Anti-aging composition and application thereof

Abstract

The invention discloses an anti-aging composition which comprises the following components or consists of the following components in parts by weight: 10-20 parts of pyrroloquinoline quinone or derivatives thereof; 2 to 30 parts by weight of ergothioneine; 0.2 to 10 parts by weight of spermidine; and 10-650 parts by weight of a pomegranate extract. The invention also discloses an application of the anti-aging composition. According to the anti-aging composition disclosed by the invention, the pyrroloquinoline quinone or the derivative thereof, the ergothioneine, the spermidine and the pomegranate extract contained in the anti-aging composition can generate a synergistic effect, so that the anti-aging composition has good anti-oxidation and anti-aging effects. The anti-aging composition can be applied to food, health care products and medicines, and has the effects of resisting skin oxidation and delaying aging.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a composition with antioxidant effects and anti-aging properties and its application. Background Art

[0002] As living standards improve, people are increasingly concerned about their health and appearance, and anti-aging has become a popular research topic in the life sciences. Aging is a complex physiological process involving the decline of body functions, cellular aging, oxidative stress damage, inflammatory response, and other aspects.

[0003] In the prior art, common anti-aging methods include the use of antioxidants, cytokines, growth factors, etc. Antioxidants such as vitamin C, vitamin E, coenzyme Q10, etc. can neutralize free radicals in the body and reduce the damage of oxidative stress to cells. However, the effect of a single antioxidant is often limited, and long-term and large-scale use may have certain side effects. Although cytokines and growth factors can promote cell proliferation and repair, they have poor stability and a short half-life in the body, requiring frequent administration, which is inconvenient in practical applications.

[0004] Furthermore, most anti-aging products currently on the market target only one or a few aging-related factors, failing to comprehensively and effectively address the complexities of the aging process. For example, some products focus solely on the skin's external appearance, such as reducing wrinkles and increasing skin elasticity, while ignoring the decline in internal body functions. Furthermore, existing anti-aging technologies still face numerous unresolved challenges in terms of their mechanisms of action, safety, and effectiveness. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an anti-aging composition, wherein the pyrroloquinoline quinone or its derivatives, ergothioneine, spermidine and pomegranate extract contained in the anti-aging composition can produce a synergistic effect, significantly enhancing the antioxidant and anti-aging effects.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] The first aspect of the present invention provides an anti-aging composition comprising or consisting of the following ingredients:

[0008] 10-20 parts by weight of pyrroloquinoline quinone or its derivatives;

[0009] 2 to 30 parts by weight of ergothioneine;

[0010] 0.2 to 10 parts by weight of spermidine; and

[0011] 10 to 650 parts by weight of pomegranate extract.

[0012] Pyrroloquinoline quinone (PQQ) is widely present in a variety of common foods, including fruits, vegetables, grains, and animal products, but its content is very low, measured in micrograms per kilogram. It is a powerful antioxidant. PQQ participates in cellular energy metabolism, helping to maintain normal cell function and vitality and delaying cell aging. It has excellent free radical scavenging ability, reducing the accumulation of oxidative damage, thereby exerting anti-aging effects. It can also stimulate nerve cells to secrete neurotrophic factors such as nerve growth factor (NGF), promoting axonal growth and dendritic branching of nerve cells, strengthening connections between neurons, and maintaining normal function of the nervous system, thereby delaying the aging of the nervous system.

[0013] Ergothioneine (EGT) is a natural antioxidant with powerful free radical scavenging ability. It can effectively remove various free radicals produced in cells, protect cells from oxidative damage, and delay cell aging. Ergothioneine can also regulate inflammatory signaling pathways, inhibit the production and release of inflammatory mediators, reduce the damage of inflammation to tissues and cells, and delay the aging process. In addition, ergothioneine also participates in some metabolic processes in cells, affects the energy metabolism and substance synthesis of cells, maintains the normal physiological functions and vitality of cells, and helps to delay cell aging.

[0014] Spermidine is a polyamine compound that can participate in cell proliferation, differentiation and autophagy, and has anti-aging and life-extending effects; secondly, spermidine has a certain free radical scavenging ability, which can protect cells from oxidative damage and play an anti-aging role; in addition, spermidine can also inhibit the activation of inflammatory signaling pathways, reduce inflammatory damage to tissues and organs, and thus play an anti-aging role.

[0015] Pomegranate extract is a natural ingredient extracted from the peel or seeds of pomegranate. It is rich in 30-50% polyphenols, mostly ellagic acid, which has strong oxidative ability, can effectively scavenge free radicals and inhibit oxidative stress. Pomegranate extract also has significant anti-inflammatory activity, can inhibit the release of inflammatory mediators and the activation of inflammatory signaling pathways. In addition, pomegranate extract can also promote the synthesis of collagen by skin fibroblasts. It can activate signaling pathways related to collagen synthesis, increase the expression of collagen genes, and thus increase the amount of collagen synthesis.

