Application of astragalus smicus glycoside preparation for promoting collagen synthesis

Abstract

The invention discloses application of an astragalus smicus glycoside preparation for promoting collagen synthesis, and belongs to the field of food and biological medicine cosmetics. The invention provides application of astragalus smicus glycoside or a plant extract containing astragalus smicus glycoside in preparation of drugs, cosmetics or food for preventing and / or delaying skin aging. The skin aging is caused by loss of collagen. Mechanism research shows that the astragalin promotes synthesis of mitochondrial NADPH by activating mitochondrial autophagy genes LGG-1 and DCT-1 and further promotes synthesis of collagen precursor proline, so that synthesis of collagen is promoted, loss of the collagen is reduced, and the effects of resisting skin aging and keeping young and elastic states are achieved. The invention discloses the new application of the astragalus smicus glycoside in promotion of collagen synthesis for the first time, and the application range of the astragalus smicus glycoside is expanded.

Description

Technical Field

[0001] This invention relates to the fields of food and cosmetics, specifically to the application of a collagen-promoting astragaloside preparation in the preparation of anti-skin aging drugs, cosmetics, or food. Background Technology

[0002] Collagen is an important component of the extracellular matrix of skin tissue. It is the most abundant and widely distributed functional protein in mammals and a major component of animal connective tissue, accounting for up to 80% of its composition. Human skin collagen is mainly composed of type I and type III collagen. Type I collagen is mainly found in the skin, bones, tendons, and ligaments, playing a crucial role in maintaining skin structure, elasticity, and resilience. Type III collagen usually coexists with type I collagen, especially in the skin, blood vessels, and organs, primarily maintaining organ structure and morphology. It also plays a role in skin healing and elasticity. Type IV collagen in human skin is mainly found in the basement membrane, a thin membrane structure located between the epidermis and dermis. It supports and connects the upper and lower dermal cells, and is essential for stabilizing skin tissue structure and morphology and maintaining elasticity.

[0003] Collagen plays a vital role in maintaining firm, smooth, and elastic skin. However, as we age, the natural synthesis of collagen gradually decreases, causing skin to become loose, saggy, and develop wrinkles and fine lines. This pervasive skin aging problem has sparked a global demand for anti-aging measures. To address the challenges of skin aging, scientific research is constantly exploring new strategies to slow collagen loss and promote its synthesis.

[0004] Mitochondrial-specific autophagy (mitochondrial autophagy) is the process by which damaged mitochondria are specifically encapsulated into autophagosomes and fused with lysosomes to complete the degradation of damaged mitochondria, thereby promoting mitochondrial quality and function. Studies have shown that mitophagy can promote the conversion of NADP(H) in mitochondrial energy metabolism, thereby promoting the synthesis of proline, an essential precursor of collagen (Bai J, et al. Autophagy loss impedes cancer-associated fibroblast activation via downregulating proline biosynthesis. Autophagy,. 2023,19(2):632-643.), indirectly promoting collagen synthesis and thus playing a role in delaying aging.

[0005] Therefore, in-depth research on various components that can combat skin aging by promoting collagen synthesis is of extremely important practical significance.

[0006] Compared to other model organisms, *Caenorhabditis elegans* has many advantages: a short lifespan and easily controlled growth conditions; high reproduction rate and easy survival; the nematode can easily survive at temperatures between 16°C and 25°C; its transparent body facilitates observation; and its epidermis contains a large amount of collagen, including type IV collagen homologous to human collagen, making it an excellent biological model for studying collagen changes during natural growth.

[0007] Kaempferol glycoside, also known as kaempferol 3-β-D-glucopyranoside, with the chemical name 3,5,7,4-tetrahydroxyflavone-3-glucoside, is one of the flavonoid compounds found in various natural plants such as kaempferol, eucommia, water lily, mulberry leaf, dodder seed, astragalus, apocynum leaf, moringa leaf, lotus leaf, tea, rose, persimmon leaf, cassia seed, and goldenrod. Its molecular formula is C2. 21 H 20 O 11 The structural formula is as follows:

[0008] Numerous in vitro and in vivo experiments and clinical studies have shown that astragaloside possesses significant anti-inflammatory, anti-cancer, antioxidant, neuroprotective, anti-diabetic, cardioprotective, anti-ulcer, and anti-fibrotic effects. However, the role of astragaloside in promoting collagen synthesis has not yet been clearly reported. Summary of the Invention

[0009] The purpose of this invention is to discover substances from natural compounds that can promote collagen synthesis and delay skin aging, and to apply them to the preparation of anti-skin aging drugs, cosmetics or food.

[0010] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides the use of astragalin or plant extracts containing astragalin in the preparation of pharmaceuticals, cosmetics, or food products for preventing and / or delaying skin aging. Skin aging is caused by collagen loss.

[0011] Furthermore, the manifestations of skin aging include: decreased skin elasticity, skin laxity, and the formation of wrinkles.

[0012] Studies have shown that decreased collagen synthesis leads to a gradual loss of skin elasticity, luster, and vitality—signs of aging. Nematode experiments have demonstrated that supplementing with astragaloside can significantly increase collagen levels in the body, effectively alleviating skin aging problems.

[0013] Furthermore, the astragalin is derived from plant extracts. In this invention, astragalin can be extracted from edible flowers and other plants, and will subsequently be used as an ingredient in pharmaceuticals, foods, and cosmetics. The extraction solvent can be a commonly used solvent in the art, preferably water.

[0014] In this invention, pure astragalin obtained from plant extracts or plant extracts containing astragalin can be used to prepare anti-skin aging pharmaceuticals, cosmetics or food.

[0015] Furthermore, the edible flower is tea tree flower or golden camellia. This invention is not limited thereto.

[0016] As a specific embodiment of the present invention, the method for preparing the plant extract containing astragalin includes: mixing edible flowers with water at a material-to-liquid ratio of 1 kg: 10-15 L, extracting at 50-60°C for 4-6 h, collecting the extract, and evaporating it at 50-55°C to obtain the edible flower extract.

