Use of a trimethylamine liver metabolite in evaluating the anti-aging ability of food

Abstract

The present application relates to the technical field of food and cosmetic efficacy evaluation, and particularly relates to application of trimethylamine liver metabolite in evaluation of anti-aging ability of food. The present application firstly induces HSF and HaCaT cells by using trimethylamine liver metabolite to obtain an oxidative damage cell model; then extracts a mother liquor by using extracts of anti-aging functional food, dilutes the mother liquor after sterilization to obtain a diluted mother liquor; pretreats HSF and HaCaT cells by using the diluted mother liquor; after the pretreatment, discards the diluted mother liquor, washes HSF and HaCaT cells by using PBS, then adds a mixed solution of trimethylamine liver metabolite and complete culture medium to act on the cells, and finally evaluates the anti-aging effect of food by detecting cell toxicity, cell antioxidant enzyme activity, cell oxidative stress level and cell inflammatory factor and collagen secretion level, so that a more extensive anti-aging evaluation method is provided.

Description

Technical Field

[0001] This invention relates to the field of food and cosmetic efficacy evaluation technology, and in particular to the application of a trimethylamine liver metabolite in evaluating the anti-aging ability of food. Background Technology

[0002] Choline, acetylcholine, L-carnitine, and lecithin are substances abundant in red meat, egg yolks, dairy products, and seafood. These substances are metabolized in the digestive tract to produce the precursor trimethylamine, which is then released as trimethylamine by trimethylamine lyase. After being absorbed by the animal's body, the resulting trimethylamine is rapidly transported to the liver via the portal vein for further metabolism. Its metabolites induce oxidative stress in cells, promote the release of inflammatory factors, induce cellular senescence, and cause damage to the body.

[0003] Cellular senescence, especially skin cell senescence, is the most direct external manifestation of aging, a process caused by both endogenous and exogenous changes. The skin serves as a protective layer for internal organs; exposure to various damaging factors, including free radicals, can lead to oxidative damage to various macromolecules, disrupting vital cellular processes and increasing mutations. Oxidative stress occurs when the amount of oxidants exceeds the capacity of the antioxidant defense system. Oxidative stress is considered a contributing factor to the aging process and the pathogenesis of various skin diseases. Free radicals, as byproducts of the mitochondrial aerobic metabolism electron transport chain, are continuously produced and are considered a major cause of aging besides genetic factors. Free radicals activate numerous signaling pathways, leading to reduced collagen production, activation and synthesis of matrix metalloproteinases (MMPs), connective tissue degradation, and the secretion of age-related secretory phenotypes, ultimately promoting skin aging. Aging skin becomes dry, dysfunctional, and increases the risk of skin diseases and malignant skin tumors.

[0004] Currently, the efficacy evaluation of anti-aging foods and cosmetics mainly uses animal models, cell models, and 3D skin models. Since 2013, the European Union has completely banned the sale of any cosmetics that have undergone safety testing on animals. Although animal testing has not been abolished in my country's cosmetics industry, it is strictly controlled by a series of regulations such as the "Regulations on the Management of Experimental Animals of the People's Republic of China." Balancing scientific objectives and animal welfare, reducing animal testing is an inevitable trend. 3D skin models have high requirements for modeling technology and are complex to operate. Cell models are relatively simple and easy to operate. However, the establishment of these models usually uses ultraviolet light, hydrogen peroxide, and D-galactose for induction, which has certain limitations in induction methods. Therefore, it is necessary to further understand the numerous induction factors that affect skin health. Summary of the Invention

[0005] To address the aforementioned problems, the present invention aims to provide an application of trimethylamine liver metabolites in evaluating the anti-aging capabilities of food products. The invention first induces oxidative damage in HSF and HaCaT cells using trimethylamine liver metabolites. Then, an extract from an anti-aging functional food product is used to prepare a stock solution. This stock solution is sterilized and diluted to obtain a diluted stock solution. HSF and HaCaT cells are pretreated with this diluted stock solution. After pretreatment, the diluted stock solution is discarded, and the HSF and HaCaT cells are washed with PBS. A mixture of trimethylamine liver metabolites and complete culture medium is then added to the cells. The anti-aging effects of the food product are evaluated by detecting cytotoxicity, cellular antioxidant enzyme activity, cellular oxidative stress levels, and cellular inflammatory factors and collagen secretion levels, providing a broader method for evaluating anti-aging effects.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] This invention provides an application of trimethylamine liver metabolites in evaluating the anti-aging ability of food, comprising the following steps:

[0008] (1) Trimethylamine liver metabolites were used to induce HSF and HaCaT cells to obtain an oxidative damage cell model.

