, cherry blossom polyphenols, and wheat polar lipids was even more pronounced, with significantly higher NAD+ levels in cells compared to experimental groups treated with ergothioneine alone, combined with cherry blossom polyphenols / wheat polar lipids, or a combination thereof.

[0087] Experiment Example 2: Zebrafish Experiment

[0088] Skin aging is a complex process, encompassing both internal aging processes and aging caused by external factors. Ultraviolet (UV) radiation is the most important exogenous factor causing photoaging of the skin. In this embodiment, a photoaging model was established in zebrafish induced by simulated sunlight (including UVA, UVB, and visible light).

[0089] Zebrafish skin structure is similar to that of humans, consisting of the epidermis, dermis, and subcutaneous tissue. The epidermis is composed of the superficial, intermediate, and basal layers. Located on the outermost layer of the skin, the epidermis is susceptible to damage from UV radiation, which can disrupt the structure of proteins and other biomolecules, thus affecting the normal growth and development of cells and tissues. In zebrafish, this manifests as roughness and wrinkling of the tail fin. The histological manifestations of photoaging of the skin are often irregular thickening of the epidermis and inflammatory cell infiltration in the dermis.

[0090] The mechanism of skin photoaging is complex and closely related to the formation of reactive oxygen species induced by UV radiation. Reactive oxygen species can cause oxidative stress, autophagy, DNA damage, inflammation, and excessive melanin synthesis. Simultaneously, the oxidative stress response triggered by UV radiation can further induce non-enzymatic glycation in the skin, producing advanced glycation end products (AGEs), including glycated hemoglobin. UV radiation then utilizes the properties of AGEs to promote oxidative stress, causing oxidative damage to the skin and accelerating photoaging.

[0091] 1. Experimental Grouping

[0092] The experiment included a normal control group, a model control group, a positive control group (approximately 0.06 mg / day of resveratrol), experimental group 1 (the composition of Comparative Example 1), and experimental group 2 (the composition of Example 5).

[0093] Wheat polar lipids, L-ergothioneine, and cherry blossom polyphenols were all used in ultrapure water as solvent. Positive control: resveratrol, white powder, batch number F2121344, Shanghai Aladdin Biochemical Technology Co., Ltd., in DMSO solvent.

[0094] 2. Experimental animals

[0095] Zebrafish were all raised in fish culture water at 28°C (Water quality: 200 mg of instant sea salt was added to every 1 L of reverse osmosis water, with a conductivity of 450 - 550 μS / cm; pH of 6.5 - 8.5; hardness of 50 - 100 mg / L CaCO3). They were provided by the Biological Innovation Experiment Center of a certain biological technology Co., Ltd. in Hangzhou. The license number for the use of experimental animals is: SYXK(Zhe)2022 - 0004. The feeding management complies with the requirements of international AAALAC accreditation (Accreditation number: 001458), and the IACUC ethical review number is: IACUC - 2025 - 11190 - 01.

[0096] 3. Instruments, consumables and reagents

[0097] Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Visual Technology Co., Ltd., China); Precision electronic balance (CP214, OHAUS, USA); Simulated sunlight (SOL500, Hoenle, Germany); Constant temperature oscillator (HZ - 9211K, Taicang Hualida Experimental Equipment Co., Ltd., China); Automatic sample rapid grinder (JXFSTPRP - 24L, Shanghai Jingxin Experimental Equipment Technology Department, China); High - speed refrigerated centrifuge (Heraeus Fresco17, ThermoFisher, Germany); Full - wavelength multifunctional microplate reader (SPARK, TECAN, Austria); Automatic nucleic acid extractor (Auto - Pure32A, Hangzhou Ausheng Instrument Co., Ltd., China); Ordinary PCR amplifier (T100, BIO - RAD, Singapore); Fluorescent quantitative PCR instrument (CFX Connect, BIO - RAD, Singapore); High - speed refrigerated centrifuge (Heraeus Fresco17, ThermoFisher, Germany); UV - visible spectrophotometer (Nanodrop2000, Thermo, USA); Microplate mini - centrifuge (BE - 6100, Haimen其林贝尔仪器制造有限公司, China); Optical adhesive sealing film B (MSB1001, Bio - rad, USA); Low - skirt 96 - well plate (transparent) (HSP9601, Bio - rad, USA); 96 - well plate (Nest Biotech, China); 96 - well microplate (Costar, China); 6 - well plate (Zhejiang Beilanbo Biotechnology Co., Ltd., China).