[0017] As a specific embodiment of the present invention, the preparation method of the astragaloside includes: dissolving the edible flower extract in ten times the mass of ultrapure water, filtering through a 0.45 μm microporous membrane, and loading the sample onto a macroporous resin chromatography column for chromatography; first eluting with 5 column volumes of ultrapure water and discarding the impurity fraction, then eluting with a 40% (v / v) ethanol aqueous solution and collecting the target fraction; the collected fraction is first evaporated by rotary evaporation and then dried by nitrogen blowing, and then separated by high performance liquid chromatography using a C18 semi-preparative column with an 80% (v / v) formic acid aqueous solution as the mobile phase, and freeze-dried to obtain high-purity astragaloside.

[0018] Mechanism studies show that astragaloside can promote collagen synthesis and reduce collagen loss, thus achieving anti-aging effects on the skin and maintaining a youthful and elastic state.

[0019] Further mechanistic studies showed that astragaloside further promotes the synthesis of collagen precursor proline by increasing the level of NADPH in the body.

[0020] Furthermore, the characteristic feature is that astragaloside activates the mitochondrial autophagy gene. LGG-1 and DCT-1 This promotes the synthesis of mitochondrial NADPH, providing reducing power for proline.

[0021] This invention uses astragaloside as the sole or one of the active ingredients in the preparation of pharmaceuticals, cosmetics or food for preventing and / or delaying skin aging.

[0022] Furthermore, the drug also includes a pharmaceutically acceptable carrier. The drug is formulated as an oral preparation.

[0023] Furthermore, the cosmetic also includes carriers or excipients acceptable in the cosmetic field. The cosmetic is formulated as a cream, lotion, liquid, gel, oil, aerosol, or sheet mask. Specifically, the cosmetic can be, but is not limited to, face cream, lotion, gel, toner, serum, mask, eye cream, aerosol cleansing foam, spray, shower gel, or facial cleanser.

[0024] Furthermore, the food product also includes a food science-acceptable carrier. The food product is in the form of an oral liquid, tablet, capsule, or solid beverage.

[0025] The beneficial effects of this invention are as follows: This invention discloses for the first time a novel application of astragalin in promoting collagen synthesis, expanding its application scope. In vivo experimental studies have found that astragalin can promote collagen production in *Caenorhabditis elegans* and reduce collagen loss. Mechanistic studies show that astragalin activates mitochondrial autophagy genes. LGG-1 and DCT-1 It promotes the synthesis of mitochondrial NADPH, which in turn promotes the synthesis of proline, a collagen precursor. Therefore, astragaloside has application value in the preparation of pharmaceuticals, cosmetics, or foods that prevent and / or delay skin aging and / or wrinkle formation. Attached Figure Description

[0026] Figure 1 The image shows the secondary mass spectrum of the astragaloside preparation in anion mode. The m / z of the daughter ion fragments of astragaloside are 227.0342, 255.0294, 284.0322 and 447.0932.

[0027] Figure 2 This is a high-performance liquid chromatogram of the astragaloside preparation.

[0028] Figure 3 To investigate the effect of Camellia chrysantha extract on collagen content in Caenorhabditis elegans on days 1, 3, and 5 of growth.

[0029] Figure 4 To investigate the effect of tea tree flower extract on collagen content in *C. elegans* on days 1, 3, and 5 of growth.

[0030] Figure 5 The effect of using 100 μM astragaloside on collagen content in *C. elegans* on days 1, 3, and 5 of growth.

[0031] Figure 6 The effect of using 150 μM astragaloside on collagen content in *C. elegans* on days 1, 3, and 5 of growth.

[0032] Figure 7 The effect of using 200 μM astragaloside on collagen content in *C. elegans* on days 1, 3, and 5 of growth.

[0033] Figure 8 The effect of using 200 μM astragaloside on ATP production in Caenorhabditis elegans on the third day of growth.

[0034] Figure 9 The effect of using 200 μM astragaloside on ROS production in Caenorhabditis elegans on day 3 of growth.

[0035] Figure 10 To investigate the effect of 200 μM astragaloside on the mitochondrial autophagy gene of Caenorhabditis elegans on the third day of growth. LGG-1 The effect of fluorescence intensity.

[0036] Figure 11 To investigate the effect of 200 μM astragaloside on the mitochondrial autophagy gene in the head of Caenorhabditis elegans on the third day of growth. LGG-1 The impact of expression levels.

[0037] Figure 12 To investigate the effect of 200 μM astragaloside on the mitochondrial autophagy gene in the tail of Caenorhabditis elegans on the third day of growth. LGG-1 The impact of expression levels.

[0038] Figure 13 To investigate the effect of 200 μM astragaloside on the whole mitochondrial autophagy gene of Caenorhabditis elegans on the third day of growth. LGG-1 The impact of expression levels.

[0039] Figure 14 The effect of using 200 μM astragaloside on NADPH levels in the mitochondria of Caenorhabditis elegans on day 3 of growth.

[0040] Figure 15 The effect of using 200 μM astragaloside on the NADP+ content in the mitochondria of Caenorhabditis elegans on the third day of growth.

[0041] Figure 16 The effect of using 200 μM astragaloside on the NADP+ / NADPH ratio in the mitochondria of Caenorhabditis elegans on the third day of growth.

[0042] Figure 17To investigate the effect of mitochondrial autophagy genes in Caenorhabditis elegans on the third day of growth during the use of 200 μM astragaloside LGG-1 The effect of mRNA expression levels.

[0043] Figure 18 To investigate the effect of mitochondrial autophagy genes in Caenorhabditis elegans on the third day of growth during the use of 200 μM astragaloside DCT-1 The effect of mRNA expression levels.

[0044] Figure 19 To investigate the effect of mitochondrial autophagy genes in Caenorhabditis elegans on the third day of growth during the use of 200 μM astragaloside PINK-1 The effect of mRNA expression levels.

[0045] Figure 20 To investigate the collagen synthesis gene in Caenorhabditis elegans on day 3 of growth during the use of 200 μM astragaloside COL-12 The effect of mRNA expression levels.

[0046] Figure 21 To investigate the collagen synthesis gene in Caenorhabditis elegans on day 3 of growth during the use of 200 μM astragaloside COL-120 The effect of mRNA expression levels.