[0016] Pyrroloquinoline quinone, ergothioneine, spermidine and red pomegranate extract, these four ingredients all have antioxidant and anti-aging effects. The inventors found that when these four ingredients are compounded and used in a certain ratio, they can produce a synergistic effect between each other, significantly enhancing the antioxidant and anti-aging effects. Specifically, pyrroloquinoline quinone can promote mitochondrial biosynthesis, enhance ATP generation, and when it is used in combination with spermidine, it can synergistically activate autophagy and remove damaged mitochondria; simultaneously, pyrroloquinoline quinone can also reduce the damage of oxidative stress to cells when used in combination with ergothioneine. And ergothioneine itself has a strong antioxidant effect, can enhance the stability of polyphenols in red pomegranate extract, and reduce inflammation by regulating the Nrf2 pathway. Spermidine can activate cell autophagy, and red pomegranate extract is rich in ellagic acid, and ellagic acid metabolite (urolithin A) can enhance mitochondrial autophagy and form "autophagy dual pathway" with spermidine; and polyphenols therein can synergistically inhibit AGEs (advanced glycation end products) generation with ergothioneine. Therefore, when pyrroloquinoline quinone, ergothioneine, spermidine and pomegranate extract are used in combination, they can synergistically enhance the antioxidant effect and improve the anti-aging effect.

[0017] Specifically, the anti-aging composition can significantly increase the glutathione (GSH) content and superoxide dismutase (SOD enzyme) activity in cells, effectively inhibit the reactive oxygen species (ROS) content in cells, promote the production of type I and type III collagen, and inhibit the expression of skin aging markers, matrix metalloproteinases MMP-1 and MMP-3.

[0018] Furthermore, in some preferred embodiments, the anti-aging composition comprises or consists of the following ingredients:

[0019] 12-20 parts by weight of pyrroloquinoline quinone or its derivatives;

[0020] 2 to 30 parts by weight of ergothioneine;

[0021] 0.2 to 6 parts by weight of spermidine; and

[0022] 20 to 100 parts by weight of pomegranate extract.

[0023] Under this ratio, the four ingredients of pyrroloquinoline quinone, ergothioneine, spermidine and pomegranate extract in the anti-aging composition can produce a better synergistic effect, and the antioxidant effect and the effect of promoting fibroblasts to produce type I and type III collagen in the composition are more prominent, thereby having a better anti-aging effect.

[0024] Furthermore, in some preferred embodiments, the anti-aging composition comprises or consists of the following ingredients:

[0025] 20 parts by weight of pyrroloquinoline quinone or its derivatives;

[0026] 5 weight portions of ergothioneine;

[0027] 0.21 parts by weight of spermidine; and

[0028] 20 parts by weight of pomegranate extract.

[0029] Under this ratio, the four ingredients of pyrroloquinoline quinone, ergothioneine, spermidine and pomegranate extract in the anti-aging composition can produce the best synergistic effect, so that the composition has the best antioxidant and anti-aging effects.

[0030] Furthermore, the pyrroloquinoline quinone derivatives include but are not limited to pyrroloquinoline quinone disodium salt.

[0031] In the present invention, the source of spermidine is not limited. Preferably, the spermidine is derived from wheat germ extract, and the content of spermidine in the wheat germ extract is 1 wt%.

[0032] The second aspect of the present invention provides the use of the anti-aging composition in anti-skin oxidation.

[0033] The third aspect of the present invention provides the use of the anti-aging composition in promoting skin fibroblasts to produce type I and type III collagen and / or inhibiting the expression of skin aging markers such as matrix metalloproteinases MMP-1 and MMP-3.

[0034] The fourth aspect of the present invention provides the use of the anti-aging composition in the preparation of a preparation for resisting skin oxidative damage.

[0035] The fifth aspect of the present invention provides the use of the anti-aging composition in the preparation of anti-aging preparations.

[0036] Furthermore, the preparation is a medicine, which can be prepared into various dosage forms, including but not limited to tablets, capsules, oral solutions, and injections.

[0037] Furthermore, the preparation is a food or a health product, and the types of the food or health product include but are not limited to oral liquid, capsules, tablets, liquid beverages, solid beverages and soft candies.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] 1. The anti-aging composition provided by the present invention, wherein the pyrroloquinoline quinone or its derivatives, ergothioneine, spermidine and pomegranate extract contained therein can produce a synergistic effect, can significantly increase the glutathione (GSH) content and superoxide dismutase (SOD) enzyme activity in cells, effectively inhibit the reactive oxygen species (ROS) in cells, promote the production of type I and type III collagen, inhibit the expression of skin aging markers metalloproteinases MMP-1 and / or MMP-3, and significantly enhance the antioxidant and anti-aging effects.

[0040] 2. The anti-aging composition provided by the present invention can effectively improve oxidative damage and delay aging. The composition can be applied to products such as food, health products, and medicines, and has broad application prospects. DETAILED DESCRIPTION

[0041] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative efforts shall fall within the scope of protection of the present invention.

