Composition capable of improving inflammatory aging and application thereof

Abstract

The invention provides a composition for improving inflammatory aging and application thereof. The composition is formed by compounding ergothioneine and verbascoside according to the mass ratio of 1: 10-10: 1, the interaction of a binary mixture is calculated by adopting a joint index (CI) method, and the synergistic anti-inflammatory effect of ergothioneine and verbascoside is determined to relieve inflammatory aging. The composition can improve inflammation by inhibiting the levels of cell inflammatory factors IL-1beta, IL-6 and TNF-alpha; the influence on inflammation is weakened by inhibiting the antioxidant activity (ROS, MDA and GSSG) of cells. Furthermore, the composition can inhibit cell ferroptosis in a targeted manner, reduce the lipid peroxidation level and the cell ferrous iron content, reduce cell aging and maintain the redox state of cells. The composition provided by the invention has a remarkable targeted inhibition effect on cell ferroptosis, and has important significance on inhibition of senescence related to ferroptosis and product development.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and anti-aging formulation technology, specifically relating to a composition that improves inflammatory aging and its application. Background Technology

[0002] Numerous studies have shown that aging is a systemic inflammatory process. In other words, during aging, cells secrete large amounts of inflammatory factors such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), generating an inflammatory response. Inflammation, in turn, leads to oxidative damage and DNA damage. Especially when the human body is stimulated by various factors such as ultraviolet radiation, air pollution, and poor dietary habits, cells in various tissues and organs are stimulated to produce inflammatory factors and reactive oxygen species, further accelerating aging and forming a vicious cycle. Therefore, delaying aging through anti-inflammatory pathways has always been an important entry point for researchers in anti-aging studies.

[0003] A novel, regulated type of cell death, distinct from apoptosis, necrosis, and autophagy, has been discovered in recent years and is called ferroptosis. Characterized by iron-dependent accumulation of lipid reactive oxygen species and glutathione depletion, it is closely associated with many age-related inflammatory diseases, such as neurodegenerative diseases and cardiovascular diseases. Studies have shown that ferroptosis reduces glutathione (GSH) levels, converting it to oxidized glutathione (GSSG), producing lipid peroxidation products such as malondialdehyde (MDA) and lipid peroxides (LPO), as well as reactive oxygen species (ROS). Increasing research indicates that iron accumulation occurs in target tissues and organs of many inflammatory diseases, and ferroptosis inhibitors can significantly inhibit cell damage and death in relevant pathological models. Therefore, inhibiting ferroptosis holds promise as an effective strategy for preventing or treating these inflammatory age-related diseases.

[0004] Ergothioneine is a sulfur-containing small-molecule histidine derivative, first isolated from rye grains infected with fungi. In recent years, it has gained more attention due to its significant accumulation in human blood and liver. Ergothioneine is well-known as a potent antioxidant with regulatory and protective functions similar to vitamins in the body. Oxidative damage is often a key factor leading to abnormal metabolism, aging, and apoptosis. Ergothioneine can rapidly remove abnormally elevated ROS in mitochondria to maintain normal levels in response to oxidative stress, inflammation, apoptosis, and aging. Simultaneously, ergothioneine can rapidly chelate metal ions in cells, such as iron, copper, and zinc, without generating additional free radicals through the Fenton reaction, thereby preventing cell and DNA damage.

[0005] Verbascoside belongs to the phenylethanoid glycoside class of compounds and is a widely distributed natural water-soluble compound in the plant kingdom, mostly isolated from medicinal plants such as osmanthus and cistanche. Verbascoside possesses various biological activities, including anti-inflammatory and antioxidant effects. However, whether the combination of these two substances has a synergistic effect in anti-inflammatory aging has not been reported. In view of this, the present invention provides a composition that improves inflammatory aging, requires a reduced dosage, and can be used in food, pharmaceuticals, or cosmetics. Summary of the Invention

[0006] The problem the invention aims to solve

[0007] The purpose of this invention is to provide an application of a composition that improves inflammatory aging, wherein the composition can effectively inhibit the occurrence of intracellular ferroptosis, thereby reducing inflammation, improving cellular antioxidant activity, reducing cellular aging, and maintaining the redox state of cells.

[0008] Solution for solving the problem

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A composition comprising ergothioneine and verbascoside in a mass ratio of 1:10 to 10:1.

[0011] Application of a composition for improving inflammatory aging, said composition being a compound of ergothioneine and verbascoside in a mass ratio of 1:10 to 10:1.

