An anti-aging oral liquid based on ergothioneine and pyrroloquinoline quinone disodium salt and a preparation method thereof

Abstract

The present application belongs to the technical field of health food, and particularly relates to an anti-aging oral liquid based on ergothioneine and pyrroloquinoline quinone disodium salt and a preparation method thereof. The anti-aging oral liquid based on ergothioneine and pyrroloquinoline quinone disodium salt comprises the following components: per 1000 mL, ergothioneine 0.2-0.8 g, pyrroloquinoline quinone disodium salt 0.05-0.2 g, sodium hyaluronate 2-6 g, fish skin small molecule collagen peptide 20-60 g, L-ascorbic acid 3-8 g, olive leaf and Anemarrhena asphodeloides extract 0.5-8.0 g, and the rest is water. The anti-aging oral liquid prepared by the present application can simultaneously consider multi-target anti-aging of exogenous antioxidant, endogenous mitochondrial activation and dermal matrix repair, and through scientific compounding and process optimization, the synergistic effect of various active ingredients is ensured.

Description

Technical Field

[0001] This invention belongs to the field of health food technology, specifically relating to an anti-aging oral liquid based on ergothioneine and pyrroloquinoline quinone disodium salt and its preparation method. Background Technology

[0002] Human aging involves two core pathways: exogenous and endogenous. Exogenous aging is mainly caused by the continuous attack of skin cells by oxidative stress factors such as ultraviolet radiation, environmental pollutants, and endogenous free radicals, leading to DNA damage, lipid peroxidation, and the release of inflammatory factors, ultimately manifesting as photoaging, dullness, and decreased elasticity of the skin. Endogenous aging is closely related to the decline of mitochondrial function, energy metabolism disorders, and a significant decrease in the ability of the dermal extracellular matrix to synthesize with age, resulting in loose skin, deeper wrinkles, weakened water retention capacity, and overall decline in bodily functions.

[0003] Currently, the health food and functional food industries have a variety of active ingredients claiming anti-aging effects, such as ergothioneine, disodium pyrroloquinoline quinone, sodium hyaluronate, and collagen peptides. Among them, ergothioneine is a rare natural chiral amino acid with extremely strong free radical scavenging and metal ion chelating abilities, and it can be efficiently accumulated in skin tissue through the OCTN1 transporter protein in keratinocytes and melanocytes; disodium pyrroloquinoline quinone, as a novel vitamin-like cofactor, can activate mitochondrial biogenesis-related pathways, promoting mitochondrial regeneration and energy metabolism.

[0004] To enhance anti-aging effects, existing patents disclose the use of compound compositions containing ergothioneine and disodium pyrroloquinoline quinone. For example, Chinese patent application CN117959297A discloses a whitening and anti-aging composition containing disodium pyrroloquinoline quinone, whose components include disodium pyrroloquinoline quinone and ergothioneine in a mass ratio of 1~6:0.5~6, and optionally ginseng extract, sodium hyaluronate, nicotinamide, vitamin C, vitamin E, and collagen peptides. This composition achieves whitening and anti-aging by synergistically inhibiting tyrosinase activity and scavenging free radicals. Another Chinese patent application CN120478534A discloses a composition for repairing DNA and delaying aging, which contains 10~30% Rhodiola rosea extract, 5~15% ergothioneine, 1~5% disodium pyrroloquinoline quinone, acai extract, astaxanthin, lycopene, and B vitamins. It achieves anti-aging effects at the gene level by activating Sirtuin family proteins, repairing DNA damage, and scavenging free radicals.

[0005] However, the above-mentioned existing technologies still have the following shortcomings: (1) Although they all mention the combination of ergothioneine and pyrroloquinoline quinone disodium salt, there is a lack of systematic synergistic effect verification data, resulting in low raw material utilization and room for improvement in anti-aging efficiency; (2) Existing compositions mostly focus on single or limited pathways such as skin whitening or DNA repair, and fail to simultaneously cover the three core intervention links of aging: exogenous antioxidant protection, endogenous mitochondrial function enhancement and dermal matrix replenishment, making it difficult to achieve a systemic and comprehensive anti-aging effect; (3) Commercially available oral products are mostly simple physical mixtures of active ingredients, lacking a systematic design for compatibility stability and oral absorption and utilization.