[0042] The present invention will be further described below with reference to specific embodiments and comparative examples.

[0043] In the following specific embodiments, the wheat germ extract contains 1 wt% spermidine.

[0044] Example 1

[0045] According to parts by weight, 20 parts of pyrroloquinoline quinone disodium salt, 5 parts of ergothioneine, 21 parts of wheat germ extract, and 20 parts of pomegranate extract were uniformly mixed to obtain the composition of this embodiment.

[0046] Example 2

[0047] According to parts by weight, 10 parts of pyrroloquinoline quinone disodium salt, 2 parts of ergothioneine, 21 parts of wheat germ extract, and 10 parts of pomegranate extract were mixed uniformly to obtain the composition of this embodiment.

[0048] Example 3

[0049] According to parts by weight, 15 parts of pyrroloquinoline quinone disodium salt, 25 parts of ergothioneine, 100 parts of wheat germ extract, and 100 parts of pomegranate extract were mixed uniformly to obtain the composition of this embodiment.

[0050] Example 4

[0051] According to parts by weight, 12 parts of pyrroloquinoline quinone disodium salt, 10 parts of ergothioneine, 800 parts of wheat germ extract, and 10 parts of pomegranate extract were mixed uniformly to obtain the composition of this embodiment.

[0052] Example 5

[0053] According to parts by weight, 10 parts of pyrroloquinoline quinone disodium salt, 3 parts of ergothioneine, 20 parts of wheat germ extract, and 500 parts of pomegranate extract were mixed uniformly to obtain the composition of this embodiment.

[0054] Comparative Example 1

[0055] According to parts by weight, 20 parts of pyrroloquinoline quinone disodium salt and 5 parts of ergothioneine were uniformly mixed to obtain the composition of this comparative example.

[0056] Comparative Example 2

[0057] According to parts by weight, 20 parts of pyrroloquinoline quinone disodium salt, 5 parts of ergothioneine, and 21 parts of wheat germ extract were uniformly mixed to obtain the composition of this comparative example.

[0058] Comparative Example 3

[0059] According to parts by weight, 20 parts of pyrroloquinoline quinone disodium salt, 5 parts of ergothioneine, and 20 parts of pomegranate extract were uniformly mixed to obtain the composition of this comparative example.

[0060] Comparative Example 4

[0061] According to parts by weight, 5 parts of ergothioneine, 21 parts of wheat germ extract and 20 parts of pomegranate extract were uniformly mixed to obtain a composition of this comparative example.

[0062] Comparative Example 5

[0063] According to parts by weight, 20 parts of pyrroloquinoline quinone disodium salt, 21 parts of wheat germ extract, and 20 parts of pomegranate extract were uniformly mixed to obtain the composition of this comparative example.

[0064] Comparative Example 6

[0065] 8 parts of pyrroloquinoline quinone disodium salt, 5 parts of ergothioneine, 21 parts of wheat germ extract and 20 parts of pomegranate extract were uniformly mixed according to parts by weight to obtain the composition of this comparative example.

[0066] Comparative Example 7

[0067] According to parts by weight, 20 parts of pyrroloquinoline quinone disodium salt, 5 parts of ergothioneine, 15 parts of wheat germ extract, and 20 parts of pomegranate extract were uniformly mixed to obtain the composition of this comparative example.

[0068] Comparative Example 8

[0069] According to parts by weight, 20 parts of pyrroloquinoline quinone disodium salt, 1.5 parts of ergothioneine, 21 parts of wheat germ extract, and 20 parts of pomegranate extract were uniformly mixed to obtain the composition of this comparative example.

[0070] Comparative Example 9

[0071] According to parts by weight, 20 parts of pyrroloquinoline quinone disodium salt, 5 parts of ergothioneine, 21 parts of wheat germ extract, and 8 parts of pomegranate extract were uniformly mixed to obtain the composition of this comparative example.

[0072] Comparative Example 10

[0073] 8 parts of pyrroloquinoline quinone disodium salt, 1 part of ergothioneine, 10 parts of wheat germ extract and 10 parts of pomegranate extract were uniformly mixed according to parts by weight to obtain the composition of this comparative example.

[0074] The specific formulas of the compositions in Examples 1 to 5 and Comparative Examples 1 to 10 are shown in Table 1.

[0075] Table 1

[0076]

[0077]

[0078] Test Example 1: Antioxidant efficacy of the composition

[0079] 1.1. Effects on GSH

[0080] 1. Instrument

[0081] Inverted microscope, cell counter, multifunctional microplate reader, carbon dioxide incubator, centrifuge.

[0082] 2. Experimental Materials

[0083] Table 2

[0084] Complete medium DMEM high glucose medium + 10% serum MTT working solution Dilute MTT to 1 mg / mL MTT working solution with complete culture medium and use it immediately sample Efficacy test experiments were conducted on samples with different ratios Positive control L-ascorbic acid as a positive control Cells and passages HaCaT cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. The cytotoxicity was determined at passage 7, and the efficacy was determined at passage 14.