[0012] Furthermore, the composition improves inflammatory aging through synergistic anti-inflammatory effects.

[0013] Furthermore, the composition can synergistically inhibit the levels of cellular inflammatory factors IL-1β, IL-6, and TNF-α.

[0014] Furthermore, the composition can synergistically reduce the level of lipid peroxidation in cells.

[0015] Furthermore, the composition is able to target and inhibit ferroptosis.

[0016] Furthermore, the verbascoside is extracted from plant extracts.

[0017] Furthermore, the verbascoside is extracted from Cistanche deserticola or Osmanthus fragrans.

[0018] Furthermore, the composition is used to prepare food, pharmaceutical or cosmetic products that inhibit inflammatory aging.

[0019] Furthermore, the food is in the form of an oral liquid, tablet, capsule, or solid beverage.

[0020] The effects of the invention

[0021] The beneficial effects of this invention are as follows:

[0022] This invention provides an application of a composition for improving inflammatory aging. The composition is a compound of ergothioneine and verbascoside in a mass ratio of 1:10 to 10:1, and the interaction of the binary mixture is calculated using the co-indices (CI) method to determine their synergistic anti-inflammatory effect in improving inflammatory aging. The composition can synergistically improve inflammation by inhibiting the levels of cellular inflammatory factors IL-1β, IL-6, and TNF-α; it can reduce cellular senescence and maintain cellular redox status by increasing cellular antioxidant activity (ROS, MDA, GSSG). Furthermore, the composition can target and inhibit cellular ferroptosis, reduce lipid peroxidation levels, and decrease cellular ferrous iron content. Attached Figure Description

[0023] Figure 1 This study presents the cytotoxicity analysis of ergothionein and verbascoside and their different ratios in Example 1. Figure 1 The AG values ​​were used for cytotoxicity analysis of ergothionein, verbascoside, composition (10:1), composition (5:1), composition (1:1), composition (1:10), and composition (1:5).

[0024] Figure 2 Example 1: Effects of ergothioneine and verbascoside and their different ratios on the levels of cellular secretion and inflammatory factors, and CI values. Figure 2 A and B represent the effects of ergothionein and verbascoside and their different ratios on the level of TNF-α secreted by cells, and their CI values, respectively. Figure 2 C and D represent the effects of ergothionein and verbascoside and their different ratios on the level of IL-1β secreted by cells, and the CI values, respectively. Figure 2 E and F represent the effects of ergothionein and verbascoside, and their different ratios, on the level of IL-6 secreted by cells, and the CI value, respectively.

[0025] Figure 3 The effects of ergothionein, verbascoside, and their different ratios on cellular antioxidant activity were investigated. Figure 3 The AC values ​​represent the effects of ergothionein and verbascoside, and their different ratios, on ROS, MDA, and GSSG.

[0026] Figure 4 The effects of ergothionein, verbascoside, and their different ratios on cellular lipid peroxidation levels.

[0027] Figure 5The effects of ergothionein, verbascoside, and their different combinations on Erastin-induced ferrous iron levels in cells. Detailed Implementation

[0028] The ergothioneine of this invention is commercially available.

[0029] The verbascoside of this invention is commercially available. On the other hand, verbascoside is also widely distributed among natural water-soluble compounds in the plant kingdom and can be isolated from medicinal plants, such as osmanthus and cistanche. Extracts from these plants also contain high levels of verbascoside.

[0030] The above-mentioned extracts can be obtained by conventional methods, such as by soaking or refluxing the plant as the source with the extraction solvent at room temperature or under heating, followed by filtration and concentration. As the extraction solvent, any solvent commonly used in extraction can be used, either alone or in combination: for example, aqueous solvents such as water, physiological saline, phosphate buffer, borate buffer; or organic solvents such as alcohols such as ethanol, propylene glycol, 1,3-butanediol, glycerol, hydrous alcohols, chloroform, dichloroethane, carbon tetrachloride, acetone, ethyl acetate, hexane, etc. Water is preferred as the solvent. The extracts obtained using the above solvents can be used directly, or by using an extract concentrated through methods such as freeze-drying. Alternatively, as needed, extracts can be used after removing impurities through adsorption methods, such as ion exchange resins, and / or extracts obtained by adsorption using a porous polymer column, followed by dissolution with the desired solvent and then concentration.