[0006] Therefore, there is an urgent need to develop an anti-aging oral liquid based on ergothioneine and pyrroloquinoline quinone disodium salt to meet consumers' demand for comprehensive anti-aging. Summary of the Invention

[0007] The purpose of this invention is to provide an anti-aging oral liquid based on ergothionein and pyrroloquinoline quinone disodium salt and its preparation method. The anti-aging oral liquid prepared by this invention can simultaneously achieve exogenous antioxidant effects, endogenous mitochondrial activation and dermal matrix repair. Through scientific formulation and process optimization, the synergistic effect of each active ingredient is ensured.

[0008] To achieve the above objectives, the present invention provides the following technical solution: An anti-aging oral liquid based on ergothioneine and disodium pyrroloquinoline quinone, comprising the following components per 1000mL: ergothioneine 0.2~0.8g, disodium pyrroloquinoline quinone 0.05~0.2g, sodium hyaluronate 2~6g, fish skin small molecule collagen peptide 20~60g, L-ascorbic acid 3~8g, olive leaf and anemarrhena asphodeloides extract 0.5~8.0g, with the balance being water.

[0009] Preferably, per 1000 mL, it comprises the following components: ergothioneine 0.4~0.6 g, pyrroloquinoline quinone disodium salt 0.1~0.2 g, sodium hyaluronate 3~5 g, fish skin small molecule collagen peptide 30~50 g, L-ascorbic acid 4~5 g, olive leaf and anemarrhena extract 3~5 g, and the balance being water.

[0010] Preferably, per 1000 mL, it comprises the following components: 0.5 g ergothioneine, 0.16 g disodium pyrroloquinoline quinone, 4 g sodium hyaluronate, 44 g fish skin small molecule collagen peptide, 4.7 g L-ascorbic acid, 4.3 g olive leaf and Anemarrhena asphodeloides extract, with the remainder being water.

[0011] This invention scientifically combines ergothioneine, disodium pyrroloquinoline quinone, and fermented extracts of olive leaves and Anemarrhena asphodeloides to create a three-pronged anti-aging effect: exogenous antioxidant protection, endogenous mitochondrial activation, and inflammatory matrix repair. The components exhibit clear complementary and synergistic relationships in their mechanisms of action. At the antioxidant level, a multi-layered, end-to-end free radical scavenging system is formed, acting on different spatial levels within the extracellular fluid, cytoplasm, and mitochondria, with complementary coverage of free radical types and scavenging ranges, achieving full-spectrum and full-space coverage of oxidative stress. At the mitochondrial function regulation level, a synergistic repair chain is formed, clearing damage, promoting regeneration, and stabilizing membrane structure through joint intervention. At the dermal matrix repair level, a synergistic effect of promoting synthesis, inhibiting degradation, and providing anti-inflammatory protection is achieved, restoring collagen fibers from a thin, broken state to a robust, continuous, densely arranged, and orderly structure, with significantly better results than combinations of individual components.

[0012] Preferably, the preparation method of the olive leaf and anemarrhena extract includes the following steps: mixing olive leaves and anemarrhena, fermenting with compound bacteria, and then sequentially passing through microfiltration, ultrafiltration, and drying.

[0013] Preferably, the compound bacteria include Lactobacillus delbrueckii, Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus casei.

[0014] Preferably, the ratio of viable bacteria of Lactobacillus delbrueckii, Lactobacillus plantarum, Lactobacillus rhamnosus and Lactobacillus casei in the compound bacteria is (1~2):(3~4):(1~2):(2~3).

[0015] Preferably, the ratio of viable bacteria of Lactobacillus delbrueckii, Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus casei in the compound bacteria is 2:4:1:3.

[0016] Preferably, the mass ratio of olive leaves to Anemarrhena asphodeloides is (2~3):1.

[0017] Preferably, the pore size of the filter membrane used in the microfiltration is 0.45~0.80μm.

[0018] Preferably, the ultrafiltration membrane used in the ultrafiltration process has a molecular weight cutoff of 5kDa to 10kDa.

[0019] This invention utilizes a specific ratio of olive leaves and Anemarrhena asphodeloides fermented with specific microbial strains, and retains active components at specific molecular weights to achieve excellent synergistic effects with ergothioneine and disodium pyrroloquinoline quinone. Analysis shows that the mass ratio of olive leaves to Anemarrhena asphodeloides (2-3:1) ensures a scientifically balanced ratio of polyphenols and steroidal saponins in the fermentation substrate. The former provides phenolic hydroxyl radical scavenging ability, while the latter provides anti-inflammatory signaling pathway regulation ability. Directed fermentation by specific complex microorganisms can convert large-molecule bound polyphenols and saponins into small-molecule free aglycones, while simultaneously producing bioactive extracellular polysaccharides, significantly enhancing antioxidant activity. The molecular weight cutoff precisely enriches specific active oligophenols and saponins, forming spatial complementarity and synergistic pathways with ergothioneine and disodium pyrroloquinoline quinone at the mitochondrial and nuclear levels.