[0085] 3 Experimental steps

[0086] 3.1. Cell Culture, Toxicity Testing, and Modeling: Cell recovery and culture were performed according to standard cell culture standard operating procedures. Cytotoxicity experiments were performed. Based on the cytotoxicity results, an appropriate sample concentration was selected for subsequent antioxidant efficacy experiments. Specifically, a 0.16 mg / mL sample was selected for subsequent efficacy testing experiments. UVB irradiation was performed to establish the model. After irradiation, the cell plate was transferred to a biosafety cabinet for sample loading.

[0087] 3.2 Cell Sampling, Collection, and Detection: Prepare solutions containing different ratios for each experimental group. Add 0.16 mg / mL of sample in complete culture medium to the sample wells, complete culture medium containing the positive control to the positive control wells, complete culture medium to the blank control wells, and complete culture medium or complete culture medium containing solvent to the model wells, with 1 mL added to each well. After addition, place the 12-well plate in a CO2 incubator and incubate for 12 ± 4 hours. After incubation, centrifuge and perform sample preparation and detection according to the GSH detection kit instructions.

[0088] 4. Data Analysis

[0089] Quantitative data were expressed as mean ± standard deviation (X ± s). The data were subjected to one-way analysis of variance, and the P value was used to determine the statistical difference. P < 0.05 indicated statistical significance.

[0090] Calculation of GSH content in cells: refer to the kit instructions.

[0091] 5. Experimental Results

[0092] Table 3

[0093]

[0094]

[0095] Glutathione (GSH) is a low molecular weight scavenger that can remove O2 - , H2O2, and LOOH. GSH levels are an important factor in measuring the body's antioxidant capacity; higher GSH values indicate the strongest antioxidant capacity. Therefore, the antioxidant efficacy of a composition can be evaluated by measuring the GSH content in cells.

[0096] As shown in Table 3, under these test conditions, the GSH content in the blank control group was 286.72 μmol / mg, a 21.04% increase compared to the model group (M), with a significant difference (P < 0.05). This indicates that after UVB irradiation, the GSH content in the model group (M) cells was significantly reduced, and the senescent cell model was successfully established, allowing for subsequent efficacy testing. The GSH content in the positive control group (PC) cells was 294.56 μmol / mg, a 24.35% increase compared to the model group (M), with a significant difference (P < 0.05), indicating that the test system is effective and the experimental results are in line with expectations.

[0097] A composition prepared using specific ratios of pyrroloquinoline quinone disodium salt, ergothioneine, spermidine, and pomegranate extract can significantly increase GSH content in cells. Referring to Examples 1-5, the GSH content increase rate of the compositions all reached 15% or more; in Example 1, the composition with a ratio of pyrroloquinoline quinone disodium salt, ergothioneine, spermidine, and pomegranate extract of 20:5:0.21:20 showed the most significant increase in GSH content, reaching 26.81%.

[0098] When any one of the components pyrroloquinoline quinone disodium salt, ergothioneine, spermidine, and pomegranate extract was missing from the composition, the rate of increase in GSH content was significantly reduced. Referring to Comparative Examples 1-5, when one or two of the components were missing from the composition, the rate of increase in GSH content was below 8%, a significant decrease compared to Example 1.

[0099] When the mass ratios of the four components in the composition are outside the range defined by the present invention, the synergistic effect is significantly reduced, resulting in a significantly lower increase in GSH content. For Comparative Examples 6-10, the GSH content increases in the compositions were all below 10%, significantly lower than those in Example 1, despite the fact that the content of at least one component in the compositions was outside the range defined by the present invention.

[0100] The above results show that when pyrroloquinoline quinone disodium salt, ergothioneine, spermidine and pomegranate extract are used in a specific proportion, they can produce a certain synergistic effect, which is more conducive to increasing the GSH content.

[0101] 1.2. In vitro epithelial cell SOD content test

[0102] The instruments, experimental materials, and experimental procedures are all as described in 1.1. After sample collection, sample preparation and testing were performed according to the instructions of the SOD detection kit.

[0103] 4. Data Analysis

[0104] Calculation of SOD inhibition rate in cells:

[0105] SOD enzyme activity unit = inhibition percentage / (1-inhibition percentage) Units Cell SOD enzyme activity enhancement rate:

[0106] Improvement rate (%) = (T / C-1) × 100%

[0107] Where:

[0108] T—average value of SOD enzyme activity of test cells;

[0109] C—Average value of SOD enzyme activity in cells of model group.

[0110] 5. Experimental Results

[0111] Table 4

[0112]

[0113]

[0114] Superoxide dismutase (SOD) catalyzes the dismutation of superoxide anions into hydrogen peroxide (H2O2) and oxygen (O2). It is an important antioxidant enzyme in living organisms and plays a crucial role in the body's oxidative and antioxidant balance. This enzyme scavenges superoxide anion free radicals, protecting cells from damage. Therefore, measuring SOD activity in treated cells can be used to evaluate the antioxidant efficacy of a composition.