[0047] Figure 22 To investigate the collagen regulatory genes in Caenorhabditis elegans on day 3 of growth during the use of 200 μM astragaloside EMB-9 The effect of mRNA expression levels.

[0048] Figure 23 To investigate the collagen regulatory genes in Caenorhabditis elegans on day 3 of growth during the use of 200 μM astragaloside LET-2 The effect of mRNA expression levels.

[0049] Figure 24 To investigate the matrix metallocollagenase synthesis gene in Caenorhabditis elegans on day 3 of growth during the use of 200 μM astragaloside ZMP-3 The effect of mRNA expression levels.

[0050] Figure 25 To investigate the matrix metallocollagenase synthesis gene in Caenorhabditis elegans on day 3 of growth during the use of 200 μM astragaloside ZMP-4 The effect of mRNA expression levels. Detailed Implementation

[0051] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0052] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0053] Example 1 1. Preparation of tea tree flower extract 1000 g of dried tea tree flowers (moisture content 10.83%, purchased from Huangshan, Anhui) were extracted three times with ten times the weight of the tea tree flowers, using 10 L of ultrapure water each time. The extraction temperature was 55℃, and the extraction time was 6 hours, including 2 hours of ultrasonic treatment (ultrasonic power 500 W) and 4 hours of immersion extraction. The extracts from the three extractions were combined and evaporated to dryness using a rotary evaporator at 50℃, yielding 99.45 g of crude extract.

[0054] 2. Isolation of astragalin from crude extract of tea flower 99.45 g of the crude extract obtained after rotary evaporation was dissolved in 1000 mL of ultrapure water (ten times the volume of the crude extract). After filtration through a 0.45 μm microporous membrane, the extract was separated by macroporous adsorption resin column chromatography. Impurities were removed with ultrapure water, and the target fraction was eluted with an equal volume of 40% ethanol aqueous solution (v / v). This separation process using macroporous adsorption resin column chromatography was repeated three times, and the eluted fractions were collected three times. The macroporous adsorption resin was a moderately polar macroporous adsorption resin (moderately polar (or weakly polar) macroporous adsorption resins are adsorption resins with ester groups in their molecular skeleton, and their surfaces have both hydrophobic and hydrophilic parts, which can adsorb non-polar substances from polar solvents and polar substances from non-polar solvents).

[0055] The chromatographic extract was first dried by rotary evaporation and then by nitrogen blowing to obtain 33.68 g of purified extract containing polyphenolic compounds such as astragaloside. Finally, it was separated and prepared by high performance liquid chromatography (C18 semi-preparative column (5 μm, 250×4.6 mm)) with 80% formic acid water as the mobile phase (water to formic acid volume ratio of 2:8) to obtain purified astragaloside product with a total amount of 7.02 g.

[0056] 3. Identification of the purified product of astragaloside extracted and enriched by ultra-high performance liquid chromatography-mass spectrometry The purified product prepared in step 2 was redissolved in ethanol, filtered through a 0.22 μm filter, and then analyzed by UHPLC-QE-MS. Instrument control, data acquisition, and analysis were performed using Xcalibur software. Additionally, the raw data files were uploaded to Compound Discoverer™ for compound identification through matching with the mzCloud mass spectrometry library and manual verification. The absolute value of the mass error between the theoretical and actual molecular weights should be less than 2 mmu.

[0057] The injection conditions included: injecting 10 μL of sample solution into a UltiMate™ 3000 ultra-high performance liquid chromatography system and performing chromatographic separation at 40 °C on a UHPLC BEH C18 column (2.1 × 100 mm, 1.7 μm). 0.1% formic acid aqueous solution (A) and 0.1% formic acid acetonitrile solution (B) were used as the water and organic mobile phases, respectively. The gradient elution system was set as follows: 0–3 min, 5–25% B; 3–4 min, 25–65% B; 4–10 min, 65% B; 10–10.1 min, 65–5% B; 10.1–13 min, 5% B, at a flow rate of 0.3 mL / min. A Q Exactive orbital trap high-resolution mass spectrometer was used, operating in positive / negative ion mode, to achieve full scan analysis in the range of 100–1000 m / z. The optimized parameters are as follows: sheath flow rate 40 L / min, auxiliary gas flow rate 10 L / min, spray voltage 3 kV, capillary temperature 320℃, probe heater temperature 350℃, and S-lens RF level 50%.

[0058] like Figure 1 As shown, UHPLC-MS / MS analysis revealed the following ion information for astragalin: the parent ion m / z is 447.039, and the characteristic ion fragments m / z for secondary mass spectrometry are 447.0932, 284.0322, 255.0294, and 227.0342. The purified product prepared in step 2 was identified as astragalin by UHPLC-MS / MS.

[0059] 4. Purity analysis of the extracted and enriched astragaloside was performed by high performance liquid chromatography. The purified product prepared in step 2 was redissolved in ethanol, filtered through a 0.22 μm filter, and then subjected to HPLC purity analysis. The injection conditions included injecting 10 μL of sample solution into an E2695 HPLC system equipped with a 2998 diode array detector, and performing chromatographic separation at 30±5℃ on a reversed-phase ODS-2 Hypersil C18 column (4.6×250 nm, 5 μm). 0.1% formic acid aqueous solution (A) and acetonitrile (B) were used as the water and organic mobile phases, respectively. The gradient elution system was set as follows: 0–30 min, 5–20% B; 30–55 min, 20–40% B, at a flow rate of 1.0 mL / min. The detection wavelength was 360 nm.

[0060] like Figure 2 As shown, HPLC analysis revealed that the retention time of astragalin extracted from tea flowers after extraction and enrichment was 29.84 min, and the purity was 99%.

[0061] Example 2 1. Extracting astragaloside from Camellia chrysanthemi (purchased from Fangchenggang, Guangxi) using the same extraction method as in Example 1.

[0062] 2. The extracted and enriched astragaloside was identified by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS), using the same method as in Example 1. The purified product extracted and separated from Camellia chrysanthemi was identified as astragaloside by UHPLC-MS / MS.

[0063] 3. The purity of the extracted and enriched astragalin was analyzed by high performance liquid chromatography (HPLC), using the same method as in Example 1. HPLC analysis revealed that the retention time of astragalin in Camellia chrysantha after extraction and enrichment was 29.84 min, and the content was 99.0%.