[0020] Preferably, the anti-aging oral liquid based on ergothioneine and pyrroloquinoline quinone disodium salt further includes oral pharmaceutical excipients.

[0021] The method for preparing the anti-aging oral liquid based on ergothioneine and pyrroloquinoline quinone disodium salt includes the following steps: adding water to ergothioneine, pyrroloquinoline quinone, sodium hyaluronate, fish skin small molecule collagen peptides, ascorbic acid, olive leaf and anemarrhena extract, and oral pharmaceutical excipients, and stirring evenly to make the particle size of the solid raw materials ≤100 mesh, thereby obtaining an oral liquid rich in ergothioneine.

[0022] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: 1. This invention discloses an oral liquid rich in ergothioneine with anti-aging properties. By scientifically combining ergothioneine, disodium pyrroloquinoline quinone, olive leaf and anemarrhena extract, fish skin small molecule collagen peptides, and sodium hyaluronate, a significant synergistic effect is produced, thereby achieving multiple beneficial effects.

[0023] 2. The compound formula of this invention can more comprehensively intervene in key pathways of cellular aging, including forming a complementary and synergistic anti-aging system. It simultaneously addresses the two core pain points of aging: mitochondrial loss and insufficient mitochondrial numbers. It also opens up three pathways: exogenous antioxidant protection, endogenous mitochondrial regeneration, and inhibition of inflammatory aging. It can not only achieve surface skin care, but also comprehensively delay the aging process at the cellular and molecular levels and enhance the body's vitality. It provides an effective solution to the technical problem of existing anti-aging products having a single mechanism of action and limited effects.

[0024] 3. This invention constructs a multi-level, three-dimensional anti-aging system covering the extracellular, cytoplasmic, mitochondrial, and nucleus levels through the synergistic compounding of three components: ergothioneine, disodium pyrroloquinoline quinone, and fermented extracts of olive leaves and Anemarrhena asphodeloides. It produces synergistic effects on multiple key anti-aging targets, such as free radical scavenging efficiency, mitochondrial function recovery, telomerase activity activation, collagen regeneration, and inhibition of inflammatory factors. It breaks through the superficial bottleneck of existing anti-aging products with single pathways of action and limited effects, and achieves comprehensive and deep-level anti-aging intervention from the molecular, cellular, and tissue levels. Attached Figure Description

[0025] Figure 1 The images show sample images of the anti-aging oral liquids from Examples 1-3 and Comparative Examples 1-8. Detailed Implementation

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] All raw materials used in the following embodiments of the present invention are commercially available products.

[0028] Lactobacillus rhamnosus, catalog number CICC22173, China Industrial Microbial Culture Collection Center.

[0029] Lactobacillus casei (product number HZB116347), Lactobacillus delbrueckii (product number HZB139506), and Lactobacillus plantarum (product number HZB116349) are all from Wuhan Gray Algae Biotechnology Co., Ltd.

[0030] Fish skin small molecule collagen peptides, product number GC-01-260, Shaanxi Angxu Biotechnology Co., Ltd.

[0031] Example 1 This embodiment provides an anti-aging oral liquid based on ergothioneine and disodium pyrroloquinoline quinone, comprising the following components per 1000mL: 0.2g ergothioneine, 0.2g disodium pyrroloquinoline quinone, 2g sodium hyaluronate, 60g fish skin small molecule collagen peptide, 3g L-ascorbic acid, 8.0g olive leaf and Anemarrhena asphodeloides extract, with the remainder being water.

[0032] The preparation method of the olive leaf and anemarrhena extract includes the following steps: (1) After washing the fresh olive leaves, air-dry them until the moisture content is 6%, and then grind them to less than 50 mesh. Grind Anemarrhena asphodeloides to less than 50 mesh. Mix the ground olive leaf powder and Anemarrhena asphodeloides powder at a mass ratio of 2.5:1. Sterilize at 121℃ for 30 minutes and cool to room temperature to obtain the mixed raw material powder. (2) Mix the raw material powder with water and adjust the moisture content to 55 wt% to obtain the fermentation raw material. Add compound bacteria to the fermentation raw material. The amount of compound bacteria is 1×10 9 The fermentation material was prepared with CFU / mL of fermentation medium. The ratio of viable bacteria of Lactobacillus delbrueckii, Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus casei in the compound bacteria was 2:4:1:3. Anaerobic fermentation was carried out at 35℃ for 40 hours. After fermentation, the mixture was sterilized to obtain the fermentation mixture.