[0115] As shown in Table 4, under these test conditions, the SOD enzyme activity in the blank control group (BC) was 1.59 units, which was 125.94% higher than that in the model group (M) (an increase of ≥15%). This indicates that after UVB irradiation, the SOD enzyme activity in the model group (M) cells decreased significantly, indicating that the cell model was successfully established and suitable for subsequent efficacy testing.

[0116] The SOD enzyme activity in the positive control group (PC) cells was 1.24 Units, which was 76.79% higher than that in the model group (M). There was a significant difference (P<0.05) in the SOD enzyme activity compared with that in the model group (M), indicating that the test system was effective.

[0117] Please refer to Table 4. The composition prepared by using pyrroloquinoline quinone disodium salt, ergothioneine, spermidine and pomegranate extract in a specific ratio can produce a synergistic effect and significantly improve the SOD enzyme activity in the cell. With reference to Examples 1-5, the SOD enzyme activity improvement rate reached 40% and above; among them, in Example 1, the composition with a ratio of pyrroloquinoline quinone disodium salt, ergothioneine, spermidine and pomegranate extract of 20:5:0.21:20 was the most significant for improving SOD enzyme activity, reaching 101.15%.

[0118] When the composition lacks any one of pyrroloquinoline quinone disodium salt, ergothioneine, spermidine, and pomegranate extract, the SOD enzyme activity enhancement rate is significantly reduced. Referring to Comparative Examples 1-5, when the composition lacks one or two of the ingredients, the SOD enzyme activity enhancement rate is below 20%, significantly lower than that in Example 1.

[0119] When the mass ratios of the four components in the composition are outside the range defined by the present invention, the synergistic effect is significantly reduced, resulting in a significant decrease in the rate of increase in SOD enzyme activity. Referring to Comparative Examples 6-10, the SOD enzyme activity increases were all below 25%, also significantly lower than in Example 1, when the content of at least one component in the composition was outside the range defined by the present invention.

[0120] The above results show that when pyrroloquinoline quinone disodium salt, ergothioneine, spermidine and pomegranate extract are used in a specific proportion, they can produce a synergistic effect and are more conducive to increasing the rate of SOD enzyme activity.

[0121] 1.3. ROS content detection

[0122] 1. Instrument

[0123] Inverted microscope, cell counter, carbon dioxide incubator, centrifuge, fluorescence microscope, and biological safety cabinet.

[0124] 2. Experimental Materials

[0125] Table 5

[0126]

[0127] 3. Experimental Procedure

[0128] The experimental procedures are largely the same as those in 1.1. For the drug addition procedure, complete culture medium containing different ratios of the combination was added to the sample wells, complete culture medium containing the positive control well (PC) was added to the positive control wells, complete culture medium was added to the blank control wells (BC), and complete culture medium was added to the model wells (M), with a total volume of 2 mL per well. After drug addition, the 6-well plate was placed in a CO2 incubator and incubated for 1 hour ± 15 minutes. After incubation, the culture medium was discarded, the plate was washed once with DPBS, and 1 mL of DCFH-DA dye working solution was added to each well. The plate was incubated in the dark for 20 minutes.

[0129] 3.2. ROS content detection

[0130] After incubation, the cells in each well were washed three times with DPBS, and 300 μL of DPBS and 3 μL of nuclear dye Hoechst were added to each well. The cell plate was transferred to a dark room and photographed with a fluorescence microscope. The collected photos were analyzed for fluorescence intensity using ImageJ software.

[0131] 4. Data Processing

[0132] 4.1. Calculation formula for ROS absolute fluorescence intensity

[0133] ROS absolute fluorescence intensity value = ROS fluorescence intensity average value / nuclear fluorescence intensity average value.

[0134] 4.2. Calculation formula for ROS relative fluorescence intensity

[0135] ROS relative fluorescence intensity (%) = T / C × 100%

[0136] T—absolute fluorescence intensity of ROS in the test sample;

[0137] C—The average of three times of absolute fluorescence intensity of ROS in the negative control group.

[0138] 4.3. Calculation formula for ROS inhibition rate

[0139] Inhibition rate (%) = (1-T / C) × 100%

[0140] T—the average of three times of the relative fluorescence intensity of ROS of the test sample;

[0141] C—The average of three times of ROS relative fluorescence intensity in the negative control group.

[0142] 5. Experimental Results

[0143] Table 6

[0144]

[0145] 2',7'-Dichlorodi-hydrofluoresceindiacetate (DCFH-DA) exhibits no autofluorescence and can freely cross cell membranes. Once inside the cell, it is hydrolyzed by esterases to form DCFH, which cannot penetrate the cell membrane and accumulates inside the cell. Intracellular reactive oxygen species can oxidize the non-fluorescent DCFH to produce fluorescent DCF. The intensity of green fluorescence is proportional to the level of intracellular reactive oxygen species. Exogenous oxidants such as hydrogen peroxide (H2O2), ultraviolet light, and blue light can induce elevated levels of intracellular reactive oxygen species (ROS). By comparing the changes in fluorescence intensity of the intracellular fluorescent dye DCFH-DA after administration of the negative control and the test substance, the relative fluorescence intensity and inhibition rate of ROS were calculated.