[0098] D-(+)-glucose (batch number D2518345, Shanghai Aladdin Biochemical Technology Co., Ltd., China); methylcellulose (batch number C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd., China); dimethyl sulfoxide (DMSO, batch number 20240619, Sinopharm Chemical Reagent Co., Ltd., China); PBS (catalog number BL601A, Biosharp, China); CM-H2DCFDA (batch number 2800340, Invitrogen, USA); Universal SYBR qPCR Master Mix (batch number 027E2201CD, Vazyme, China); FastKing cDNA First Strand Synthesis Kit (Genomic De-generated) (batch number X1213, Tiangen Biotech (Beijing) Co., Ltd., China); Universal RNA Extraction TL Kit C (catalog number TL643-03C, Foshan Aowei Biotechnology Co., Ltd., China); zebrafish COI I ELISA KIT (batch number 202505, Shanghai Enzyme-Linked Biotechnology Co., Ltd., China).

[0099] 4. Detection Method

[0100] 4.1 Determination of Maximum Test Concentration (MTC)

[0101] Wild-type AB strain zebrafish, 3 days post-fertilization (3 dpf), were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples were administered in water solution (concentrations shown in Table 3), and a normal control group and a model control group were also included. Each well contained 3 mL of sample. After treatment at 28℃ for 3 h, except for the normal control group, all other experimental groups underwent photoaging induced by simulated sunlight. After another day of treatment at 28℃, the MTC of the samples on the model zebrafish was measured.

[0102] 4.2 Evaluation of anti-photoaging efficacy (tail fin area)

[0103] Wild-type AB strain zebrafish (3dpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples were administered in water (concentrations shown in Table 4), with resveratrol at a positive control concentration of 10.0 μg / mL. A normal control group and a model control group were also included, with a volume of 3 mL per well. After treatment at 28℃ for 3 hours, all experimental groups except the normal control group underwent photoaging induced by simulated sunlight. After another day of treatment at 28℃, 10 zebrafish from each experimental group were randomly selected and photographed under a dissecting microscope. Data were analyzed and collected using NIS-ELEMENTSD 3.20 advanced image processing software. The caudal fin area of ​​the zebrafish was analyzed, and the statistical analysis results of this index were used to evaluate the anti-photoaging efficacy of the samples. Statistical results are expressed as mean ± SE. Statistical analysis was performed using statistical software; p < 0.05 indicated statistical significance.

[0104] 4.3 Evaluation of anti-photoaging efficacy (AGEs)

[0105] Wild-type AB strain zebrafish (3dpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples were administered via water-soluble administration (concentrations shown in Table 5), with resveratrol at a positive control concentration of 10.0 μg / mL. A normal control group and a model control group were also included, with a volume of 3 mL per well. After treatment at 28℃ for 3 h, except for the normal control group, all other experimental groups underwent photoaging induced by simulated sunlight combined with water-soluble glucose administration. Zebrafish from each group were transferred to 1.5 mL centrifuge tubes, with 10 zebrafish treated in each tube (experimental group). After shaking treatment at 60℃ for 1 day, the supernatant was collected by centrifugation, and data were collected using a multi-functional microplate reader. The AGEs fluorescence values ​​of each experimental group were analyzed, and the statistical analysis results of this index were used to evaluate the anti-photoaging efficacy of the samples. Statistical results are expressed as mean ± SE. Statistical analysis was performed using statistical software, and p < 0.05 was considered statistically significant.

[0106] 4.4 Evaluation of anti-photoaging efficacy (ROS)

[0107] Wild-type AB strain zebrafish (3dpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples were administered in water (concentrations shown in Table 6), with resveratrol at a positive control concentration of 10.0 μg / mL. A normal control group and a model control group were also included, with a volume of 3 mL per well. After treatment at 28℃ for 3 h, all experimental groups except the normal control group underwent photoaging induced by simulated sunlight. After another day of treatment at 28℃, the samples were washed, and CM-H2DCFDA was added to each experimental group. After another day of treatment at 28℃, data were collected using a multi-mode microplate reader to analyze the ROS fluorescence values ​​in the zebrafish. The anti-photoaging efficacy of the samples was evaluated based on the statistical analysis results of this index. Statistical results are expressed as mean ± SE. Statistical analysis was performed using statistical software; p < 0.05 indicated statistical significance.