[0033] (3) The mixture after fermentation was first microfiltered using a microfiltration membrane with a pore size of 0.45 μm, and the microfiltration permeate was collected. Then the microfiltration permeate was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 8 kDa to retain small molecule active ingredients. The ultrafiltration permeate was collected. The ultrafiltration permeate was concentrated and vacuum dried to obtain olive leaf and Anemarrhena asphodeloides fermentation extract.

[0034] The method for preparing the anti-aging oral liquid based on ergothioneine and pyrroloquinoline quinone disodium salt includes the following steps: adding water to ergothioneine, pyrroloquinoline quinone disodium salt, sodium hyaluronate, fish skin small molecule collagen peptides, ascorbic acid, olive leaf and Anemarrhena asphodeloides extracts and stirring evenly to obtain an oral liquid rich in ergothioneine.

[0035] Example 2 This embodiment provides an anti-aging oral liquid based on ergothioneine and disodium pyrroloquinoline quinone, comprising the following components per 1000mL: 0.5g ergothioneine, 0.16g disodium pyrroloquinoline quinone, 4g sodium hyaluronate, 44g fish skin small molecule collagen peptide, 4.7g L-ascorbic acid, 4.3g olive leaf and Anemarrhena asphodeloides extract, with the remainder being water.

[0036] The preparation method of the olive leaf and anemarrhena extract includes the following steps: (1) After washing the fresh olive leaves, air-dry them until the moisture content is 6%, and then grind them to less than 50 mesh. Grind Anemarrhena asphodeloides to less than 50 mesh. Mix the ground olive leaf powder and Anemarrhena asphodeloides powder at a mass ratio of 2.5:1. Sterilize at 121℃ for 30 minutes and cool to room temperature to obtain the mixed raw material powder. (2) Mix the raw material powder with water and adjust the moisture content to 55 wt% to obtain the fermentation raw material. Add compound bacteria to the fermentation raw material. The amount of compound bacteria is 1×10 9The fermentation material was prepared with CFU / mL of fermentation medium. The ratio of viable bacteria of Lactobacillus delbrueckii, Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus casei in the compound bacteria was 2:4:1:3. Anaerobic fermentation was carried out at 35℃ for 40 hours. After fermentation, the mixture was sterilized to obtain the fermentation mixture.

[0037] (3) The mixture after fermentation was first microfiltered using a microfiltration membrane with a pore size of 0.45 μm, and the microfiltration permeate was collected. Then the microfiltration permeate was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 8 kDa to retain small molecule active ingredients. The ultrafiltration permeate was collected. The ultrafiltration permeate was concentrated and vacuum dried to obtain olive leaf and Anemarrhena asphodeloides fermentation extract.

[0038] The method for preparing the anti-aging oral liquid based on ergothioneine and pyrroloquinoline quinone disodium salt includes the following steps: adding water to ergothioneine, pyrroloquinoline quinone, sodium hyaluronate, fish skin small molecule collagen peptides, ascorbic acid, olive leaf and Anemarrhena asphodeloides extracts and stirring evenly to obtain an oral liquid rich in ergothioneine.

[0039] Example 3 This embodiment provides an anti-aging oral liquid based on ergothioneine and disodium pyrroloquinoline quinone, comprising the following components per 1000mL: 0.4g ergothioneine, 0.2g disodium pyrroloquinoline quinone, 3g sodium hyaluronate, 50g fish skin small molecule collagen peptide, 4g L-ascorbic acid, 5g olive leaf and Anemarrhena asphodeloides extract, with the remainder being water.

[0040] The preparation method of the olive leaf and anemarrhena extract includes the following steps: (1) After washing the fresh olive leaves, air-dry them until the moisture content is 6%, and then grind them to less than 50 mesh. Grind Anemarrhena asphodeloides to less than 50 mesh. Mix the ground olive leaf powder and Anemarrhena asphodeloides powder at a mass ratio of 2.5:1. Sterilize at 121℃ for 30 minutes and cool to room temperature to obtain the mixed raw material powder. (2) Mix the raw material powder with water and adjust the moisture content to 55 wt% to obtain the fermentation raw material. Add compound bacteria to the fermentation raw material. The amount of compound bacteria is 1×10 9 The fermentation material was prepared with CFU / mL of fermentation medium. The ratio of viable bacteria of Lactobacillus delbrueckii, Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus casei in the compound bacteria was 2:4:1:3. Anaerobic fermentation was carried out at 35℃ for 40 hours. After fermentation, the mixture was sterilized to obtain the fermentation mixture.