[0146] Please refer to Table 6. Under these test conditions, the relative fluorescence intensity of ROS in the blank control group (BC) cells was 69.71%, a 30.29% inhibition compared to the model group (M), with a significant difference in relative fluorescence intensity between the two groups (P < 0.05). This indicates that after UVB irradiation, the ROS content in the model group (M) cells was significantly increased, and the senescent cell model was successfully established, allowing for subsequent efficacy testing. The relative fluorescence intensity of ROS in the positive control group (PC) cells was 73.12%, a 26.88% inhibition compared to the model group (M), with a significant difference in relative fluorescence intensity between the two groups (P < 0.05), indicating that the test system is effective and the experimental results are in line with expectations.

[0147] Please refer to Table 6. The composition prepared using a specific ratio of pyrroloquinoline quinone disodium salt, ergothioneine, spermidine and pomegranate extract can effectively inhibit cellular ROS. Please refer to Examples 1-5. The inhibition rate of the composition for ROS is 30% or more. Among them, in Example 1, the composition with a ratio of pyrroloquinoline quinone disodium salt, ergothioneine, spermidine and pomegranate extract of 20:5:0.21:20 has the most significant improvement in the inhibition rate of ROS, reaching 39.54%.

[0148] When any one of pyrroloquinoline quinone disodium salt, ergothioneine, spermidine, and pomegranate extract was missing from the composition, the ROS inhibition rate was significantly reduced. Referring to Comparative Examples 1-5, when one or two of the components were missing from the composition, the ROS inhibition rate was below 15%, significantly lower than that in Example 1.

[0149] When the ratios of the four components in the composition are outside the range defined by the present invention, the synergistic effect is significantly reduced, resulting in a significant decrease in ROS inhibition rate. For Comparative Examples 6-10, the ROS inhibition rates of all compositions were below 20%, also significantly lower than those in Example 1, even though the content of at least one component was outside the range defined by the present invention.

[0150] The above results show that when pyrroloquinoline quinone disodium salt, ergothioneine, spermidine and pomegranate extract are used in a specific proportion, they can produce a certain synergistic effect and are more conducive to inhibiting the production of ROS.

[0151] Test 2: Effects of the composition on collagen type I and collagen type III

[0152] 1. Instrument

[0153] Inverted microscope, cell counter, carbon dioxide incubator, centrifuge, microplate reader, and biological safety cabinet.

[0154] 2. Experimental Materials

[0155] Table 7

[0156]

[0157]

[0158] 3. Experimental Procedure

[0159] 3.1. Cell Culture and Toxicity Testing: Cells were revived and cultured according to standard cell culture operating procedures, and cytotoxicity testing was performed. Based on the cytotoxicity results, an appropriate sample concentration was selected for subsequent antioxidant efficacy testing. Specifically, a 0.16 mg / mL sample was selected for subsequent efficacy testing.

[0160] 3.2. Cell sampling, collection, and detection

[0161] Select cells in the logarithmic growth phase, collect the cells after digestion, make a cell suspension and count. Dilute the cells to the appropriate inoculation density with cell culture medium. After inoculation, place them in a CO2 incubator for culture. Discard the culture medium in the well plate and carry out the dosing operation. Add culture medium containing different ratios of 0.16 mg / ml samples to the sample group wells, add culture medium containing positive control to the positive control wells, and add normal cell culture medium to the blank control wells, 200 μL per well, and set up 4 parallel wells for each group. Place the 96-well plate in a CO2 incubator and culture for 24h±2h. After the incubation and culture is completed, centrifuge and place in a -80℃ ultra-low temperature refrigerator for storage. ELISA detection should be performed according to the instructions of the human type I and type III collagen enzyme-linked immunosorbent assay kit.

[0162] 4. Data Analysis

[0163] 4.1. Increase rate (%) = (T / C-1) × 100%

[0164] Where:

[0165] T—average value of type I and III collagen content of the test substance;

[0166] C—Average value of type I and III collagen content in blank control.

[0167] 4.2. Relative content of type I and III collagen (%) = (T / C) × 100%

[0168] Where:

[0169] T—average value of type I and III collagen content of the test substance;

[0170] C—Average value of type I and III collagen content in blank control.

[0171] 5. Experimental Results

[0172] Table 8

[0173]

[0174] Please refer to Table 8. Under the experimental conditions of this study, the expression rate of type I collagen in the positive control group was significantly increased by 43.95% compared with the blank group. The test system was effective, and the coefficient of variation CV value between replicate wells in each group was ≤20%, indicating that the test parallelism was effective.