[0064] Example 3: Determination of Collagen Content 1. Construction of a natural growth model of Caenorhabditis elegans Several adult Caenorhabditis elegans containing eggs were collected and synchronized to obtain nematodes of the same life cycle from the same batch. Specific steps included: (1) Washing: Collect the oviposition nematodes into a 1.5 mL centrifuge tube with M9 buffer, centrifuge quickly and remove the supernatant. Wash with M9 buffer 2-3 times to remove culture medium, Escherichia coli and other substances from the surface of the nematodes.

[0065] (2) Lysis: Nematode lysis buffer was prepared fresh and used immediately. The preparation method was as follows: a sodium hypochlorite solution containing 6-14% active chlorine was diluted 10-fold with deionized water and then mixed with an equal volume of 1 M sodium hydroxide solution. 1 mL of the lysis buffer was applied to oviposition-stage nematodes for 5 min. During this time, the solution was gently and repeatedly poured, followed by immediate and rapid centrifugation. The nematodes were washed 2-3 times with M9 buffer to remove the lysis buffer. Finally, less than 100 μL of the residual solution was added to an appropriate amount of M9 buffer and incubated overnight at 20°C. The next day, the solution was collected in a 1.5 mL centrifuge tube, rapidly centrifuged, and the supernatant was removed. The L1 stage nematode precipitate was added to fresh NGM containing food. After incubation at 20°C for 36 h, L4 stage nematodes were obtained.

[0066] 2. Intervention strategies The changes in collagen content of L4-stage Caenorhabditis elegans were compared under natural growth conditions (1, 3, 5 days) by feeding L4-stage Caenorhabditis elegans with crude extracts of tea flower prepared in Example 1 (10, 20, 50 mg / mL), crude extracts of golden camellia flower prepared in Example 2 (10, 20, 50 mg / mL), pure astragaloside prepared in Example 1 (100 μM, 150 μM, 200 μM), a VC positive control, and a buffer blank control (1 mL).

[0067] 3. Determination of hydroxyproline content Sample pretreatment for hydroxyproline content determination: Collect 1000 Caenorhabditis elegans worms each on days 1, 3, and 5. Wash 2-3 times with M9 buffer to remove residual OP50 (Escherichia coli). After leaving 100 μL, add 1 mL of extraction buffer (6 mol / L hydrochloric acid), boil or bake in a 110℃ oven for 2-6 hours until transparent. After cooling, adjust the pH to 6-8 with 10 mol / L NaOH (approximately 0.5 mL) (do not make it too acidic or too alkaline), bring the volume to 2 mL with distilled water, centrifuge at 16000 rpm, 25℃ for 20 min, and collect the supernatant for analysis.

[0068] Sample pretreatment for protein concentration determination: Take 1000 Caenorhabditis elegans larvae from the same period, wash 2-3 times with M9 buffer to remove residual OP50, leaving only 100 μL. Add 10 μL of protease inhibitor (PMSF) and 2% SDS at a 1:1 ratio and mix well. After repeated freeze-thaw cycles in liquid nitrogen, boil in a metal bath at 100℃ for 10 min, then centrifuge at 13000 rpm at 4℃ for 10 min. Collect the supernatant for analysis.

[0069] Hydroxyproline (HYP) content assay kit for measuring collagen content in *C. elegans*: Dilute the kit standards with distilled water to prepare standard solutions with concentrations of 30, 15, 7.5, 3.75, 1.875, 0.938, 0.469, and 0.234 μg / mL, respectively, for later use. Follow the specific procedures outlined in Table 1 below.

[0070] Table 1

[0071] BCA protein concentration assay kit measures sample protein concentration: Preparation of protein standards: a. Add 1.2 mL of protein standard preparation solution to one tube of protein standard (30 mg BSA), dissolve thoroughly, and prepare a 25 mg / mL protein standard solution; b. Take an appropriate amount of 25 mg / mL protein standard and dilute it with PBS to a final concentration of 0.5 mg / mL. Preparation of BCA working solution: According to the number of samples, prepare an appropriate amount of BCA working solution by adding 1 volume of BCA reagent B to 50 volumes of BCA reagent A (50:1) and mix thoroughly.

[0072] Protein concentration detection: a. Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of the standard to the wells of a 96-well plate, and then add standard diluent to bring the total volume to 20 μL. This corresponds to standard concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL, respectively. b. Add an appropriate volume of sample to the wells of the 96-well plate. If the sample volume is less than 20 μL, add standard diluent to bring it to 20 μL, and record the sample volume. c. Add 200 μL of BCA working solution to each well and incubate at 37°C for 30 minutes; d. Measure A562 using an ELISA reader; e. Calculate the protein concentration (Cpr) of the sample based on the standard curve and the sample volume used.

[0073] Formulas for calculating hydroxyproline content and collagen: a. Plotting the standard curve: Plot the standard solution concentration as the x-axis and ΔAstandard (ΔAstandard = Astandard tube - Ablank tube) as the y-axis to obtain the equation y = kx + b. Substitute ΔAmeasured (ΔAmeasured = Ameasured tube - Ablank tube) into the equation to obtain x (μg / mL); b. Calculation of hydroxyproline content: Hydroxyproline content (µg / mg prot) = x × V sample ÷ (Cpr × V sample) = x ÷ Cpr.

[0074] Collagen content calculation formula: Hydroxyproline (Hyp is a non-essential amino acid, one of the main components of collagen, and is generally considered to be a unique amino acid in collagen. It contains 12% hydroxyproline by mass), collagen = hydroxyproline × 8.33.

[0075] 4. Results Analysis like Figure 3 As shown, 10 mg / mL, 20 mg / mL and 50 mg / mL Camellia chrysantha flower extracts can increase the collagen content level in Caenorhabditis elegans, with an effect 2.2-2.7 times that of the blank control group.

[0076] like Figure 4 As shown, tea tree flower extracts at concentrations of 10 mg / mL, 20 mg / mL, and 50 mg / mL increased collagen levels in *C. elegans* by 2.5–2.9 times compared to the control group.