[0108] 4.5 Evaluation of anti-photoaging efficacy (overall type I collagen content)

[0109] Wild-type AB strain zebrafish (3dpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples were administered in water (concentrations shown in Table 7), with resveratrol at a positive control concentration of 10.0 μg / mL. A normal control group and a model control group were also included, with a volume of 3 mL per well. The experiment was conducted in triplicate. After treatment at 28℃ for 3 hours, all experimental groups except the normal control group underwent photoaging induced by simulated sunlight. After another day of treatment at 28℃, zebrafish samples were collected according to the zebrafish COIIELISA KIT instructions. Data were collected using a multi-mode microplate reader to analyze the overall type I collagen content of the zebrafish. The anti-photoaging efficacy of the samples was evaluated using statistical analysis of this indicator. Statistical results are expressed as mean ± SE. Statistical analysis was performed using statistical software; p < 0.05 indicated statistical significance.

[0110] 4.6 Evaluation of anti-photoaging efficacy (genes)

[0111] Wild-type AB strain zebrafish (3dpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples were administered in water (concentrations shown in Table 8), with a positive control of resveratrol at a concentration of 10.0 μg / mL. A normal control and a model control group were also included, with a volume of 3 mL per well. The experiment was repeated in triplicate. After treatment at 28℃ for 3 hours, all experimental groups except the normal control group underwent photoaging induced by simulated sunlight. After another day of treatment at 28℃, zebrafish samples were collected from each experimental group. Total RNA was extracted from the zebrafish using the Universal RNA Extraction TL Kit C, and the concentration and purity of the total RNA were determined using a UV-Vis spectrophotometer. 2.00 μg of total RNA from the zebrafish sample was used to synthesize 20.0 μL of cDNA according to the instructions of the cDNA first-strand synthesis kit. The expression of β-actin, tnf-α, il-6, and il-1β genes was detected by q-PCR. β-actin was used as an internal control for gene expression. The relative RNA expression levels of TNF-α, IL-6, and IL-1β genes were calculated, and the anti-photoaging efficacy of the samples was evaluated based on the statistical analysis results of this index. Statistical results are expressed as mean ± SE. Statistical analysis was performed using statistical software, and p < 0.05 was considered statistically significant.

[0112] At the experimental endpoint, total RNA was extracted from zebrafish, and the RNA concentration was determined using a UV-Vis spectrophotometer.

[0113] The A260 / A280 ratios (Table 8) are all between 1.8 and 2.2, indicating that the extracted zebrafish total RNA is of good quality and can be used for subsequent q-PCR experiments. Primer sequences are shown in Table 2.

[0114] Table 2 Primer sequence information

[0115]

[0116] 5. Test Results

[0117] 5.1 Maximum Test Concentration (MTC) Detection Results

[0118] Table 3 Results of the concentration exploratory experiment on the anti-photoaging efficacy of samples (n=30)

[0119]

[0120]

[0121] Under the experimental conditions, the anti-photoaging efficacy MTC of experimental group 1 (a combination of ergothioneine, wheat polar lipids, and cherry blossom polyphenols) and experimental group 2 (wheat polar lipids) were 2000 μg / mL and 1000 μg / mL, respectively. The specific results are shown in Table 3.

[0122] 5.2 Evaluation of anti-photoaging efficacy (tail fin area)

[0123] Table 4. Experimental results of the anti-photoaging efficacy evaluation of samples (tail fin area) (n=10)

[0124] Group Concentration (μg / mL) Tail fin area (pixels, mean ± SE) Normal control group - 411743±8626*** Model control group - 286829±8538 Resveratrol 10.0 319448±9662* Test group 1 2000 371774±8095*** Test group 2 250 293015±7739

[0125] Note: Compared with the model control group, *p<0.05, ***p<0.001.

[0126] Under the experimental conditions, (1) Group 1 (a combination of ergothioneine, wheat polar lipids, and cherry blossom polyphenols) showed anti-photoaging effects, specifically in repairing tail fin damage; Group 2 (wheat polar lipids) showed no significant anti-photoaging effects. (2) Compared at the same concentration, the absolute value of the tail fin area in Group 1 was significantly larger than that in the positive control, indicating that the larger the absolute value of the tail fin area, the better the anti-photoaging effect. Therefore, Group 1 showed better anti-photoaging effects than the positive control. See Table 4 for details. Figure 3 .