[0009] (2) Take the extract of anti-aging functional food to prepare the mother liquor, and dilute the mother liquor after sterilization to obtain the diluted mother liquor;

[0010] (3) Use the diluted mother liquor obtained in step (2) to pretreat HSF and HaCaT cells;

[0011] (4) After the pretreatment was completed, the diluted mother solution was discarded and HSF and HaCaT cells were washed with PBS;

[0012] (5) The mixture of trimethylamine liver metabolites and complete culture medium was applied to washed HSF and HaCaT cells.

[0013] (6) Untreated HSF and HaCaT cells were used as the control group, the oxidative damage cell model obtained in step (1) was used as the experimental group, and the HSF and HaCaT cells obtained in step (5) were collected as the intervention group. The cellular antioxidant capacity, the secretion level of cellular inflammatory factors TNF-α and IL-6, and the secretion level of cellular CoL I and MMP-1 were detected in each group to evaluate the anti-aging ability of the food.

[0014] In one embodiment of the present invention, in step (1), during the induction process, the concentration of trimethylamine liver metabolites is 150-300 mM.

[0015] In one embodiment of the present invention, in step (1), the induction time is 18-36 hours.

[0016] In one embodiment of the present invention, in step (2), the mother liquor is a mixed solution of 10 mg of extract and 10 mL of complete culture medium;

[0017] The complete culture medium is a mixture of 45 mL DMEM basal medium, 5 mL fetal bovine serum, and 500 μL penicillin-streptomycin solution.

[0018] In one embodiment of the present invention, in step (2), the concentration of the mother liquor is 1 mg / mL.

[0019] In one embodiment of the present invention, in step (2), the concentration of the diluted mother liquor is 1.56-6.25 μg / mL.

[0020] In one embodiment of the present invention, in step (3), the pretreatment time is 12-18 hours.

[0021] In one embodiment of the present invention, in step (5), the volume ratio of trimethylamine liver metabolites to complete culture medium is 3:10.

[0022] In one embodiment of the present invention, the action time in step (5) is 18-36 hours.

[0023] In one embodiment of the present invention, in step (6), during evaluation, when the experimental group has lower cellular antioxidant capacity, higher levels of cellular inflammatory factors TNF-α and IL-6, lower levels of cellular CoL I secretion and higher levels of MMP-1 secretion compared with the control group, the anti-aging ability of the intervention group is further evaluated.

[0024] Compared with the experimental group, the intervention group showed stronger cellular antioxidant capacity, lower levels of cellular inflammatory factors TNF-α and IL-6, higher levels of cellular CoL I secretion, and lower levels of MMP-1 secretion, indicating that the food had strong anti-aging ability.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) Trimethylamine liver metabolites are closely related to the food people consume in their daily diet, especially people who consume a high proportion of phospholipid foods such as fish, meat and soy products. The experimental operation based on cell models is simple and easy to control, effectively avoiding environmental pollution and pain, and also effectively reducing the harm to the health of experimental operators.

[0027] (2) The method of the present invention screens out trimethylamine liver metabolites at a concentration of 150-300mM through cytotoxicity experiments, and evaluates the effect by detecting cytotoxicity, cellular antioxidant enzyme activity, cellular oxidative stress level, and cellular inflammatory factors and collagen secretion level, providing a broader evaluation method for anti-aging. Attached Figure Description

[0028] Figure 1 The following graphs show the cell viability of HSF cells after treatment with different concentrations of trimethylamine liver metabolites for 24 hours, as determined by the MTT assay: a. HSF cell viability after H2O2 treatment; b. HSF cell viability after treatment with trimethylamine liver metabolites.

[0029] Figure 2 The following graphs show the cell viability of HaCaT cells after treatment with different concentrations of trimethylamine liver metabolites for 24 hours, as determined by the MTT assay: a. HaCaT cell viability after H2O2 treatment; b. HaCaT cell viability after treatment with trimethylamine liver metabolites.

[0030] Figure 3 HSF cells were treated with three concentrations of trimethylamine liver metabolites (low, medium, and high) selected in this invention for 24 hours. The activities of several antioxidant enzymes, total antioxidant capacity, and the content of lipid metabolite MDA were then detected using a multifunctional microplate reader. The graphs show: a. Reduced glutathione (GSH) content; b. Superoxide dismutase (SOD) activity; c. Catalase (CAT) activity; d. Total antioxidant capacity (T-AOC); e. Malondialdehyde (MDA) content.

[0031] Figure 4 After treating HaCaT cells with three concentrations (low, medium, and high) of trimethylamine liver metabolites selected in this invention for 24 hours, the activities of several antioxidant enzymes, total antioxidant capacity, and the content of lipid metabolite MDA in the cells were detected using a multifunctional microplate reader. The graphs show: a. Reduced glutathione (GSH) content; b. Superoxide dismutase (SOD) activity; c. Catalase (CAT) activity; d. Total antioxidant capacity (T-AOC); e. Malondialdehyde (MDA) content.