[0041] (3) The mixture after fermentation was first microfiltered using a microfiltration membrane with a pore size of 0.45 μm, and the microfiltration permeate was collected. Then the microfiltration permeate was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 8 kDa to retain small molecule active ingredients. The ultrafiltration permeate was collected. The ultrafiltration permeate was concentrated and vacuum dried to obtain olive leaf and Anemarrhena asphodeloides fermentation extract.

[0042] The method for preparing the anti-aging oral liquid based on ergothioneine and pyrroloquinoline quinone disodium salt includes the following steps: adding water to ergothioneine, pyrroloquinoline quinone disodium salt, sodium hyaluronate, fish skin small molecule collagen peptides, ascorbic acid, olive leaf and Anemarrhena asphodeloides extracts and stirring evenly to obtain an oral liquid rich in ergothioneine.

[0043] Comparative Example 1 The difference between this comparative example and Example 2 is that it does not contain disodium pyrroloquinoline quinone.

[0044] An anti-aging oral liquid based on ergothioneine and pyrroloquinoline quinone disodium salt, comprising the following components per 1000mL: 0.5g ergothioneine, 4g sodium hyaluronate, 44g fish skin small molecule collagen peptide, 4.7g L-ascorbic acid, 4.3g olive leaf and Anemarrhena asphodeloides extract, with the remainder being water.

[0045] Comparative Example 2 The difference between this comparative example and Example 2 is that it does not contain ergothioneine.

[0046] An anti-aging oral liquid based on ergothioneine and disodium pyrroloquinoline quinone, comprising the following components per 1000mL: 0.1~0.2g disodium pyrroloquinoline quinone, 4g sodium hyaluronate, 44g fish skin small molecule collagen peptide, 4.7g L-ascorbic acid, 4.3g olive leaf and Anemarrhena asphodeloides extract, with the remainder being water.

[0047] Comparative Example 3 The difference between this comparative example and Example 2 is that it does not contain olive leaf and Anemarrhena asphodeloides extract.

[0048] An anti-aging oral liquid based on ergothioneine and disodium pyrroloquinoline quinone, comprising the following components per 1000mL: 0.5g ergothioneine, 0.1-0.2g disodium pyrroloquinoline quinone, 4g sodium hyaluronate, 44g fish skin small molecule collagen peptides, 4.7g L-ascorbic acid, with the remainder being water.

[0049] Comparative Example 4 The difference between this comparative example and Example 2 is that the olive leaf and anemarrhena extracts were replaced with an equal amount of ginseng extract.

[0050] Comparative Example 5 The difference between this comparative example and Example 2 is that the olive leaf and anemarrhena extracts were replaced with an equal amount of rhodiola rosea extract.

[0051] Comparative Example 6 The difference between this comparative example and Example 2 is that the crushed olive leaf powder and Anemarrhena asphodeloides powder were mixed evenly at a mass ratio of 1:2.5.

[0052] Comparative Example 7 The difference between this comparative example and Example 2 is that the compound bacteria does not include Lactobacillus casei, and the ratio of live bacteria of Lactobacillus delbrueckii, Lactobacillus plantarum, and Lactobacillus rhamnosus in the compound bacteria is 2:4:1.

[0053] Comparative Example 8 The difference between this comparative example and Example 2 is that the ultrafiltration membrane with a molecular weight cutoff of 8 kDa was replaced with an ultrafiltration membrane with a molecular weight cutoff of 3 kDa.

[0054] Performance testing 1. Using three classic in vitro antioxidant models—DPPH free radical scavenging test, ABTS total antioxidant capacity test, and FRAP ferric reducing power test—the basic antioxidant efficacy of Examples 1-3 and Comparative Examples 1-8 was determined to identify the optimal synergistic formulation of ergothionein and pyrroloquinoline quinone disodium salt. Figure 1 The images show sample images of the anti-aging oral liquids from Examples 1-3 and Comparative Examples 1-8.