[0175] As can be seen from Table 8, the composition prepared using a specific ratio of pyrroloquinoline quinone disodium salt, ergothioneine, spermidine and pomegranate extract can significantly promote the production of type I and type III collagen. Please refer to Examples 1-5. The composition has an upregulation rate of more than 15% for type I collagen and an upregulation rate of more than 20% for type III collagen. Among them, in Example 1, the composition with a ratio of pyrroloquinoline quinone disodium salt, ergothioneine, spermidine and pomegranate extract of 20:5:0.21:20 has the most significant effect on promoting the production rate of type I and type III collagen, reaching more than 30%.

[0176] When the composition lacks any one of pyrroloquinoline quinone disodium salt, ergothioneine, spermidine, and pomegranate extract, the promotion effect on type I and type III collagen is not obvious. Please refer to Comparative Examples 1-5. When one or two of the ingredients are missing from the composition, the promotion effect on type I and type III collagen is limited, and the upregulation rate of type I and type III collagen is less than 10%, which is significantly lower than that of Example 1.

[0177] When the ratios of the four components in the composition are outside the range defined by the present invention, the synergistic effect is significantly reduced, resulting in a significant decrease in the promotion effect on type I and type III collagen. Please refer to Comparative Examples 6-10. The content of at least one component in the composition is outside the range defined by the present invention, and the upregulation rates of type I and type III collagen are both less than 10%, which is also significantly lower than that of Example 1.

[0178] The above results show that when pyrroloquinoline quinone disodium salt, ergothioneine, spermidine and pomegranate extract are used in a specific proportion, they can produce a certain synergistic effect and are more conducive to promoting the production of type I and type III collagen.

[0179] The human dermis has the ability to automatically produce type I collagen, though this capacity decreases with age. Type III collagen, on the other hand, is less readily produced and difficult to supplement. The composition of the present invention can simultaneously promote the production of both type I and type III collagen, thereby enhancing the skin's anti-aging capabilities.

[0180] Test 3: Effects of the composition on MMP1 and MMP3 in cells

[0181] 1. Instrument

[0182] Inverted microscope, cell counter, carbon dioxide incubator, centrifuge, microplate reader, and biological safety cabinet.

[0183] 2. Experimental Materials

[0184] Table 9

[0185]

[0186]

[0187] 3. Experimental Procedure

[0188] 3.1. Cell Culture, Toxicity Testing, and Modeling: Cell recovery and culture were performed according to standard cell culture standard operating procedures. Cytotoxicity experiments were performed. Based on the cytotoxicity results, an appropriate sample concentration was selected for subsequent antioxidant efficacy experiments. Specifically, a 0.16 mg / mL sample was selected for subsequent efficacy testing experiments. UVB irradiation was performed to establish the model. After irradiation, the cell plate was transferred to a biosafety cabinet for sample loading.

[0189] 3.2 Cell sampling, collection and detection: Complete culture medium containing 0.16 mg / ml samples in different ratios was added to the sample group wells, complete culture medium containing positive control was added to the positive control wells, and complete culture medium was added to the blank control wells and model wells, 1 mL per well. After the addition of the drug, the 12-well plate was placed in a CO2 incubator and cultured for 24 ± 4 hours. After the incubation and culture, centrifugation was performed for cell RNA extraction experiments. The collected cell samples were subjected to RNA extraction and cDNA synthesis according to the operating procedures for detecting gene expression in cells by fluorescence quantitative method. The synthesized cDNA product was diluted with DEPC water at a ratio of 1:1 and then subjected to qRT-PCR to detect the expression of MMPs in the cells.

[0190] 4. Data Processing

[0191] Gene expression was calculated using 2 -△△CT Methods The data obtained from the qRT-PCR experiment were processed and statistically analyzed.

[0192] 5. Experimental Results

[0193] Table 10

[0194]

[0195]

[0196] Matrix metalloproteinases (MMPs) are widely distributed in plants, vertebrates, and invertebrates. They are essential enzymes for the degradation of the extracellular matrix, degrading nearly all components of the extracellular matrix, including collagen and elastin. For example, MMP1 hydrolyzes types I, II, and III collagen in cells; MMP-3 hydrolyzes type IV collagen and gelatin, and activates MMP1 / 7 / 9.

[0197] The expression level of MMPs in normal stable tissues is extremely low, but its expression level increases under the stimulation of inflammatory cytokines, hormones, growth factors, etc. and during cell aging. Therefore, UVB stimulation can be used to induce increased expression of MMPs in human fibroblasts to establish a senescent cell model. By measuring the downregulation of MMPs content in senescent cells after administration, the efficacy of the composition in inhibiting MMPs expression can be evaluated.

[0198] Under the experimental conditions of this study, the relative gene expression of MMP1 in the blank control group (BC) cells was 0.56, which was 45.25% lower than that in the model group (M). This indicates that after UVB irradiation, the content of MMP1 in the model group (M) cells was significantly increased, and the aging cell model was successfully established, allowing for subsequent efficacy testing. The relative gene expression of MMP1 in the positive control group (PC) cells was 0.11, which was 89.16% lower than that in the model group (M), indicating that the test system is effective.