[0077] like Figure 5 As shown, 100 μM astragaloside can increase the collagen content level in *C. elegans*, with an effect 4.6-5.5 times that of the blank control group and 3.6-4.7 times that of the positive control group (100 μM VC). Astragaloside can increase the collagen content level in *C. elegans*, with the level on day 3 being 1.2 times that of day 1 and on day 5 being 2.0 times that of day 3.

[0078] like Figure 6 As shown, 150 μM astragaloside can increase the collagen content level in *C. elegans*, with an effect 5.7-6.1 times that of the blank control group and 3.1-3.9 times that of the positive control group (150 μM VC). Astragaloside can increase the level of collagen content in *C. elegans*, with the level on day 3 being 1.3 times that of day 1 and on day 5 being 2.1 times that of day 3.

[0079] like Figure 7 As shown, 200 μM astragaloside can increase the collagen content level in *C. elegans*, with an effect 6.2-7.5 times that of the blank control group and 3.0-3.4 times that of the positive control group (200 μM VC). Astragaloside can increase the level of collagen content in *C. elegans*, with the level on day 3 being 1.2 times that of day 1 and on day 5 being 2.4 times that of day 3.

[0080] Example 4: Measurement of ATP production 1. The construction of the natural growth model of Caenorhabditis elegans is the same as in Example 3.

[0081] 2. Intervention strategy: The amount of mitochondrial ATP produced in nematodes under natural growth conditions (day 3) was compared by feeding L4 stage Caenorhabditis elegans with 200 μM astragaloside (pure astragaloside prepared in Example 1), VC positive control and buffer blank control 1 mL.

[0082] 3. Measurement of ATP production Sample pretreatment for ATP production determination: Collect 100 nematodes from different groups using a needle and add them to 50 μL of M9 solution. After centrifugation and precipitation, remove as much M9 solution as possible. Add 100–200 μL of ATP lysis buffer, grind thoroughly, and freeze in liquid nitrogen. Storage at -80°C is possible. The collected nematodes were boiled in a 100°C metal bath for 15 min, followed by 5 min on ice. Centrifuged at 14800 g for 10 min at 4°C, and the supernatant was collected for analysis. ATP in the nematodes was detected using a reference ATP detection kit.

[0083] Sample pretreatment for protein concentration determination is the same as in Example 3.

[0084] The BCA protein concentration assay kit was used to measure the protein concentration of the sample, using the same method as in Example 3.

[0085] ATP assay kit test: a. Preparation for standard curve determination: Thaw the reagents to be used on an ice bath, and dilute the ATP standard solution with ATP detection lysis buffer to a gradient concentration of 0.01, 0.03, 0.1, 0.3, 1, 3 and 10 μM; b. Preparation of ATP detection working solution: Prepare an appropriate amount of ATP detection working solution according to the ratio of 100 μL required for each sample or standard. Thaw the reagent to be used on ice. Take an appropriate amount of ATP detection reagent and dilute it with ATP detection reagent diluent at a ratio of 1:4. The diluted ATP detection reagent is the ATP detection working solution used for subsequent experiments, and should be temporarily stored on ice. c. Determination of ATP concentration: Add 100 μL of ATP detection working solution to a 96-well plate. Incubate at room temperature for 3-5 minutes to deplete background ATP, then add 20 μL of sample or standard to each well. Mix quickly with a pipette. After at least 2 seconds, measure the RLU value using a chemiluminescence analyzer. d. Calculate the concentration of ATP in the sample based on the standard curve; e. Based on the protein concentration in the measured sample, convert the ATP concentration into nmol / mg protein.

[0086] 4. Results Analysis like Figure 8As shown, 200 μM astragaloside can increase the level of ATP production in mitochondria of Caenorhabditis elegans, with an effect 1.4 times that of the blank control group and 1.1 times that of the positive control group of 200 μM VC.

[0087] Example 5: ROS Level Measurement 1. The construction of the natural growth model of Caenorhabditis elegans is the same as in Example 3.

[0088] 2. Intervention strategy and method are the same as in Example 4.

[0089] 3. Sample pretreatment for ROS level determination: Collect nematodes in M9 buffer at a rate of 45 nematodes per 10 μL.

[0090] ROS Level Determination with Reactive Oxygen Species (ROS) Detection Kit: Prepare DCFH-DA Stock Solution: Dissolve 24.365 mg DCFH-DA in 1 mL DMSO to form a 50 mM stock solution. Aliquot into foil-wrapped centrifuge tubes and store at -20°C for at least 3 months. Dilute the DCFH-DA stock solution to 50 μM working solution with M9 buffer. Add 40 μL of M9 buffer and 10 μL of the above nematode suspension to each well of a 96-well plate (black background, black border). Then add 50 μL of probe working solution. Using the fluorescence analysis mode of a multi-mode plate reader, gently shake the plate at room temperature for 30 seconds, and then measure the fluorescence signal intensity at an excitation wavelength of 490 nm and an emission wavelength of 540 nm. Measure the fluorescence signal hourly (maximum 4 h). The slope of the fitted straight line reflects the change in ROS levels in nematodes over a certain period, i.e., the ROS treatment capacity of nematodes in different treatment groups.

[0091] 4. Results Analysis like Figure 9 As shown, 200 μM astragaloside can reduce the level of ROS production in mitochondria of Caenorhabditis elegans. After 4 h, it decreased by 0.4 times compared with the blank control group and by 0.1 times compared with the positive control 200 μM VC group.

[0092] Example 6: Autophagy Activity Assay 1. The construction of the natural growth model of Caenorhabditis elegans is the same as in Example 3.

[0093] 2. Intervention strategy and method are the same as in Example 4.