[0031] First, it should be clarified that the combined index method is based on the intermediate-effect equation and is derived from the principle of the law of mass action. The law of mass action is a unified theory that provides common relationships between single entities and multiple entities, including first-order and higher-order dynamics. The resulting combined index (CI) theorem defines additive effects (CI = 1), synergistic effects (CI < 1), and antagonistic effects (CI > 1). This method requires the results to be expressed in a dose-effect manner, and the effect value must be between 0 and 1.

[0032] Example

[0033] 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.

[0034] The main sources of experimental reagents in the examples are:

[0035] Ergothioneine was purchased from Shenzhen Zhongke Xinyang Biotechnology Co., Ltd.

[0036] Verbena glycoside, purity ≥98%, purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0037] The other reagents or instruments in the examples are all commercially available conventional products, sourced as follows:

[0038] Palmitic acid, Xi'an Kunchuang Biotechnology Co., Ltd.;

[0039] Erastin, MedChemexpress Biotechnology Co., Ltd., USA;

[0040] DMEM culture medium, Shanghai Yuanpei Biotechnology Co., Ltd.;

[0041] Fetal bovine serum, penicillin / streptomycin (100X), 0.25% trypsin-EDTA solution, Sigma-Aldrich, USA;

[0042] Dimethyl sulfoxide (DMSO) (sterile), Shandong Sikejie Biotechnology Co., Ltd.;

[0043] PBS buffer (sterile), Wuhan Saiweier Biotechnology Co., Ltd.

[0044] CCK-8, Beijing Aoqing Biotechnology Co., Ltd.;

[0045] GSSG assay kit, ROS fluorescent probe DCFH-DA, BCA protein concentration assay kit, C11-BODIPY581 / 591, Shanghai Beyotime Biotechnology Co., Ltd.

[0046] TNF-α, IL-1β, IL-6 ELISA kit, Wuhan Boster Biological Technology Co., Ltd.

[0047] MDA Detection Kit, Beijing Solarbio Biotechnology Co., Ltd.

[0048] BSA124S-CW electronic analytical balance, Sartorius GmbH, Germany;

[0049] MJX-150S Constant Temperature Incubator, Ningbo Saif Experimental Instrument Co., Ltd.

[0050] ZQTY-70E Shaking Incubator, Shanghai Zhichu Instrument Co., Ltd.

[0051] ECLIPSE Ci-L upright biological microscope, Nikon Corporation, Japan;

[0052] YXQ-LS-50SII Vertical Pressure Steam Sterilizer, Shanghai Boxun Medical Bio-Instrument Co., Ltd.;

[0053] SW-CJ-1FD Medical Clean Bench, Suzhou Antai Air Technology Co., Ltd.

[0054] Eon microplate reader, BioTek Instruments, Inc., USA;

[0055] Healforce pure water system, Shanghai Peisheng Instrument Equipment Co., Ltd.;

[0056] IMS-20 ice maker, Xiamen Guoyi Scientific Instruments Co., Ltd.;

[0057] MX-F Vortex Mixer, Wuhan Saiweier Biotechnology Co., Ltd.;

[0058] HH-2 Digital Display Thermostatic Water Bath, Shanghai Lichen Bangxi Instrument Technology Co., Ltd.

[0059] Where no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in this field or the product manual shall apply.

[0060] First, it should be clarified that the combined index method is based on the intermediate-effect equation and is derived from the principle of the law of mass action. The law of mass action is a unified theory that provides common relationships between single entities and multiple entities, including first-order and higher-order dynamics. The resulting combined index (CI) theorem defines additive effects (CI = 1), synergistic effects (CI < 1), and antagonistic effects (CI > 1). This method requires the results to be expressed in a dose-effect manner, and the effect value must be between 0 and 1.

[0061] 1. Cytotoxicity test

[0062] Cytotoxicity was determined using the CCK-8 assay. Cells were sputtered at a concentration of 1 × 10⁻⁶. 4 Cells were seeded at a density of 80% or higher in 96-well plates until the cell density reached 80%. Complete culture medium containing different formulations was then added. After 24 hours, the medium was discarded, and the cells were washed 2-3 times with PBS. 100 μl of fresh complete culture medium and 10 μl of CCK-8 were added to each well. After incubation at 37°C for 10-20 minutes, the absorbance (OD) value, representing cell viability, was measured at 450 nm using a microplate reader. The untreated control group served as the baseline.