[0127] 5.3 Evaluation of Anti-Photoaging Efficacy (AGEs)

[0128] Table 5. Experimental results of the anti-photoaging efficacy evaluation of samples (AGEs) (n=3)

[0129] Group Concentration (μg / mL) AGEs fluorescence value (mean ± SE) Normal control group - 43751±375*** Model control group - 49458±524 Resveratrol 10.0 45596±833* Test group 1 2000 31516±526*** Test group 2 250 48987±308

[0130] Compared with the model control group, *p<0.05, ***p<0.001.

[0131] Under the experimental conditions, (1) Group 1 (a combination of ergothioneine, wheat polar lipids, and cherry blossom polyphenols) showed anti-photoaging effects, specifically by reducing AGEs fluorescence values; Group 2 (wheat polar lipids) showed no significant anti-photoaging effects. (2) Compared at the same concentration, the absolute value of AGEs fluorescence in Group 1 was significantly lower than that in the positive control, indicating that the lower the absolute value of AGEs fluorescence, the better the anti-photoaging effect. Therefore, Group 1 showed better anti-photoaging effects than the positive control. See Table 5 for details.

[0132] 5.4 Evaluation of anti-photoaging efficacy (ROS)

[0133] Table 6. Experimental results of the anti-photoaging efficacy evaluation of samples (ROS) (n=10)

[0134] Group Concentration (μg / mL) ROS fluorescence value (mean ± SE) Normal control group - 2243±59.6*** Model control group - 3099±86.0 Resveratrol 10.0 1572±51.3*** Test group 1 2000 2637±58.4*** Test group 2 250 3013±111

[0135] Compared with the model control group, ***p<0.001.

[0136] Under the experimental conditions, (1) experimental group 1 (a combination of ergothioneine, wheat polar lipids, and cherry blossom polyphenols) showed anti-photoaging effects, specifically by reducing ROS fluorescence values; experimental group 2 (wheat polar lipids) showed no significant anti-photoaging effects. (2) At the same concentration, there was no significant difference in ROS fluorescence values ​​between experimental group 1 and the positive control. See Table 6 for details.

[0137] 5.5 Evaluation of anti-photoaging efficacy (overall type I collagen content)

[0138] Table 7. Experimental results of the anti-photoaging efficacy evaluation of samples (overall type I collagen content) (n=3)

[0139]

[0140]

[0141] Compared with the model control group, **p<0.01, ***p<0.001.

[0142] Under the experimental conditions, (1) Experimental group 1 (a combination of ergothioneine, wheat polar lipids, and cherry blossom polyphenols) showed anti-photoaging effects, specifically by increasing the content of type I collagen; Experimental group 2 (wheat polar lipids) did not show significant anti-photoaging effects. (2) Compared with the same concentration, the absolute value of type I collagen content in experimental group 1 was significantly higher than that in the positive control, indicating that the higher the absolute value of type I collagen content, the better the anti-photoaging effect. Therefore, the anti-photoaging effect of experimental group 1 was better than that of the positive control, as detailed in Table 7.

[0143] 5.6 RNA extraction results

[0144] TNF-α is the earliest and most important inflammatory cytokine to appear during the inflammatory response, and it can activate neutrophils. Interleukin-1β (IL-1β) and interleukin-6 (IL-6) are mediators of inflammation. Interleukins play an important role in the inflammatory response by transmitting information, activating and regulating immune cells, and mediating the activation, proliferation and differentiation of T and B cells.

[0145] Table 8. Total RNA concentration and A260 / A280 ratio (n=3)

[0146]

[0147] Under the experimental conditions, (1) both experimental group 1 (a combination of ergothionein, wheat polar lipids, and cherry blossom polyphenols) and experimental group 2 (wheat polar lipids) downregulated the relative expression levels of TNF-α, IL-6, and IL-1β genes. (2) At the same concentration, there was no significant difference in the expression levels of each gene between experimental group 1 and experimental group 2. See Table 9 for details.