[0094] 3. Autophagy activity assay a. Preparation of nematode agarose pads: Weigh 0.5 g of agarose and dissolve it in 50 mL of 1×TAE buffer solution. Microwave heat until clear and transparent, then place in an 80℃ water bath. Transfer 200 μL to the side of the coverslip and the top of the slide. Use tweezers to hold the coverslip down slowly. After cooling, store in a 4℃ refrigerator for later use. b. Collection of Caenorhabditis elegans: Different treatment groups were... LGG-1 The strains of nematodes were collected and washed on the 3rd day. They were then anesthetized by adding 1 mL of M9 buffer solution containing 5 mM levamisole hydrochloride. After rapid centrifugation, a small amount of the above solution containing nematodes was dropped onto an agarose pad, covered with a coverslip and labeled for microscopic observation. c. Laser confocal microscopy: Using a 40× objective lens (water microscope), observe the overall fluorescence production of the nematode under the green fluorescent protein channel (excitation and emission wavelengths are 488 nm and 510~540 nm, respectively). Take photos of the nematode's head and tail separately for comparison.

[0095] 4. Results Analysis like Figure 10 As shown, 200 μM astragaloside can activate the level of mitochondrial autophagy in Caenorhabditis elegans.

[0096] like Figure 11 As shown, 200 μM astragaloside can activate the level of mitochondrial autophagy in the head of Caenorhabditis elegans.

[0097] like Figure 12 As shown, 200 μM astragaloside can activate mitochondrial autophagy in the tail of Caenorhabditis elegans.

[0098] like Figure 13 As shown, 200 μM astragaloside can activate the overall mitochondrial autophagy level of Caenorhabditis elegans.

[0099] Example 7: NADPH, NADP + NADP + / NADPH level measurement 1. The construction of the natural growth model of Caenorhabditis elegans is the same as in Example 3.

[0100] 2. Intervention strategy and method are the same as in Example 4.

[0101] 3. NADPH, NADP + NADP + Sample pretreatment for NADPH level determination: Collect 4000 Caenorhabditis elegans worms on day 3, wash 2-3 times with M9 buffer to remove residual OP50, leaving only 100 μL, then add 200 μL of NADP. + The NADPH extract was homogenized on ice. Then, it was centrifuged at 12000×g at 4℃ for 5-10 minutes, and the supernatant was collected as the sample to be tested.

[0102] Sample pretreatment for protein concentration determination: The method is the same as in Example 3.

[0103] NADP + / NADPH detection kit measures NADPH and NADP in samples + NADP + / NADPH level: a. Preparation of NADPH standard: Dissolve 5 mg of NADPH provided in this kit in 6 mL of ultrapure water to obtain 1 mM NADPH standard. Aliquot the 1 mM NADPH standard appropriately and store at -80°C protected from light. b. Setting up the NADPH standard curve: Mix 1 mM NADPH standard with NADP... + The NADPH extract was diluted to concentration gradients of 0, 0.25, 0.5, 1, 2, and 4 μM, and 50 μL of each was added to a 96-well plate, equivalent to 0, 12.5, 25, 50, 100, and 200 pmol of NADPH per well. The point with a concentration of 0 μM served as a blank control. Note: Because NADPH is very unstable, it should be used as soon as possible after preparation. c. Preparation of G6PDH working solution: Dilute G6PDH 50 times with reaction buffer, add 100 μL of G6PDH working solution to each standard or sample, and prepare fresh according to the number of standards and samples to be tested.

[0104] NADPH, NADP + NADP + / NADPH level measurement: a. Pipette 50 μL of the sample to be tested, diluted with NADP+ / NADPH extraction buffer, into a 96-well plate and set up duplicate wells for the sample.

[0105] b. NADP in the sample + The content of NADPH or NADP + Determination of the NADPH ratio: Pipette 100-200 μL of the sample to be tested into a centrifuge tube and heat in a 60℃ water bath for 30 minutes to decompose NADP. + The remaining sample should be stored at 4°C and equilibrated to room temperature before analysis. If insoluble matter is produced after heating, centrifuge at 10000×g at 4°C for 5 minutes, and then take 50 μL and dilute with NADP. + The supernatant after dilution of the NADPH extract was used as the sample to be tested in a 96-well plate, and duplicate wells were set up for the sample. c. Incubate at 37°C in the dark for 10 minutes to remove NADP from the sample. + Converted to NADPH; d. Mix the colorimetric solution appropriately, then add 10 μL of the solution to each well, mix well, and incubate at 37°C in the dark for 10–20 minutes. An orange-yellow formazan will form at this time. Measure the absorbance at 450 nm. Follow the specific procedures outlined in Table 2 below.

[0106] Table 2

[0107] The BCA protein concentration assay kit was used to measure the protein concentration of the sample, using the same method as in Example 3.

[0108] Calculation of NADP+ / NADPH levels in the sample: a. Calculate the average absorbance of each point in the standard group, subtract the absorbance of the blank control group, and the result is the absorbance of each standard. b. Plot a standard curve with NADPH concentration on the x-axis and absorbance on the y-axis; c. Calculate the total concentration of NADP+ and NADPH in the sample (NADPtotal) or the concentration of NADPH based on the standard curve, and calculate the concentration of NADP+ based on the detected concentration and the volume of the sample; d. The calculation formula is [NADP] + ] = [NADPtotal] - [NADPH]; [NADP + ] / [NADPH]=([NADP total ]-[NADPH]) / [NADPH]; e. The obtained NADPH and NADP + The content is compared to the protein concentration of the corresponding sample, and finally expressed as NADP per unit protein mass. + It is expressed as the total amount of NADPH or the individual content of each component.

[0109] 4. Results Analysis like Figure 14 As shown, the mitochondria of *C. elegans* treated with 200 μM astragaloside produced the highest level of NADPH, which was 0.4 times higher than the 200 μM VC positive control group and 2.9 times higher than the blank control group.

[0110] like Figure 15 As shown, NADP produced by the mitochondria of *C. elegans* after being administered 200 μM astragaloside. + The level was the lowest, 0.3 times lower than the 200 μM VC positive control group and 1.0 times lower than the blank control group.

[0111] like Figure 16 As shown, NADP in the mitochondria of *C. elegans* treated with 200 μM astragaloside +The NADPH ratio was the lowest, 1.4 times lower than the 200 μM VC positive control group and 17.4 times lower than the blank control group.

[0112] Example 8: Detection of expression of genes related to mitophagy 1. The construction of the natural growth model of Caenorhabditis elegans is the same as in Example 3.

[0113] 2. Intervention strategy and method are the same as in Example 4.