[0032] Figure 5 The graphs above show the fluorescence intensity of intracellular free radicals after HSF cells were treated with trimethylamine liver metabolites for 24 hours, as measured by an inverted fluorescence microscope after loading the DCFH-DA fluorescent probe. The graphs are: a) Blank control group; b) Damage control group; c) 200mM trimethylamine liver metabolite treatment group; d) 250mM trimethylamine liver metabolite treatment group; e) 300mM trimethylamine liver metabolite treatment group.

[0033] Figure 6 The following is a graph showing the fluorescence intensity of intracellular free radicals after HaCaT cells were treated with trimethylamine liver metabolites for 24 hours, using an inverted fluorescence microscope to detect the fluorescence intensity of the DCFH-DA fluorescent probe: a. Blank control group; b. Damage control group; c. 150mM trimethylamine liver metabolite treatment group; d. 200mM trimethylamine liver metabolite treatment group; e. 250mM trimethylamine liver metabolite treatment group.

[0034] Figure 7 The free radical levels in HSF cells were detected by flow cytometry after 24 h of treatment with trimethylamine liver metabolites in this invention; a. DCFH-DA fluorescence intensity map; b. Free radical fluorescence intensity map of each group relative to the blank control.

[0035] Figure 8 The free radical levels in HaCaT cells were detected by flow cytometry after 24 h of treatment with trimethylamine liver metabolites in this invention; a. DCFH-DA fluorescence intensity map; b. Free radical fluorescence intensity map of each group relative to the blank control.

[0036] Figure 9 The images show the secretion levels of intracellular inflammatory factors TNF-α and IL-6 in HSF cells after 24 hours of treatment with trimethylamine liver metabolites, as determined by ELISA. a. IL-6 secretion level; b. TNF-α secretion level.

[0037] Figure 10 The images show the secretion levels of intracellular inflammatory factors TNF-α and IL-6 in HaCaT cells after treatment with trimethylamine liver metabolites for 24 hours, as determined by ELISA. a. IL-6 secretion level; b. TNF-α secretion level.

[0038] Figure 11 The following diagrams show the secretion levels of type I collagen (CoL I) and matrix metalloproteinase 1 (MMP-1) in HSF cells after 24 hours of treatment with trimethylamine liver metabolites in this invention, obtained by ELISA: a. Type I collagen (CoL I) secretion level; b. MMP-1 secretion level.

[0039] Figure 12 The following diagrams show the secretion levels of type I collagen (CoL I) and matrix metalloproteinase 1 (MMP-1) in HaCaT cells after treatment with trimethylamine liver metabolites for 24 hours in this invention, obtained by ELISA: a. Type I collagen (CoL I) secretion level; b. MMP-1 secretion level.

[0040] Figure 13 The graphs show the activities of several antioxidant enzymes, total antioxidant capacity, and the content of lipid metabolite MDA in HSF cells after OPC intervention: a. Reduced glutathione (GSH) content; b. Superoxide dismutase (SOD) activity; c. Catalase (CAT) activity; d. Total antioxidant capacity (T-AOC); e. Malondialdehyde (MDA) content.

[0041] Figure 14 The graphs show the activities of several intracellular antioxidant enzymes, total antioxidant capacity, and the content of lipid metabolite MDA in HaCaT cells after OPC intervention: a. Reduced glutathione (GSH) content; b. Superoxide dismutase (SOD) activity; c. Catalase (CAT) activity; d. Total antioxidant capacity (T-AOC); e. Malondialdehyde (MDA) content.

[0042] Figure 15 The graphs show the intracellular free radical fluorescence intensity after OPC intervention in HSF cells: a. Free radical fluorescence intensity graph; b. Free radical fluorescence intensity graph (relative to blank control).

[0043] Figure 16 The graphs show the intracellular free radical fluorescence intensity after OPC intervention in HaCaT cells: a. Free radical fluorescence intensity; b. Free radical fluorescence intensity (relative to the blank control).

[0044] Figure 17 The diagram shows the secretion levels of intracellular inflammatory factors TNF-α and IL-6 in HSF cells after OPC intervention; a. IL-6 secretion level; b. TNF-α secretion level.

[0045] Figure 18 The graphs show the secretion levels of intracellular inflammatory factors TNF-α and IL-6 in HaCaT cells after OPC intervention; a. IL-6 secretion level; b. TNF-α secretion level.

[0046] Figure 19 Figure 1 shows the secretion levels of type I collagen (CoLⅠ) and matrix metalloproteinase 1 (MMP-1) in HSF cells after OPC intervention; a. Secretion level of type I collagen (CoLⅠ); b. Secretion level of matrix metalloproteinase 1 (MMP-1).