[0055] (1) Determination of DPPH free radical scavenging rate: The anti-aging oral liquid and 0.2 mmol / L DPPH ethanol solution were mixed at a volume ratio of 1:1 and reacted in the dark for 30 min. The absorbance was measured at a wavelength of 517 nm, and the free radical scavenging rate of each group was calculated. The higher the scavenging rate, the stronger the antioxidant activity. (2) Determination of total antioxidant capacity of ABTS: The anti-aging oral liquid and ABTS free radical stock solution (with a diluted absorbance of 0.70±0.02 at 734nm) were reacted in the dark at a volume ratio of 1:4 for 10 min. The absorbance was measured at 734nm and the total antioxidant capacity Trolox equivalent was calculated. (3) FRAP reducing power test: The anti-aging oral liquid and (freshly prepared) FRAP working solution are mixed at a volume ratio of 1:10, incubated at 37℃ for 15 min, and the absorbance is measured at 593nm. The higher the absorbance, the stronger the iron ion reducing power and endogenous antioxidant activity of the raw material.

[0056] The test results are shown in Table 1.

[0057] Table 1 Comparison of basic antioxidant activities of different formulations

[0058] As shown in Table 1, the DPPH free radical scavenging rate, ABTS total antioxidant capacity, and FRAP iron ion reducing power of Examples 2-3 were significantly higher than those of other groups, indicating that the oral liquid with this ratio had the strongest basic antioxidant activity, which was superior to Example 1.

[0059] In Comparative Example 1, the lack of the recycling mechanism of pyrroloquinoline quinone disodium salt significantly weakened the antioxidant activity of ergothionein and its extract due to the lack of sustained regeneration activity, resulting in synergistic chain breakage and a decrease in overall antioxidant efficiency.

[0060] Comparative Example 2 lacks the efficient scavenging of specific free radicals by ergothionein and lacks the active skin enrichment effect mediated by OCTN1 transporter. The disodium pyrroloquinoline quinone and extract cannot fill this target gap, resulting in a poorer synergistic effect.

[0061] In Comparative Example 3, the antioxidant effects of ergothionein and pyrroloquinoline quinone disodium salt were weakened due to the loss of microenvironment regulation after the loss of metal chelation and anti-inflammatory synergy of plant polyphenols and saponins.

[0062] Comparative examples 4-5 show that the specific phenolic acids and steroidal saponins contained in olive leaves and anemarrhena are irreplaceable, and other plant extracts cannot provide the same metal chelation and Nrf2 pathway activation capabilities, resulting in a significant decrease in synergistic effects.

[0063] Comparative Example 6 altered the mass ratio of olive leaves to Anemarrhena asphodeloides. The substrate imbalance led to an imbalance in the relative ratio of polyphenols and saponins after fermentation, which prevented them from simultaneously exerting their antioxidant and anti-inflammatory functions and weakened their synergistic effect with ergothioneine and pyrroloquinoline quinone disodium salt.

[0064] In Comparative Example 7, incomplete bacterial strains lead to insufficient conversion of macromolecular bound components into small free aglycones during fermentation, resulting in reduced release of active ingredients and bioavailability, thus affecting the synergistic quality of the final extract.

[0065] In Comparative Example 8, the small molecular weight cutoff resulted in the removal of some effective active oligophenols and saponins, while the retained fragments had a larger molecular weight, affecting their transmembrane transport and intracellular distribution, and preventing them from forming effective spatial synergy with ergothionein and pyrroloquinoline quinone disodium salt at the organelle level.

[0066] 2. Validation assay for cell senescence markers Experimental cells: human skin fibroblasts (HSF), using D-galactose to construct a premature cell aging model.

[0067] Experimental groups: blank control group, model group, Examples 1-3 and Comparative Examples 1-8.

[0068] Drug administration system: The oral solution of each group was uniformly diluted to the theoretical equivalent of ergothioneine in the system, 0.3 μg / mL, for cell intervention, with 6 replicates per group.

[0069] (1) β-galactosidase senescence staining assay: β-galactosidase staining was used as the gold standard for cell senescence, and the percentage of senescent positive cells was counted. The results are shown in Table 2.

[0070] Table 2 Percentage of β-galactosidase-positive senescent cells in each group ( ±s, n=6 )

[0071] As can be seen from Table 2, the proportion of senescent cells in the model group increased significantly, and the proportion of senescent positive cells in Examples 2-3 was the lowest, which was significantly better than all comparative examples and Example 1 (P<0.05), proving that the compound ratio of Examples 2-3 has the best synergistic inhibitory effect on premature cell aging.