[0114] 3. Sample pretreatment for detecting mitophagy-related gene expression: Mitophagy-related genes were collected through literature review, as follows: LGG-1 It is a core protein in the autophagy process, mainly involved in the formation and expansion of autophagosomes. It is homologous to LC3 protein (microtubule-associated protein 1A / 1B-light chain 3, LC3).

[0115] DCT-1 It plays a crucial role in mitophagy, responsible for recognizing damaged mitochondria and guiding them to the autophagic pathway for degradation. It shares homology with mammalian BNIP3 and NIX proteins.

[0116] PINK1 PTEN-induced kinase 1 (PTEN-1) is a mitochondrial-associated serine / threonine kinase that plays a crucial role in maintaining mitochondrial quality control and protecting cells from mitochondrial damage. In human cells, its function is closely related to autophagy and mitophagy, particularly playing a vital role in the clearance of damaged mitochondria.

[0117] Primer sequence information is shown in Table 3 below. The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. The expression levels of autophagy-related gene mRNAs were determined using RT-qPCR.

[0118] Nearly 1000 adult *C. elegans* worms per group were collected after 3 days of treatment with 200 μM VC and 200 μM astragaloside (with NGM containing 200 μM FUDR used to inhibit progeny reproduction). The worms were then centrifuged in M9 buffer, and the supernatant was removed. The tubes were washed with M9 buffer at least three times until no obvious flocculent matter remained in the supernatant, and the supernatant was removed as much as possible. Subsequent operations were performed on ice. After rapid centrifugation, the tubes were transferred to enzyme-free EP tubes, 1 mL of RNAiso Plus was added, vortexed for 30 s, and then placed in liquid nitrogen. The tubes were thawed at 40–60 °C and then placed in liquid nitrogen, repeated several times. Finally, the tubes were thawed for 30 s and placed on ice for 30 s. This process was repeated until completely thawed. After placing the EP tubes at room temperature for 5 min, 200 μL of chloroform was added to a fume hood, vortexed for 30 s, incubated for 3 min, and then centrifuged at 12000 g for 15 min at 4 °C. At this point, the sample separates into three layers: a bottom layer of Trizol (pink), a middle layer of protein (white), and an upper clear layer (RNA layer). Transfer the supernatant (approximately 400–450 μL) to a new enzyme-free EP tube, being careful not to aspirate the protein layer. In a clean bench, add an equal volume of isopropanol reagent to the supernatant, mix gently, and incubate for 10 min. Centrifuge at 12000 g for 15 min at 4°C. Carefully aspirate the supernatant from the inside of the enzyme-free EP tube off-axis. Wash the precipitate with 1 mL of ice-cold 75% ethanol (prepared with DEPC water). Centrifuge at 12000 g for 5 min at 4°C. Collect the supernatant, invert the enzyme-free EP tube, and allow it to air dry for 10 min until a faint halo appears around the precipitate. Resuspend the precipitate in 25–100 μL of DEPC water, ensuring a concentration of 250–1000 ng / μL. Obtain absorbance readings at 260 and 280 nm using NanoDrop. OD 260 / 280 A value between 1.9 and 2.1 indicates that the RNA purity is relatively good.

[0119] Table 3

[0120] Detection of mRNA expression of genes related to mitochondrial autophagy: a. Following the SPARKscript II RT Plus Kit instructions, the extracted total RNA was reverse-transcribed into cDNA. A 20 μL system was prepared on ice and subjected to temperature-controlled PCR using a standard PCR instrument. The system consisted of: 4 μL Total RNA, 1 μL gDNA Eraser, and 5 μL RNase-Free H2O. After removing the gDNA, 10 μL Mix was added for reverse transcription. The cDNA was aliquoted into 5 μL tubes and stored at -20°C for later use. b. Following the instructions for the 2×SYBR Green qPCR Mix, prepare a 13 μL qPCR system on ice: 3.6 μL LNase-Free H2O, 5 μL 2×SYBR qPCR Mix, 1.2 μL ROX Reference Dye II, 1.2 μL 10 μM forward and reverse primers, and 0.8 μL cDNA stock solution. Seal the PCR reaction plate (96 wells) and run it on the instrument. Set the PCR reaction program: select SYBR Green dye as the chemical reagent, and use the instrument's default melting curve acquisition program; pre-denaturation at 94℃ for 2.5 min. PCR cycling: denaturation at 94℃ for 10 s, annealing at 60℃ for 30 s, for 40 cycles, collecting fluorescence signals during annealing.

[0121] 4. Results Analysis like Figure 17 As shown, 200 μM astragaloside can promote the mitochondrial autophagy gene expression in *C. elegans*. LGG- 1 The expression level of mRNA was 2.0 times higher than that of the 200 μM VC positive control group and 5.6 times higher than that of the blank control group.

[0122] like Figure 18 As shown, 200 μM astragaloside can promote the mitochondrial autophagy gene expression in *C. elegans*. DCT- 1 The expression level of mRNA was 0.9 times higher than that of the 200 μM VC positive control group and 7.1 times higher than that of the blank control group.

[0123] like Figure 19 As shown, 200 μM astragaloside can promote the mitochondrial autophagy gene expression in *C. elegans*. PINK- 1 The expression level of mRNA was 0.1 times higher than that of the 200 μM VC positive control group and 0.3 times higher than that of the blank control group.

[0124] Example 9: Expression of collagen synthesis-related genes 1. The construction of the natural growth model of Caenorhabditis elegans is the same as in Example 3.

[0125] 2. Intervention strategy and method are the same as in Example 4.

[0126] 3. Sample pretreatment for detecting the expression of collagen synthesis-related genes in *Caenorhabditis elegans* was performed using the same method as in Example 8. Primer sequence information is shown in Table 4 below.

[0127] COL-12 and COL-120 Belongs to a large number of proteins that encode the structure of the stratum corneum COLThe collagen family is part of the stratum corneum and is part of the collagen trimer. Collagen plays an important role in maintaining the stratum corneum.

[0128] Table 4

[0129] The mRNA expression of collagen synthesis-related genes in Caenorhabditis elegans was detected using the same method as in Example 8.