[0063] 2. Establishment of a cellular inflammation model and drug administration

[0064] A skin inflammation model was established using palmitic acid-induced HaCaT cells. Cells were cultured at a rate of 1×10⁶. 6Cells were seeded at a density of cells / well in 6-well plates and cultured at 37°C in a 5% CO2 incubator until the cell density in the wells reached 80% or higher. Cells were then treated with palmitic acid at a final concentration of 400 μM, alone or separately with different formulations, and incubated for 24 h. Cells in complete culture medium were used as a blank control, with a solvent control.

[0065] 3. ELISA method for detecting cellular inflammatory factor levels (TNF-α, IL-6, IL-1β)

[0066] Twenty-four hours after cell drug treatment, the cell supernatant was aspirated into 1.5 mL EP tubes for analysis. Detection procedures were performed according to the TNF-α, IL-6, and IL-1β kit instructions.

[0067] 4. Calculation of anti-inflammatory interactions

[0068] The cell-cell interaction evaluation model uses CompuSyn software to calculate the CI value, and the calculation formula is as follows:

[0069] CI = C A / IC A +C B / IC B

[0070] When CI < 1, it indicates a synergistic effect; CI = 1, it indicates an additive effect; CI > 1, it indicates an antagonistic effect. CI represents the combination index of the activity of a binary mixture to achieve a certain efficiency (X%, typically 50%); C A and C B These represent the proportions of substances A and B in the binary mixture when the efficiency reaches X% activity; IC A and IC B These represent the dosages required for each of compounds A and B to achieve X% activity. The interactions between the compound compositions can be explained by comparing the experimental results with theoretically calculated values ​​obtained from analyzing the effects of the two substances individually.

[0071] The theoretical values ​​of inflammatory factor (TNF-α, IL-6, IL-1β) levels were calculated using the isoradiometric analysis formula:

[0072] Theoretical value = R A / (P1+R A / R B ×P2)

[0073] Among them, R A R BP1 and P2 represent the effect values ​​of substances A and B acting alone, respectively; P1 represents the proportion of substance A in the compound combination; P2 represents the proportion of substance B in the compound combination, and P2 = 1 - P1.

[0074] 5. Measurement of cellular ROS levels

[0075] According to the ROS detection kit instructions, logarithmic growth phase cells were seeded in 6-well plates, and ROS levels were detected using the DCFH-DA fluorescent probe. After drug intervention, the culture medium was aspirated. Serum-free medium containing 10 μM DCFH-DA was added for in-situ probe loading. After incubation at 37°C for 30 min, the cells were washed three times with serum-free medium. Fluorescence values ​​were detected using a fluorescence microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The relative fluorescence intensity (%) of the remaining groups was calculated with the blank group fluorescence value as 100%.

[0076] 6. Determination of cellular MDA and GSSG content

[0077] Cells at 6 × 10 5 Cells were seeded at a density of [number] cells / well in 6-well plates, with 2 mL of complete culture medium added to each well. The plates were incubated at 37°C with 5% CO2 until cells reached confluence (over 80%). The culture medium was discarded, and the cells were washed twice with PBS. 24 hours after drug treatment, the cell supernatant was discarded, and the cells were washed 1-2 times with PBS. 150 μL of cell lysis buffer (containing 1 mM PMSF) was added, and the cells were pipetted several times to ensure thorough contact between the lysis buffer and the cells. After complete lysis, the cells were centrifuged at 12000g for 5 min, and the supernatant was collected as the total cell protein solution. This solution was analyzed immediately or aliquoted and stored at -20°C for later analysis. BCA protein quantification was performed using the original concentration sample or a proportionally diluted sample to standardize the protein concentration across all groups. The content was then determined using MDA and GSSG assay kits.

[0078] 7. Cellular lipid peroxidation level

[0079] Cells were loaded at a rate of 1×10 4 Seed cells at a density of 1 cell / well in confocal culture dishes, discard the cell culture medium, wash once with PBS for 5 min each time, and add 1 mL of C11-BODIPY. 581 / 591 Incubate the working solution at room temperature for 30 min. Remove the supernatant and wash twice with PBS for 5 min each time. Detect the fluorescence value using a fluorescence microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 565 nm. Calculate the relative fluorescence intensity (%) of the remaining groups, using the blank group fluorescence value as 100%.

[0080] 8. Detection of iron content in cells

[0081] According to the instructions of the cell iron content assay kit, the iron content in cells was measured. 2+ Quantitative analysis at different levels. Extraction of cellular proteins was based on Method 5(1) of the kit instructions.