[0199] In terms of the effect on the relative expression of the MMP3 gene in cells, the relative expression of the MMP3 gene in the blank control group (BC) cells was 0.51, which was 49.73% lower than that in the model group (M), indicating that after UVB irradiation treatment, the MMP3 content in the model group (M) cells was significantly increased, and the aging cell model was successfully established, and subsequent efficacy testing can be carried out; the relative expression of the MMP3 gene in the positive control group (PC) cells was 0.38, which was 62.40% lower than that in the model group (M), indicating that the test system is effective.

[0200] Please refer to Table 10. The composition prepared by using a specific ratio of pyrroloquinoline quinone disodium salt, ergothioneine, spermidine and pomegranate extract can effectively inhibit the expression of MMP1 and MMP3. The compositions of Examples 1-5 were used to treat cells. Compared with the model group (M), the inhibition rates for MMP1 were all above 25%, and the inhibition rates for MMP2 were all above 20%. Among them, in Example 1, the composition with a ratio of pyrroloquinoline quinone disodium salt, ergothioneine, spermidine and pomegranate extract of 20:5:0.21:20 had an inhibition rate of more than 35% for the expression of MMP1 and MMP3, which was the highest inhibition rate.

[0201] When any one of the components pyrroloquinoline quinone disodium salt, ergothioneine, spermidine, and pomegranate extract is missing from the composition, the inhibitory effect on MMP1 and MMP3 expression is reduced. Referring to Comparative Examples 1-5, when one or two of the components are missing from the composition, the inhibition rates on MMP1 and MMP3 expression are all below 15%, significantly lower than that in Example 1.

[0202] When the ratios of the four components in the composition are outside the ranges defined by the present invention, the synergistic effect is significantly reduced, leading to a significant decrease in the inhibitory effect on the expression of MMP1 and MMP3. For Comparative Examples 6-10, the composition, in which the content of at least one component is outside the range defined by the present invention, exhibited MMP1 and MMP3 expression inhibition rates below 20%, also significantly lower than those in Example 1.

[0203] The above results show that when pyrroloquinoline quinone disodium salt, ergothioneine, spermidine and pomegranate extract are used in a specific proportion, they can produce a certain synergistic effect, which is more conducive to enhancing the effect of inhibiting the expression of MMP1 and MMP3.

[0204] The composition of the present invention can effectively inhibit the expression of matrix metalloproteinases (MMP-1) and MMP-3, two markers of skin aging. Furthermore, at specific ratios, a synergistic effect can occur, further enhancing the composition's inhibitory effect on matrix metalloproteinase expression. This effectively protects dermal fibroblasts, promotes the production of type I and type III collagen by fibroblasts, and increases the production rate of type I and type III collagen. Therefore, the composition of the present invention can effectively improve human skin elasticity, reduce wrinkles, and delay aging.

[0205] The above embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. An anti-aging composition, characterized in that Includes or consists of the following ingredients: 10-20 parts by weight of pyrroloquinoline quinone or its derivatives; 2 to 30 parts by weight of ergothioneine; 0.2 to 10 parts by weight of spermidine; and 10 to 650 parts by weight of pomegranate extract.

2. An anti-aging composition according to claim 1, characterized in that The anti-aging composition comprises or consists of the following ingredients: 12-20 parts by weight of pyrroloquinoline quinone or its derivatives; 2 to 30 parts by weight of ergothioneine; 0.2 to 6 parts by weight of spermidine; and 20 to 100 parts by weight of pomegranate extract.

3. An anti-aging composition according to claim 1, characterized in that The anti-aging composition comprises or consists of the following ingredients: 20 parts by weight of pyrroloquinoline quinone or its derivatives; 5 weight portions of ergothioneine; 0.21 parts by weight of spermidine; and 20 parts by weight of pomegranate extract.

4. An anti-aging composition according to claim 1, characterized in that The pyrroloquinoline quinone derivatives include pyrroloquinoline quinone disodium salt; and / or, The source of spermidine includes wheat germ extract.

5. Use of the anti-aging composition according to any one of claims 1 to 4 in anti-skin oxidation.

6. Use of the anti-aging composition according to any one of claims 1 to 4 in promoting the production of type I and type III collagen by skin fibroblasts and / or inhibiting the expression of matrix metalloproteinases MMP-1 and MMP-3, which are skin aging markers.

7. Use of the anti-aging composition according to any one of claims 1 to 4 in the preparation of a preparation for resisting skin oxidative damage.

8. Use of the anti-aging composition according to any one of claims 1 to 4 in the preparation of anti-aging preparations.

9. The use according to claim 7 or 8, characterized in that The preparation is a medicine, and the dosage form of the medicine includes tablets, capsules, oral liquid and injection.

10. The use according to claim 7 or 8, characterized in that The preparation is a food or a health product, and the types of the food or health product include oral liquid, capsule, tablet, liquid beverage, solid beverage and soft candy.

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