[0130] 4. Results Analysis like Figure 20 As shown, 200 μM astragaloside can promote the collagen synthesis gene in *C. elegans*. COL- 12 The expression level of mRNA was 1.6 times higher than that of the 200 μM VC positive control group and 8.6 times higher than that of the blank control group.

[0131] like Figure 21 As shown, 200 μM astragaloside can promote the collagen synthesis gene in *C. elegans*. COL- 120 The expression level of mRNA was not increased compared to the 200 μM VC positive control group, but it was increased by 1 time compared to the blank control group.

[0132] Example 10: Collagen-regulated gene expression 1. The construction of the natural growth model of Caenorhabditis elegans is the same as in Example 3.

[0133] 2. Intervention strategy and method are the same as in Example 4.

[0134] 3. Sample pretreatment for detecting the expression of collagen-regulated genes in Caenorhabditis elegans: The method is the same as in Example 8, and the primer sequence information is shown in Table 5 below.

[0135] EMB-9 The gene encodes a protein homologous to human type IV collagen, primarily encoding the α1 chain of type IV collagen. The function of type IV collagen depends on its triple helix structure composed of α1 and α2 chains. EMB-9 It is a gene that inhibits collagen production to prevent excessive deposition and accumulation of type IV collagen, which can induce diseases.

[0136] LET-2 The gene encodes a protein homologous to human type IV collagen, primarily encoding the α2 chain of type IV collagen. LET-2 It is an activating gene for the synthesis of type IV collagen.

[0137] The function of type IV collagen depends on the triple helix structure composed of α1 and α2 chains.

[0138] Table 5

[0139] The expression of mRNA related to collagen regulation in Caenorhabditis elegans was detected using the same method as in Example 8.

[0140] 4. Results Analysis like Figure 22 As shown, 200 μM astragaloside can inhibit collagen regulatory genes in Caenorhabditis elegans. EMB- 9 The expression level of mRNA did not increase the inhibition rate compared to the 200 μM VC positive control group, but it increased by 0.3 times compared to the blank control group.

[0141] like Figure 23 As shown, 200 μM astragaloside can promote the collagen regulatory gene in *C. elegans*. LET- 2 The expression level of mRNA was 1.4 times higher than that of the 200 μM VC positive control group and 10.1 times higher than that of the blank control group.

[0142] The results above indicate that the use of astragaloside can promote the synthesis of type IV collagen.

[0143] Example 11: Detection of expression of matrix metalloproteinase synthesis genes 1. The construction of the natural growth model of Caenorhabditis elegans is the same as in Example 3.

[0144] 2. Intervention strategy and method are the same as in Example 4.

[0145] 3. Sample pretreatment for detecting matrix metalloproteinase synthesis gene expression in Caenorhabditis elegans: The method is the same as in Example 8. Primer sequence information is shown in Table 6 below.

[0146] ZMP-3 This refers to the homologous genes of matrix metalloproteinases 1, 3, and 8 in *C. elegans*. ZMP-3 This gene encodes a matrix metalloproteinase that can initiate matrix metalloproteinase activity and is expected to be involved in collagen catabolism and extracellular matrix tissue processes. This gene is an ortholog of several human genes, including MMP1 (matrix metalloproteinase 1), MMP3 (matrix metalloproteinase 3), and MMP8 (matrix metalloproteinase 8).

[0147] ZMP-4 This refers to the homologous genes of matrix metalloproteinases 2, 9, and 23B in *C. elegans*. ZMP-4It encodes another matrix metalloproteinase, which can initiate matrix metalloproteinase activity and is expected to be involved in collagen catabolism and extracellular matrix tissue formation. This gene is an ortholog of several human genes, including MMP2 (matrix metalloproteinase 2), MMP9 (matrix metalloproteinase 9), and MMP23B (matrix metalloproteinase 23B).

[0148] Table 6

[0149] The mRNA expression of the matrix metallocollagenase synthesis gene in Caenorhabditis elegans was detected using the same method as in Example 8.

[0150] 4. Results Analysis like Figure 24 As shown, 200 μM astragaloside can inhibit the matrix metallocollagenase synthesis gene in Caenorhabditis elegans. ZMP-3 The expression level of mRNA was 0.4 times higher than that of the 200 μM VC positive control group and 0.8 times higher than that of the blank control group.

[0151] like Figure 25 As shown, 200 μM astragaloside can inhibit the matrix metallocollagenase synthesis gene in Caenorhabditis elegans. ZMP-4 The expression level of mRNA was 0.7 times higher than that of the 200 μM VC positive control group and 0.9 times higher than that of the blank control group.

[0152] While the specific embodiments of the present invention have been described above in conjunction with examples, they are not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that any modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection of the present invention.

Claims

1. The use of astragaloside or plant extracts containing astragaloside in the preparation of pharmaceuticals, cosmetics or food for the prevention and / or delay of skin aging.

2. The application as described in claim 1, characterized in that, The signs of skin aging include: decreased skin elasticity, sagging skin, and wrinkles.

3. The application as described in claim 1, characterized in that, The astragaloside is derived from plant extracts.

4. The application as described in claim 3, characterized in that, The plant in question is an edible flower, specifically a tea tree flower or a golden camellia.

5. The application as described in claim 1, characterized in that, Skin aging is caused by collagen loss. Astragalus glycosides promote collagen synthesis and reduce collagen loss, thus achieving an anti-aging effect on the skin.

6. The application as described in claim 5, characterized in that, Astragaloside further promotes the synthesis of proline, a collagen precursor, by increasing the level of NADPH in the body.

7. The application as described in claim 6, characterized in that, Astragaloside activates mitochondrial autophagy genes LGG-1 and DCT-1 This promotes the synthesis of mitochondrial NADPH, providing reducing power for proline.

8. The application as described in claim 1, characterized in that, The drug also includes a pharmaceutically acceptable carrier and is formulated as an oral preparation.

9. The application as described in claim 1, characterized in that, The cosmetics also include carriers or excipients acceptable in the cosmetics field, and are formulated in the form of creams, lotions, aqueous solutions, gels, oils, aerosols, or films.

10. The application as described in claim 1, characterized in that, The food also includes food science-acceptable carriers, and the formulation is in the form of oral liquid, tablets, capsules or solid beverage.

Images