[0072] (2) Detection of relative telomere length and relative TERT mRNA expression level: qPCR method. The results are shown in Table 3.

[0073] Table 3. Relative telomere length and relative TERT mRNA expression level ( ±s, n=6 )

[0074] As shown in Table 3, the model group exhibited severe telomere shortening and significantly downregulated telomerase TERT expression. The Example 2-3 group showed the longest relative telomere length and the highest TERT expression, significantly delaying telomere wear and enhancing telomerase activity, demonstrating superior effects compared to all other formulations.

[0075] Comparative Example 1 lacks disodium pyrroloquinoline quinone. Disodium pyrroloquinoline quinone is a core factor in activating SIRT1 and promoting mitochondrial regeneration. The absence of disodium pyrroloquinoline quinone results in NAD+ transcriptional regulation by TERT. + Reduced activity of the SIRT1 signaling pathway leads to downregulated telomerase expression and accelerated telomere wear.

[0076] Comparative Example 2: Ergothioneine deficiency. Ergothioneine is the only natural antioxidant that can be efficiently enriched in the cell nucleus through the OCTN1 transporter. After the deficiency of ergothioneine, the cell nucleus's ability to defend against DNA damage factors such as hydroxyl radicals decreases, and telomere DNA oxidative damage accumulates. Even if TERT expression is upregulated, it is difficult to effectively extend telomeres.

[0077] In Comparative Example 3, the absence of olive leaf and Anemarrhena asphodeloides extract resulted in the loss of the extract's metal chelating and anti-inflammatory regulatory effects, leading to increased levels of intracellular inflammatory factors, aggravated SASP secretion, deterioration of the telomere microenvironment, and accelerated telomere loss. Simultaneously, insufficient activation of the Nrf2 antioxidant pathway weakened the protective effect of telomeres.

[0078] Comparative Examples 4 and 5, when replaced with ginseng or rhodiola rosea extracts, demonstrated that the specific phenol-saponin combination provided by olive leaves and anemarrhena asphodeloides is irreplaceable in protecting nuclear DNA and activating the endogenous antioxidant system, and that other extracts could not maintain telomere homeostasis as effectively.

[0079] In Comparative Example 6, the ratio of olive leaves to Anemarrhena asphodeloides was unbalanced, resulting in an imbalance in the ratio of polyphenols to saponins in the fermentation products. Consequently, the anti-inflammatory and antioxidant functions could not work synergistically, leading to a significant decrease in the telomere protection effect.

[0080] Comparative Example 7 lacks Lactobacillus casei. Incomplete bacterial strains lead to insufficient release of active aglycones, limiting the ability of the extract to enter the cell nucleus, failing to effectively scavenge intranuclear free radicals, and providing insufficient protection against telomere DNA damage.

[0081] Comparative Example 8 showed that the small molecular weight cutoff resulted in the loss of effective small molecule active ingredients, and the cell membrane permeability of the retained components decreased, making it difficult to achieve an effective concentration in the cell nucleus to synergistically protect telomeres.

[0082] (3) Detection of SASP aging-related secretory factors IL-6 and TNF-α (ELISA), the results are shown in Table 4.

[0083] Table 4. Cell supernatant IL-6 and TNF-α content ( ±s, n=3 )

[0084] Table 4 shows that the model group released large amounts of IL-6 and TNF-α, indicating severe inflammatory senescence. Example 1 only moderately downregulated inflammatory factors. Examples 2-3 showed the lowest concentrations of IL-6 and TNF-α in the supernatant, demonstrating the best ability to inhibit the secretion of SASP inflammatory factors and significantly improving inflammatory senescence. Comparative Examples 1-8 showed varying degrees of weakened anti-inflammatory ability.

[0085] 3. Validation test of anti-aging indexes in mouse skin Experimental model and grouping D-galactose subacute aging mouse model, male ICR mice, were randomly divided into: blank control group, model group, Examples 1-3, and comparative examples 1-8; 10 mice in each group were administered the corresponding oral solution by gavage daily for 42 consecutive days; the blank group and model group were administered the same volume of physiological saline by gavage.

[0086] At the end of the experiment, skin tissue from a uniform area on the back of mice was collected and subjected to HE pathological sectioning to observe dermal thickness and fiber morphology; the hydroxyproline content in the skin tissue was detected to characterize the total amount of collagen.