[0047] Figure 20Figure 1 shows the secretion levels of type I collagen (CoLⅠ) and matrix metalloproteinase 1 (MMP-1) in HaCaT cells after OPC intervention; a. Secretion level of type I collagen (CoLⅠ); b. Secretion level of matrix metalloproteinase 1 (MMP-1). Detailed Implementation

[0048] This invention provides an application of trimethylamine liver metabolites in evaluating the anti-aging ability of food, comprising the following steps:

[0049] (1) Trimethylamine liver metabolites were used to induce HSF and HaCaT cells to obtain an oxidative damage cell model.

[0050] (2) Take the extract of anti-aging functional food to prepare the mother liquor, and dilute the mother liquor after sterilization to obtain the diluted mother liquor;

[0051] (3) Use the diluted mother liquor obtained in step (2) to pretreat HSF and HaCaT cells;

[0052] (4) After the pretreatment was completed, the diluted mother solution was discarded and HSF and HaCaT cells were washed with PBS;

[0053] (5) The mixture of trimethylamine liver metabolites and complete culture medium was applied to washed HSF and HaCaT cells.

[0054] (6) Untreated HSF and HaCaT cells were used as the control group, the oxidative damage cell model obtained in step (1) was used as the experimental group, and the HSF and HaCaT cells obtained in step (5) were collected as the intervention group. The cellular antioxidant capacity, the secretion level of cellular inflammatory factors TNF-α and IL-6, and the secretion level of cellular CoL I and MMP-1 were detected in each group to evaluate the anti-aging ability of the food.

[0055] In one embodiment of the present invention, in step (1), during the induction process, the concentration of trimethylamine liver metabolites is 150-300 mM.

[0056] In one embodiment of the present invention, in step (1), the induction time is 18-36 hours.

[0057] In one embodiment of the present invention, in step (2), the mother liquor is a mixed solution of 10 mg of extract and 10 mL of complete culture medium;

[0058] The complete culture medium is a mixture of 45 mL DMEM basal medium, 5 mL fetal bovine serum, and 500 μL penicillin-streptomycin solution.

[0059] In one embodiment of the present invention, in step (2), the concentration of the mother liquor is 1 mg / mL.

[0060] In one embodiment of the present invention, in step (2), the concentration of the diluted mother liquor is 1.56-6.25 μg / mL.

[0061] In one embodiment of the present invention, in step (3), the pretreatment time is 12-18 hours.

[0062] In one embodiment of the present invention, in step (5), the volume ratio of trimethylamine liver metabolites to complete culture medium is 3:10.

[0063] In one embodiment of the present invention, the action time in step (5) is 18-36 hours.

[0064] In one embodiment of the present invention, in step (6), during evaluation, when the experimental group has lower cellular antioxidant capacity, higher levels of cellular inflammatory factors TNF-α and IL-6, lower levels of cellular CoL I secretion and higher levels of MMP-1 secretion compared with the control group, the anti-aging ability of the intervention group is further evaluated.

[0065] Compared with the experimental group, the intervention group showed stronger cellular antioxidant capacity, lower levels of cellular inflammatory factors TNF-α and IL-6, higher levels of cellular CoL I secretion, and lower levels of MMP-1 secretion, indicating that the food had strong anti-aging ability.

[0066] The raw materials used in this invention, human skin fibroblasts (HSF) and keratinocytes (HaCaT), were donated by the Edible Fungi Research Institute of Shanghai Academy of Agricultural Sciences. Unless otherwise specified, all other materials are conventional commercial products. Unless otherwise specified, the methods used in this invention are conventional methods in the field.

[0067] Example 1

[0068] This example demonstrates the preparation of experimental reagents.

[0069] (1) Prepare a phosphate buffer solution (PBS) with a pH of 7.4.

[0070] Weigh out 8.00g of NaCl, 0.20g of KCl, 0.24g of KH2PO4, and 1.58g of Na2HPO4 into a clean 1L beaker, add 500mL of deionized water, sonicate in a water bath until completely dissolved, transfer to a volumetric flask and bring the volume to 1L. Take 200mL of each solution into a clean dispensing bottle, autoclave at 120℃, cool to room temperature, seal in a laminar flow hood, and store in a 4℃ refrigerator for later use.

[0071] (2) Preparation of complete culture medium

[0072] Take a 50mL sterile centrifuge tube, add 45mL DMEM basal medium + 5mL fetal bovine serum + 500μL penicillin-streptomycin solution (double antibiotic) (i.e., 1% double antibiotic and 10% fetal bovine serum), mix well, and the complete culture medium is stored in a 4℃ refrigerator for later use.

[0073] Example 2

[0074] This embodiment provides HSF and HaCaT cell resuscitation, passage, and cryopreservation.