[0082] 9. Statistical Analysis of Results

[0083] Plotting was performed using Origin 2024, and results are expressed as Mean ± SD. t-tests were used for comparisons between groups. * indicates P < 0.05, considered statistically significant; ** indicates P < 0.01, considered highly statistically significant; *** indicates P < 0.001, considered extremely statistically significant.

[0084] 10. Experimental Results

[0085] (1) As Figure 1 As shown, when the ergothioneine concentration is less than 5 μg / mL, the cell viability can reach over 90%. Figure 1 A); when the concentration of verbascoside is 0-300 μg / mL, the cell viability can reach over 90%. Figure 1 (B); When the concentration of different ratios of ergothioneine and verbascoside was 0-500 μg / mL, the cell viability could reach over 90%. Figure 1 (CG). Therefore, in this embodiment, 5 μg / mL was ultimately selected as the final concentration of each substance.

[0086] (2) Figure 2 As shown, both ergothioneine and verbascoside significantly inhibited the levels of TNF-α, IL-1β, and IL-6 secreted by cells when added alone. Combining ergothioneine and verbascoside at different ratios with the same concentration (5 μg / mL) also significantly inhibited the levels of inflammatory factors. This indicates that the combination of ergothioneine and verbascoside at a ratio of 10:1 to 1:10 has an inhibitory effect on cellular inflammation. The CI values ​​of the combined groups were calculated using CompuSyn software. Overall, when the ratios of ergothioneine and verbascoside were 10:1 and 5:1, the CI < 1, indicating a strong synergistic effect of ergothioneine and verbascoside in their anti-inflammatory activities at ratios of 10:1 to 5:1.

[0087] (3) Figure 3As shown, different ratios of ergothioneine and verbascoside can inhibit ROS production, MDA levels, and GSSG content, significantly improving cellular antioxidant activity. This indicates that the combination of ergothioneine and verbascoside at ratios of 10:1 to 1:10 enhances cellular antioxidant activity. Especially at ratios of 10:1 and 5:1, a synergistic inhibitory effect is observed. Ergothioneine and verbascoside at ratios of 10:1 to 5:1 exhibit a synergistic effect in enhancing cellular antioxidant activity.

[0088] (4) Figure 4 and 5 As shown, ergothioneine and verbascoside, alone or in combination, significantly inhibited Erastin-induced ferroptosis. Specifically, both ergothioneine and verbascoside, when added alone, significantly inhibited cellular lipid peroxidation and iron content; similarly, combinations of the two at different ratios also showed significant inhibitory effects on cellular lipid peroxidation and iron content. This indicates that a ergothioneine to verbascoside ratio of 10:1 to 1:10 inhibits cellular peroxidation and iron content. Especially at a 10:1 ratio, a synergistic inhibitory effect on cellular lipid peroxidation is observed.

[0089] (5) Conclusion: The composition provided by this invention can enhance the antioxidant activity (ROS, MDA, GSSG) of cells by synergistically inhibiting the levels of cellular inflammatory factors IL-1β, IL-6, and TNF-α, thereby reducing cellular senescence and maintaining the redox state of cells. Furthermore, it can target and inhibit cellular ferroptosis, reduce lipid peroxidation levels, and decrease the content of ferrous iron in cells.

Claims

1. A composition, characterized in that, The composition is prepared by compounding ergothioneine and verbascoside in a mass ratio of 1:10 to 10:

1.

2. The application of a composition that improves inflammatory aging, characterized in that, The composition is prepared by compounding ergothioneine and verbascoside in a mass ratio of 1:10 to 10:

1.

3. The application according to claim 2, characterized in that, The composition improves inflammatory aging through its anti-inflammatory effect.

4. The application according to claim 3, characterized in that, The composition can synergistically inhibit the levels of inflammatory cytokines IL-1β, IL-6, and TNF-α.

5. The application according to claim 2, characterized in that, The composition can reduce the level of lipid peroxidation in cells and / or inhibit ferroptosis.

6. The application according to claim 2, characterized in that, The composition can reduce the iron content in cells.

7. The application according to claim 2, characterized in that, The verbascoside is extracted from plant extracts.

8. The application according to claim 2, characterized in that, The verbascoside is extracted from Cistanche deserticola or Osmanthus fragrans.

9. The composition according to claim 1, characterized in that, The composition is used to prepare food, medicine or cosmetics that inhibit inflammatory aging.

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