[0087] Table 5 Results of dermal thickness detection on the back of mice in each group ( ±s, n=10 )

[0088] The results in the table above show that the dermis of the model group mice was significantly thinner, and the collagen fibers were severely aged and damaged. Examples 2-3 showed significantly higher dermal thickness than all other treatment groups, with the best collagen fiber integrity and the best improvement in skin structure aging. Example 1 only moderately increased dermal thickness, and the fibers still showed significant aging damage. Comparative Examples 1-8 showed minimal improvement in skin structure.

[0089] The results of the detection of hydroxyproline content in skin tissue (a quantitative indicator of collagen) are shown in Table 6.

[0090] Table 6. Hydroxyproline content in mouse skin (μg / mg skin tissue) ±s, n=10 )

[0091] As can be seen from the results in Table 6 above, hydroxyproline is a characteristic amino acid of collagen, and the lower the content, the more severe the loss of collagen in the skin. The hydroxyproline content in the model group decreased significantly. The hydroxyproline content in the skin of Examples 2-3 was significantly higher than that in all comparative examples and Example 1, which significantly reduced collagen degradation caused by aging and promoted collagen retention, resulting in the best anti-aging effect. The increase in hydroxyproline in Example 1 was limited, and comparative examples 1-8 only slightly increased collagen content.

[0092] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An anti-aging oral liquid based on ergothioneine and pyrroloquinoline quinone disodium salt, characterized in that, Per 1000 mL, it includes the following components: ergothioneine 0.2~0.8 g, pyrroloquinoline quinone disodium salt 0.05~0.2 g, sodium hyaluronate 2~6 g, fish skin small molecule collagen peptide 20~60 g, L-ascorbic acid 3~8 g, olive leaf and anemarrhena extract 0.5~8.0 g, and the balance is water.

2. The anti-aging oral liquid based on ergothioneine and pyrroloquinoline quinone disodium salt according to claim 1, characterized in that, Per 1000mL, it includes the following components: ergothioneine 0.4~0.6g, disodium pyrroloquinoline quinone 0.1~0.2g, sodium hyaluronate 3~5g, small molecule collagen peptides from fish skin 30~50g, L-ascorbic acid 4~5g, olive leaf and anemarrhena extract 3~5g, and the balance is water.

3. The anti-aging oral liquid based on ergothioneine and pyrroloquinoline quinone disodium salt according to claim 1, characterized in that, The preparation method of the olive leaf and anemarrhena extract includes the following steps: mixing olive leaves and anemarrhena, fermenting with compound bacteria, and then sequentially passing through microfiltration, ultrafiltration and drying.

4. The anti-aging oral liquid based on ergothioneine and pyrroloquinoline quinone disodium salt according to claim 3, characterized in that, The compound bacteria include Lactobacillus delbrueckii, Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus casei.

5. The anti-aging oral liquid based on ergothioneine and pyrroloquinoline quinone disodium salt according to claim 4, characterized in that, The ratio of viable bacteria of Lactobacillus delbrueckii, Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus casei in the compound bacteria is (1~2):(3~4):(1~2):(2~3).

6. The anti-aging oral liquid based on ergothioneine and pyrroloquinoline quinone disodium salt according to claim 3, characterized in that, The mass ratio of olive leaves to Anemarrhena asphodeloides is (2~3):

1.

7. The anti-aging oral liquid based on ergothioneine and pyrroloquinoline quinone disodium salt according to claim 3, characterized in that, The microfiltration uses a filter membrane with a pore size of 0.45~0.80μm.

8. The anti-aging oral liquid based on ergothioneine and pyrroloquinoline quinone disodium salt according to claim 7, characterized in that, The ultrafiltration membrane used in this process has a molecular weight cutoff of 5kDa to 10kDa.

9. The anti-aging oral liquid based on ergothioneine and pyrroloquinoline quinone disodium salt according to claim 1, characterized in that, The anti-aging oral liquid based on ergothioneine and pyrroloquinoline quinone disodium salt also includes oral pharmaceutical excipients.

10. A method for preparing an anti-aging oral liquid based on ergothioneine and pyrroloquinoline quinone disodium salt as described in any one of claims 1 to 9, characterized in that, The process includes the following steps: adding water to ergothioneine, disodium pyrroloquinoline quinone, sodium hyaluronate, small molecule collagen peptides from fish skin, ascorbic acid, olive leaf and anemarrhena extracts, and oral pharmaceutical excipients, and stirring until homogeneous to obtain an oral solution rich in ergothioneine.

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