[0148] Table 9. Experimental results of the anti-photoaging efficacy evaluation of samples (genes) (n=3)

[0149] Group Concentration (μg / mL) Relative expression of tnf-α gene Relative expression of il-6 gene Relative expression of il-1β gene Normal control group - 0.191(±0.020***) 0.148(±0.005***) 0.154(±0.024***) Model control group - 1.00±0.063 1.00±0.084 1.00±0.054 Resveratrol 10.0 0.222±0.021*** 0.444±0.043** 0.141±0.020*** Test group 1 2000 0.569±0.006** 0.375±0.022** 0.296±0.012*** Test group 2 250 0.708±0.052* 0.459±0.068** 0.435±0.038***

[0150] Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001.

[0151] Under the experimental conditions, experimental group 1, composed of wheat polar lipids, cherry blossom polyphenols, and ergothionein, exhibited anti-photoaging effects, specifically repairing tail fin damage, reducing AGEs fluorescence values, reducing ROS fluorescence values, increasing type I collagen content, and downregulating the relative expression levels of TNF-α, IL-6, and IL-1β genes. Wheat polar lipids could downregulate the relative expression levels of TNF-α, IL-6, and IL-1β genes, but their effects on repairing tail fin damage, reducing AGEs fluorescence values, reducing ROS fluorescence values, and increasing type I collagen content were not significant.

Claims

1. A composition, characterized in that, The composition comprises, by weight, the following components: 1 part of wheat polar lipids and 1-10 parts of cherry blossom extract; Preferably, the composition comprises, by weight, the following components: 1 part of wheat polar lipids and 4 parts of cherry blossom extract.

2. The composition according to claim 1, characterized in that, The composition may also include ergothioneine and / or PQQ.

3. The composition according to claim 2, characterized in that, The composition comprises, by weight, the following components: 1 part of wheat polar lipids, 1-10 parts of cherry blossom extract, and 1-20 parts of ergothioneine. Preferably, the composition comprises, by weight, the following components: 1 part of wheat polar lipids, 4 parts of cherry blossom extract, and 1 part of ergothioneine; Preferably, the composition comprises, by weight parts, the following components: 1 part of wheat polar lipids, 4 parts of cherry blossom extract, and 20 parts of ergothioneine; Preferably, the composition comprises, by weight, the following components: 1 part wheat polar lipids, 4 parts cherry blossom extract, and 6 parts ergothioneine.

4. The use of the composition according to any one of claims 1-3 in the preparation of products that increase the body's NAD+ levels; Preferably, increasing the body's NAD+ level refers to increasing the NAD+ content in human skin cells or tissues.

5. Use of the composition according to any one of claims 1-3 in the preparation of products for preventing, alleviating and / or treating photodamage or / or photoaging.

6. The application according to claim 5, having one or more of the following features, (1) The photodamage refers to skin redness, burning, pain and / or peeling caused by ultraviolet and / or blue light; (2) Photoaging refers to the degradation of skin collagen, formation of skin wrinkles, decrease in skin elasticity, skin pigmentation, and / or decrease in skin luster caused by ultraviolet and / or blue light; (3) The prevention, relief and / or treatment of photodamage refers to reducing the expression of inflammatory factors, accumulation of reactive oxygen species and / or accumulation of advanced glycation end products in skin tissues or cells caused by ultraviolet and / or blue light; (4) In some specific implementations, the prevention, mitigation and / or treatment of photodamage refers to reducing skin redness, burning, pain and / or peeling caused by ultraviolet and / or blue light; (5) In some specific implementations, the prevention, mitigation and / or treatment of photoaging refers to reducing skin collagen degradation, wrinkle formation, decreased skin elasticity, skin pigmentation and / or decreased skin luster caused by ultraviolet and / or blue light.

7. A food product comprising the composition of any one of claims 1-3 and an excipient acceptable in the food product; preferably, the food product is a health food, a general food, or a dietary supplement.

8. The food product according to claim 7, characterized in that, Acceptable excipients in the food include any one or more combinations of fillers, lubricants, disintegrants, thickeners, acidulants, sweeteners, and preservatives.

9. A cosmetic product comprising the composition of any one of claims 1-3 and cosmetic-acceptable additives.

10. The cosmetic product according to claim 9, characterized in that, The acceptable additives in the cosmetics include any one or more combinations of surfactants, thickeners, conditioning agents, moisturizers, preservatives, chelating agents, fragrances, and water.

Images