[0075] (1) Cell resuscitation

[0076] Preheat the water bath to 37°C to maintain a constant temperature, and place the culture flask containing 6 mL of complete culture medium in a 37°C incubator for 30 minutes. Remove the HSF cells frozen in liquid nitrogen and rapidly agitate them in the 37°C water bath to completely thaw the cryopreservation solution within 2 minutes. Wipe the walls of the cryovials thoroughly with 75% alcohol to prevent contamination. Transfer the cryopreservation solution to a 15 mL sterile centrifuge tube in a laminar flow hood, add 3 mL of complete culture medium, pipette to mix, centrifuge at 1000 rpm for 3 minutes, discard the supernatant, add 1 mL of complete culture medium to resuspend the cells, and finally transfer to the preheated culture flask, mix well, and incubate at 37°C. On the second day (within 24 hours), after the cells have adhered to the culture vessel, discard the old culture medium, add 6 mL of fresh complete culture medium, and continue culturing in the incubator.

[0077] (2) Cell passage

[0078] Observe cell growth under an inverted microscope. Once cell confluence reaches 80%, typically after 3 days, cell passage can be performed. Discard the old culture medium, add 3 mL of sterile PBS buffer solution to wash the cells three times, and discard any residual liquid. Add 1 mL of trypsin and spread it evenly on the bottom of the culture flask. Incubate in an incubator for 5 minutes. Under a microscope, observe that the cells are in a round, single-cell state. Gently tap the bottom of the culture flask to detach the cells from the bottom, and immediately add 2 mL of complete culture medium. Mix well by pipetting, transfer to a 15 mL sterile centrifuge tube, and centrifuge at 1000 rpm for 3 minutes. Discard the supernatant. Add 2 mL of complete culture medium to resuspend the cells. Take two new culture flasks, add 1 mL of cell suspension to each, then add 6 mL of complete culture medium. Mix well by pipetting and place in an incubator at 37°C for further culture.

[0079] (3) Cell cryopreservation

[0080] Prepare a cell cryopreservation solution in a 9:1 ratio (e.g., 900 μL fetal bovine serum + 100 μL dimethyl sulfoxide (DMSO)) for later use. When HSF cells are in the logarithmic growth phase and the cell confluence reaches 80%, discard the old culture medium, wash the cells three times with PBS buffer, digest with trypsin, centrifuge, discard the supernatant, and aspirate all residual liquid, retaining the cell pellet. Add 2 mL of cryopreservation solution to each bottle of cell pellet to resuspend the cells and mix well by pipetting. Take two 2 mL sterile cryopreservation tubes, transfer 1 mL of cell suspension to each tube, seal with sealing film, place in a pre-cooled 4°C programmed cooling box, loosen the cap half a turn, and quickly place in a -80°C freezer. After 24 hours, remove the cryopreservation tubes and store them in liquid nitrogen for long-term storage.

[0081] Example 3

[0082] (1) MTT assay for cell viability

[0083] HSF and HaCaT cells in the logarithmic growth phase were digested with trypsin and then subjected to a 1×10⁻⁶ solution. 5 Cells / well were seeded at a density of 100 μL per well in 96-well plates and cultured for 24 h to allow cells to reattach. The cell culture medium was removed, and 200 μL of different concentrations of H2O2 and trimethylamine liver metabolite solution were added. HSF and HaCaT cells were cultured for another 24 h. The supernatant was not discarded, and 20 μL / well of 5 mg / mL MTT solution was added. The cells were incubated at 37°C in the dark for 4 h. The supernatant was carefully discarded, and 150 μL / well of dimethyl sulfoxide (DMSO) was added. The cells were incubated at 37°C in the dark for 10 min to completely dissolve the crystals. The absorbance was measured at 490 nm using a microplate reader. Cell viability was calculated as the percentage of surviving cells in the experimental group compared to the control group.

[0084] Cell viability (%) = (OD) 试验 -OD 空白 ) / (OD 阴性 -OD 空白 )×100%

[0085] Where: OD 试验 Absorbance of the drug intervention group (cell group treated with H2O2 or trimethylamine liver metabolites);

[0086] OD 阴性 The absorbance of the negative control group (cells not treated with the drug) is shown.

[0087] OD 空白 The absorbance is for the blank control group (a blank group without drugs and cells).

[0088] (2) Detection of antioxidant enzyme CAT activity, SOD activity, GSH content, T-AOC capacity and MDA content

[0089] HSF and HaCaT cells in the logarithmic growth phase were digested with trypsin and then subjected to a 1×10⁻⁶ solution. 5 Cells were seeded at a density of 2 mL per well in 6-well plates and cultured overnight to allow for re-adhesion. The culture medium was removed, and the cells were washed twice with PBS buffer. H2O2 and trimethylamine liver metabolite solution were added at 2 mL / well, and the cells were cultured for another 24 h. The culture medium was discarded, and the cells were washed twice with PBS buffer. 1 mL of pre-chilled PBS buffer was added to each well, and adherent cells were collected into 1.5 mL sterile centrifuge tubes using a disposable sterile cell scraper. Cells were lysed using a cell disruptor and centrifuged at 12000 rpm for 10 min at 4°C. The supernatant was collected, and the activities of superoxide dismutase (SOD), reduced glutathione (GSH), catalase (CAT), total antioxidant capacity (T-AOC), and malondialdehyde (MDA) were measured according to the kit instructions.

[0090] (3) Observe the fluorescence intensity of cell free radicals using an inverted fluorescence microscope

[0091] HSF and HaCaT cells in the logarithmic growth phase were digested with trypsin and then subjected to a 1×10⁻⁶ solution. 5 Cells were seeded at a density of 2 mL per well in 6-well plates and cultured overnight to allow for re-adhesion. The culture medium was removed, and the cells were washed twice with PBS buffer. H2O2 and trimethylamine liver metabolite solution were added at 2 mL / well, and HSF cells were cultured for another 24 h. The culture medium was discarded, and the cells were washed twice with PBS buffer. A final concentration of 10 μmol / L of the DCFH-DA fluorescent probe was added to each well, and the cells were reacted at 37°C in the dark for 20 min. The probe was then removed, and the cells were washed twice with serum-free medium. The fluorescence intensity of each group was observed and photographed (20x) under blue excitation light using an inverted fluorescence microscope.

[0092] (4) Multifunctional enzyme-linked immunosorbent assay (ELISA) instrument for detecting the relative content of cell free radicals

[0093] HSF and HaCaT cells in the logarithmic growth phase were digested with trypsin and then subjected to a 1×10⁻⁶ solution. 5 Cells were seeded at a density of 100 μL per well in black 96-well plates and cultured for 24 h to allow the cells to reattach. The cell culture medium was removed, and 200 μL of different concentrations of H2O2 and trimethylamine liver metabolite solution were added. Cells were cultured for another 24 h, the culture medium was discarded, and 200 μL of DCFH-DA fluorescent probe (final concentration 10 μmol / L) was added to each well. The wells were incubated at 37°C in the dark for 20 min, the probe was removed, and each well was washed twice with serum-free medium. Fluorescence values ​​were detected using a multi-mode microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0094] (5) Flow cytometry detection of cell free radical fluorescence intensity and content

[0095] HSF and HaCaT cells in the logarithmic growth phase were digested with trypsin and then subjected to a 1×10⁻⁶ solution. 5 Cells were seeded at a density of 2 mL per well in 6-well plates and cultured overnight to allow for re-adhesion. The culture medium was removed, and the cells were washed twice with PBS buffer. H₂O₂ and trimethylamine liver metabolite solution were added at 2 mL / well, and the cells were cultured for another 24 h. The culture medium was discarded, and the cells were washed twice with PBS buffer. A final concentration of 10 μmol / L of the DCFH-DA fluorescent probe was added to each well, and the cells were reacted at 37°C in the dark for 20 min. The probe was then removed, and the cells were washed twice with serum-free medium. After trypsin digestion, PBS was added, and the cells were carefully pipetted and collected into 1.5 mL sterile centrifuge tubes. Fluorescence in each group was immediately detected using flow cytometry under the green fluorescence channel.

[0096] (7) ELISA was used to detect the secretion levels of cellular inflammatory factors TNF-α and IL-6.

[0097] HSF and HaCaT cells in the logarithmic growth phase were digested with trypsin and then subjected to a 1×10⁻⁶ solution. 5 Cells / well were seeded at a density of 2 mL per well in 6-well plates and cultured overnight to allow for re-adhesion. The culture medium was removed, and the cells were washed twice with PBS buffer. H2O2 and trimethylamine liver metabolite solution were added at 2 mL / well, and HSF cells were cultured for another 24 h. The cell culture supernatant was collected in sterile tubes, centrifuged at 300g for 5 min, and the supernatant was collected. The sample was added to an ELISA plate pre-coated with a target protein-specific monoclonal capture antibody. After incubation and washing, biotinylated antibody was added to form a sandwich immune complex. Horseradish peroxidase-labeled streptavidin was then added to specifically bind biotin. A blue substance was formed under the action of tetramethylbenzidine, which later turned yellow under the action of stop solution. The absorbance was measured at 450 nm. The TNF-α or IL-6 content in the sample was calculated using ELISACalc.

[0098] (8) ELISA method was used to detect the secretion levels of CoL I and MMP-1 in cells.

[0099] HSF and HaCaT cells in the logarithmic growth phase were digested with trypsin and then subjected to a 1×10⁻⁶ solution. 5Cells were seeded at a density of 2 mL per well in 6-well plates and cultured overnight to allow for re-attachment. The culture medium was removed, and the cells were washed twice with PBS buffer. H2O2 and trimethylamine liver metabolite solution were added at 2 mL / well, and the cells were cultured for another 24 hours. The cell culture supernatant was collected in sterile tubes and centrifuged at 3000 rpm for 20 min. The supernatant was collected again. Samples or standards were added to wells pre-coated with CoL I or MMP-1 antibodies, followed by biotin-labeled recognition antigens. The mixture was incubated at 37°C for 30 minutes. The antibodies competitively bind to the solid-phase antigens to form immune complexes. After washing with PBST, avidin-HRP was added, and the mixture was incubated at 37°C for 30 minutes. The bound HRP catalyzed the formation of a blue color in tetramethylbenzidine, which subsequently turned yellow under acidic conditions. The absorbance was measured at 450 nm. The CoL I or MMP-1 content in the sample was calculated using ELISACalc.

[0100] Example 4

[0101] This embodiment provides a method for combating aging by reducing cellular oxidative damage using grape seed extract (OPC), comprising the following steps:

[0102] (1) The final volume of the liver microsome incubation system was 1 mL, in which the final mass concentration of liver microsomes was 1 mg / mL, the concentration of trimethylamine was 1 mol / L, and the pH value of tris(hydroxymethyl)aminomethane hydrochloride buffer was 7.4. The system was pre-incubated in a water bath at 37°C for 5 min, and then NADPH coenzyme solution with a final concentration of 1 mmol / L was added. The mixture was vortexed and the reaction system was started. The system was incubated at 37°C for 8 h. The reaction was terminated by placing the incubation system in a -20°C refrigerator to obtain the trimethylamine liver metabolite.

[0103] (2) HSF and HaCaT cells were induced for 24 h using 150 mM trimethylamine liver metabolites to obtain an oxidative damage cell model.

[0104] (3) Take 10 mg of grape seed extract and mix it with 10 mL of complete culture medium to obtain a stock solution with a concentration of 1 mg / mL. Then filter it through a microporous membrane to remove bacteria, and then dilute it with complete culture medium to a concentration of 6.25 μg / mL to obtain a diluted stock solution.

[0105] (4) Use the diluted mother liquor obtained in step (3) to pretreat HSF and HaCaT cells for 12 h;

[0106] (5) After the pretreatment was completed, the diluted mother solution was discarded and HSF and HaCaT cells were washed once with PBS. Then, a mixed solution of trimethylamine liver metabolites and complete culture medium (the volume ratio of trimethylamine liver metabolites to complete culture medium was 3:10) was added and the cells were treated for 24 hours.

[0107] (6) After collecting the cells obtained in the above steps, the antioxidant capacity of each group of cells, the secretion levels of cellular inflammatory factors TNF-α and IL-6, and the secretion levels of cellular CoLⅠ and MMP-1 were detected.

[0108] The blank control group represents those not treated with grape seed extract and trimethylamine liver metabolites; the experimental group represents those treated with trimethylamine liver metabolites; and the OPC intervention group represents those treated with grape seed extract and trimethylamine liver metabolites.

[0109] like Figure 1-2 As shown, cell viability gradually decreased with increasing H2O2 and TMAO concentrations. For HSF cells, H2O2 at 0.05 mM and TMAO at 200-300 mM resulted in a cell viability of 80%. For HaCaT cells, H2O2 at 0.02 mM and TMAO at 150-250 mM resulted in a cell viability of 80%.

[0110] like Figure 3 , 4 As shown in Figures 13 and 14, after trimethylamine liver metabolites acted on HSF and HaCaT cells, compared with the blank control, the activities of GSH, SOD, and CAT enzymes in the cellular antioxidant system were significantly reduced, and the total antioxidant capacity (T-AOC) was significantly reduced in a dose-dependent manner. The content of the cellular lipid peroxidation product MDA increased with the increase of trimethylamine liver metabolite concentration, and was significantly higher than that of the blank control group. This indicates that trimethylamine liver metabolites damage the cellular antioxidant system and reduce cellular antioxidant capacity. However, after intervention with grape seed extract, the activities of GSH, SOD, and CAT enzymes in HSF and HaCaT cells were significantly increased, and the total antioxidant capacity (T-AOC) was significantly increased, while the MDA content was reduced. This indicates that grape seed extract can resist oxidative damage to cells through cellular antioxidant capacity, thereby playing an anti-aging role.

[0111] like Figure 5-6 As shown, after the liver metabolites of trimethylamine acted on HSF and HaCaT, fluorescence microscopy revealed that the liver metabolites of trimethylamine could enhance the intracellular fluorescence intensity, indicating that the liver metabolites of trimethylamine could increase the level of intracellular free radicals and regulate the cellular oxidative stress state.

[0112] like Figure 7 , 8 As shown in Figures 15 and 16, the effects of trimethylamine liver metabolites significantly enhanced the free radical fluorescence intensity of HSF and HaCaT cells, indicating that trimethylamine liver metabolites can stimulate oxidative stress by enhancing intracellular free radical levels, thereby regulating cellular antioxidant capacity. In contrast, after intervention with grape seed extract, the intracellular free radical fluorescence intensity was significantly lower than that of the experimental group, indicating that grape seed extract can improve the anti-aging capacity of cells by reducing the content of cellular free radicals.

[0113] like Figure 9 , 10 As shown in Figures 17 and 18, after trimethylamine liver metabolites acted on HSF and HaCaT, the secretion levels of cellular inflammatory factors IL-6 and TNF-α increased with the increase of trimethylamine liver metabolite concentration. However, after intervention with grape seed extract, the secretion levels of cellular inflammatory factors IL-6 and TNF-α were significantly reduced, close to the blank control. This indicates that grape seed extract improves the anti-aging ability of cells by regulating cellular inflammation levels and reducing the damage of inflammatory factors to cells.

[0114] like Figure 11 , 12 As shown in Figures 19 and 20, the effects of trimethylamine liver metabolites significantly reduced the content of collagen COLⅠ in HSF and HaCaT cells and significantly increased the activity of matrix metalloproteinase MMP-1, accelerating the decomposition of the cell matrix and collagen. However, after intervention with grape seed extract, compared with the experimental group, the content of collagen COLⅠ in cells was significantly increased, while the activity of matrix metalloproteinase MMP-1 was significantly reduced, thereby enhancing the anti-aging ability of cells.

[0115] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. The application of a trimethylamine liver metabolite in evaluating the anti-aging ability of food, characterized in that, Includes the following steps: (1) Trimethylamine liver metabolites were used to induce HSF and HaCaT cells to obtain a cell model of oxidative damage; During induction, the concentration of trimethylamine liver metabolites was 150-300 mM; (2) Prepare a mother liquor by taking the extract of anti-aging functional food, sterilize the mother liquor and dilute it to obtain a diluted mother liquor; (3) Use the diluted mother liquor obtained in step (2) to pretreat HSF and HaCaT cells; (4) After the pretreatment is completed, discard the diluted mother solution and wash HSF and HaCaT cells with PBS; (5) The mixture of trimethylamine liver metabolites and complete culture medium was applied to washed HSF and HaCaT cells; (6) Untreated HSF and HaCaT cells were used as the control group, the oxidative damage cell model obtained in step (1) was used as the experimental group, and the HSF and HaCaT cells obtained in step (5) were collected as the intervention group. The cellular antioxidant capacity, the secretion level of cellular inflammatory factors TNF-α and IL-6, and the secretion level of cellular CoL I and MMP-1 were detected in each group to evaluate the anti-aging ability of the food. During the evaluation, the anti-aging ability of the intervention group was further evaluated when the experimental group had lower cellular antioxidant capacity, higher levels of cellular inflammatory factors TNF-α and IL-6, lower levels of cellular CoL I secretion, and higher levels of MMP-1 secretion compared with the control group. Compared with the experimental group, the intervention group showed stronger cellular antioxidant capacity, lower levels of cellular inflammatory factors TNF-α and IL-6, higher levels of cellular CoL I secretion, and lower levels of MMP-1 secretion, indicating that the food had strong anti-aging ability.

2. The application of a trimethylamine liver metabolite according to claim 1 in evaluating the anti-aging ability of food, characterized in that, In step (1), the induction time is 18-36 hours.

3. The application of a trimethylamine liver metabolite according to claim 1 in evaluating the anti-aging ability of food, characterized in that, In step (2), the mother liquor is a mixture of 10 mg of extract and 10 mL of complete culture medium; The complete culture medium consisted of 45 mL DMEM basal medium, 5 mL fetal bovine serum, and 500 mL of... A mixture of penicillin and streptomycin solutions.

4. The application of a trimethylamine liver metabolite according to claim 1 in evaluating the anti-aging ability of food, characterized in that, In step (2), the concentration of the mother liquor is 1 mg / mL.

5. The application of a trimethylamine liver metabolite according to claim 1 in evaluating the anti-aging ability of food, characterized in that, In step (2), the concentration of the diluted mother liquor is 1.56-6.25 μg / mL.

6. The application of a trimethylamine liver metabolite according to claim 1 in evaluating the anti-aging ability of food, characterized in that, In step (3), the pretreatment time is 12-18 hours.

7. The application of a trimethylamine liver metabolite according to claim 1 in evaluating the anti-aging ability of food, characterized in that, In step (5), the volume ratio of trimethylamine liver metabolites to complete culture medium is 3:

10.

8. The application of a trimethylamine liver metabolite according to claim 1 in evaluating the anti-aging ability of food, characterized in that, In step (5), the treatment time is 18-36 hours.

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